table 6-2 dabrafenib + trametinib
table 6-3 dabrafenib +/or trametinib

| Table 6-4: Dabrafenib + /or Trametinib Dose Modification for HFSR                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                               |                                                                                                                                                                                            |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| CTCAE Grade                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         | Adverse Event Management                                                                      | Action and Dose Modification                                                                                                                                                               |
| Grade 1a                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | Lifestyle changes recommended.b  Initiate symptomatic treatment if  clinically appropriatec | Continue study treatment at current dose  level.                                                                                                                                           |
| Grade 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             | Lifestyle changes recommended.b                                                              |  Interrupt study treatment until recovery to                                                                                                                                              |
|  Initiate symptomatic treatment.c                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | ≤ grade 1.d                                                                                   |                                                                                                                                                                                            |
|  Recovery to ≤ grade 1 within 7 days:  restart study treatment at previous dose  level.  If no recovery to ≤ grade 1 within 7 days or  ≥ 2nd occurrence: restart with study  treatment reduced by one dose level.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                               |                                                                                                                                                                                            |
| Grade ≥3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | Lifestyle changes recommended.b  Initiate symptomatic treatment.c  Consult dermatologist.  |  Interrupt study treatment until rash  recovers to ≤ grade 1.d  Restart with study treatment reduced by  one dose level.e  If 3rd occurrence, discontinue study  treatment permanently. |
| a  A full-body skin examination and removal of pre-existing calluses and keratotic skin is  recommended prior to initiation of study treatment. b  Life-style changes: (1) reduce exposure of hands and feet to hot water; (2) avoid traumatic activity  including vigorous exercise especially in the first 4 weeks after start of study treatment; (3) avoid  constrictive footwear; (4) avoid excessive friction on the skin when applying topical treatments; (5)  wear thick cotton socks and gloves, and shoes with padded insoles. c  Symptomatic treatments: (1) use moisturizing creams frequently and especially on hands and feet;  (2) consider topical keratolytics: urea 20-40% cream, or salicylic acid 6%, or tazarotene 0.1% cream,  or fluorouracil 5% cream; (3) erythematous areas: clobetasol propionate 0.05% ointment; (4) pain:  topical lidocaine 2%, and/or systemic pain medication such as nonsteroidal anti-inflammatory drugs,  codeine, and pregabalin. d  Approval of overall PI is required to restart study treatment after ≥21 days of interruption. e  escalation of study treatment to the previous dose level is allowed if no HFSR is observed in the 4  weeks subsequent to dose reduction. |                                                                                               |                                                                                                                                                                                            |
table 6-2 dabrafenib + trametinib
6.2.5 dabrafenib + trametinib dose

Cutaneous squamous cell carcinoma (SCC) has been observed in patients treated with dabrafenib and the combination of dabrafenib and trametinib. These treatment-related SCC should be surgically removed according to institutional practice; dose modifications or interruptions of the study treatment are not required. Occurrence of SCC must be reported as an SAE.
table 6-2 dabrafenib + trametinib
6.2.6 dabrafenib +/or trametinib dose

Cases of renal insufficiency have occurred in patients receiving the combination of dabrafenib and trametinib. Prior to start of study treatment, concomitant medications should be reviewed for the potential risk of inducing nephrotoxicity and, if so, should be modified if clinically possible.  
| for Renal Insufficiency                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------|
| Serum Creatinine Level                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Management Guideline                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Action and Dose  Modification                     |
| Serum creatinine increase >0.2 mg/dL (18 mcmol/L) BUT ≤0.5 mg/dL (44 mcmol/L)  above baseline                                                                                                                                                                                                                                                                                                                                                                                                                                    |  Recheck serum creatinine within 1  week.  Serum creatinine increase >1 week:  contact Overall PI.  If pyrexia is present, treat pyrexia as  per guidelines.a                                                                                                                                                                                                                                                                                                                                                                                                           | Continue study treatment at  the same dose level. |
| Serum creatinine increase >0.5 mg/dL (44 mcmol/L) OR >2 mg/dL (>177 mcmol/L)                                                                                                                                                                                                                                                                                                                                                                                                                                                     |  Monitor serum creatinine ≥2-times per  week.  Hospitalization may be necessary if  serum creatinine cannot be monitored  frequently.  If pyrexia is present, treat pyrexia per  guidelines.  Consult nephrologist if clinically  indicated.  Perform renal biopsy if clinically  indicated, for example:  Renal insufficiency persists despite  volume repletion.  Patient has new rash or signs of  hypersensitivity (such as elevated  eosinophil count).  Interrupt study treatment  until serum creatinine  recovers to baseline.  Restart study treatment.b |                                                   |
| a  NSAIDs can induce renal insufficiency, especially in patients with dehydration; encourage oral  fluids or consider IV fluids as clinically indicated. b  Investigator may restart at either the same or a reduced dose level. Escalation of study treatment  to previous dose level is allowed if another episode of renal insufficiency does not occur after 4  weeks of dose reduction. Consultation with the overall PI is required before restarting study  treatment if there is evidence of thrombotic microangiopathy. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                   |
table 6-2 dabrafenib + trametinib
6.2.7 dabrafenib +/or trametinib dose

Decreases of the left ventricular ejection fraction (LVEF) have been observed in patients receiving trametinib. Therefore, ECHOs must be performed at intervals outlined in the Study Calendar. The same procedure (either ECHO or MUGA, although ECHO is preferred) should be performed at baseline and at Week 9.  
| Management Requirements for LVEF Decrease                                                                                                                                                       |                                                                                                         |                                                                                                                                                                                                          |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Clinic                                                                                                                                                                                          | LVEF-drop (%) or CTCAE grade                                                                            | Dose Modification                                                                                                                                                                                        |
| Asymptomatic                                                                                                                                                                                    | Absolute decrease of >10% in  LVEF compared to baseline and  ejection fraction below the  institution's LLN.  Interrupt study treatment and repeat ECHO  within 2 weeks.a  If the LVEF recovers within 4 weeks (defined  as LVEF ≥LLN and absolute decrease ≤10% compared to baseline):  Consult with the overall PI and request  approval for restart.  Restart trametinib and dabrafenib (if cohort  1) at reduced doses by one dose level.  Repeat ECHO 2, 4, 8, and 12 weeks after restart; continue in intervals of 12 weeks  thereafter.  If LVEF does not recover within 4 weeks:  Consult with cardiologist.  Permanently discontinue trametinib.   Resumption of dabrafenib (if cohort 1) may  be considered after consultation with the  overall PI.  Repeat ECHO after 2, 4, 8, 12, and 16  weeks or until resolution.                                                                                                         |                                                                                                                                                                                                          |
| Symptomaticb                                                                                                                                                                                    |  Grade 3: resting LVEF 39- 20% or >20% absolute  reduction from baseline  Grade 4: Resting LVEF ≤20%. |  Permanently discontinue trametinib.  Consult overall PI for continuation of  dabrafenib (if cohort 1). Consult with  cardiologist.  Repeat ECHO after 2, 4, 8, 12, and 16 weeks  or until resolution. |
| a  If ECHO does not show LVEF recovery after 2 weeks, repeat ECHO 2 weeks later. b  Symptoms may include: dyspnea, orthopenea, and other signs and symptoms of pulmonary  congestion and edema. |                                                                                                         |                                                                                                                                                                                                          |
table 6-2 dabrafenib + trametinib
6.2.8 dabrafenib +/or trametinib dose

Increases in blood pressure (BP) have been observed in patients receiving trametinib. Recommendations for BP monitoring and CONFIDENTIAL
This document is confidential. Do not disclose or use except as authorized.  
Monitoring: All BP assessments are to be performed as follows:
In subjects with an initial BP reading within the hypertensive range, a second reading should be taken at least 1 minute later, with the two readings averaged to obtain a final BP measurement. The averaged value should be recorded in the eCRF.  
Visits to monitor increased blood pressure can be scheduled independently from the per-protocol visits outlined in the study calendar. Ideally, subsequent blood pressure assessments should be performed within 1 week.  
Persistent hypertension is defined as an increase of systolic BP
(SBP) >140 mmHg and/or diastolic BP (DBP) >90 mmHg in three consecutive visits with blood pressure assessments from two readings.  
Asymptomatic hypertension is defined as an increase of SBP
>140 mmHg and/or diastolic BP (DBP) >90 mmHg in the absence of headache, light-headedness, vertigo, tinnitus, episodes of fainting, or other symptoms indicative of hypertension.  
| Requirements for Hypertension   |                      |                   |
|---------------------------------|----------------------|-------------------|
| Event                           | Management Guideline | Dose Modification |
| (Scenario A)  Asymptomatic and persistenta SBP of ≥140 and <160 mmHg,  or DBP ≥90 and <100 mmHg OR  Adjust current or initiate new  antihypertensive  medication(s).  Titrate antihypertensive  medication(s) during the  next 2 weeks to achieve  well-controlled BP. If BP is  not well-controlled within 2  weeks, consider referral to a  specialist and go to scenario  (B).  Continue protocol therapy.  If BP is not well-controlled within 2  weeks, consider referral to a specialist  and go to scenario (B).  C linically significant increase in  DBP of 20 mmHg (but still  below 100 mmHg) (Scenario B)  Asymptomatic SBP ≥160 mmHg, or DBP ≥100 mmHg,  OR  Adjust current or initiate new  antihypertensive  medication(s).  Titrate antihypertensive  medication(s) during the  next 2 weeks to achieve  well-controlled BP.  Interrupt trametinib if clinically  indicated.  Once BP is well-controlled, restart  trametinib reduced by one dose level.  Dabrafenib (if cohort 1) may be  continued  Failure to achieve wellcontrolled BP within 2 weeks  in Scenario A. (Scenario C)  Symptomatichypertension   Adjust current or initiate new  antihypertensive  medication(s).  Titrate antihypertensive  medication(s) during the  next 2 weeks to achieve  well-controlled BP.  Referral to a specialist for  further evaluation and  follow-up is recommended.  Interrupt trametinib. Once BP is well controlled, restart  trametinib reduced by one dose level.  Dabrafenib (if cohort 1) may be  continued. OR  Persistent SBP ≥160 mmHg, or DBP ≥100 mmHg,  despite antihypertensive  medication and dose reduction  of study treatment (Scenario D) Refractory hypertension  unresponsive to above  interventions or hypertensive  crisis. Continue follow-up per   Discontinue trametinib. protocol.  Hold dabrafenib until BP is controlled.  If the treatment delay is >21 days,  overall PI should be consulted for  resumption of dabrafenib.                                 |                      |                   |
table 6-2 dabrafenib + trametinib
6.2.9 dabrafenib +/or trametinib dose

| QTc Prolongation                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                                                                         |
|-------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Prolongation                                                                                                      | Action and Dose Modification                                                                                                                                                                                                                                                                                                                                                                                                            |
|                                                                                                                  | QTcB ≥501 msec, or                                                                                                                                                                                                                                                                                                                                                                                                                      |
|                                                                                                                  | Uncorrected QT >600 msec, or                                                                                                                                                                                                                                                                                                                                                                                                            |
|                                                                                                                  | QTcB >530 msec for subjects                                                                                                                                                                                                                                                                                                                                                                                                             |
| with bundle branch block                                                                                          |  Interrupt dabrafenib and trametinib (cohort 1) or trametinib  (cohort 2) until QTcB prolongation resolves to grade 1 or  baseline.  If the event resolves, restart dabrafenib and trametinib  (cohort 1) or trametinib (cohort 2) treatment at current dose  level.  If the event does not resolve, permanently discontinue  dabrafenib and trametinib treatment.   If the event recurs, permanently discontinue study  treatment. |
| Abbreviations: msec = milliseconds; QTcB = QT interval on electrocardiogram corrected using the  Bazett's formula |                                                                                                                                                                                                                                                                                                                                                                                                                                         |  
6.2.10 Dabrafenib +/or Trametinib Dose Modification for **Diarrhea**
Episodes of diarrhea have been observed in patients receiving dabrafenib, trametinib, or both therapies in combination. Other, frequent causes for diarrhea including concomitant medications (*e.g.*, stool softeners, laxatives, antacids, *etc.*), infections caused by *C. difficile* or other pathogens, partial bowel obstruction, *etc.*, should be clinically excluded.  
Table 6-9: Dabrafenib +/or Trametinib Treatment Modification and Management Requirements for Diarrhea CTCAE Grade Management Action and Dose Modification  
| Management Requirements for Diarrhea                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                           |                           |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------|
| CTCAE Grade                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Management                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | Action and Dose Modification                                                                                                                                                              |                           |
| Uncomplicated  Diarrhea,1 Grade 1 or 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |  Diet: Stop all lactose containing products;  eat small meals, BRAT-diet (banana, rice,  apples, toast) recommended.  Hydration: 8-10 large glasses of clear  liquids per day (e.g., Gatorade or broth). Loperamide3 : Initially 4 mg, followed by 2  mg every 4 hours or after every unformed  stool; maximum 16 mg/day. Continue until  diarrhea-free for 12 hours.  Diarrhea >24 hours: Loperamide 2 mg  every 2 hours; maximum 16 mg/day.  Consider adding oral antibiotics.  Diarrhea >48 hous: Loperamide 2 mg  every 2 hours; maximum 16 mg/day. Add  budesonide or other second-line therapies  (otreotide, or tincture of opium) and oral  antibiotics. |  Continue study treatment.  If diarrhea is grade 2 for > 48  hours, interrupt study treatment  until diarrhea resolves to grade  ≤1.  Restart study treatment at the  same dose level. |                           |
| Uncomplicated  Diarrhea,1 Grade 3 or 4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |  Clinical evaluation mandatory. Loperamide3 : Initially 4 mg, followed by 2  mg every 4 hours or after every unformed  stool; maximum 16 mg/day. Continue until  diarrhea-free for 12 hours.  Oral antibiotics and second-line therapies if  clinically indicated  Hydration: Intravenous fluids if clinically  indicated.  Antibiotics (oral or intravenous) if clinically  indicated.  Intervention should be continued until the  subject is diarrhea-free for ≥24 hours.  Intervention may require hospitalization for  subjects at risk of life-threatening  complications.                                                                               |                                                                                                                                                                                          | Interrupt study treatment |
| until diarrhea resolves to ≤ grade 1.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                           |                           |
|  Restart with study treatment  reduced by one dose level.4  If 3 dose reductions of  study treatment are clinically  indicated, permanently  discontinue study treatment.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                           |                           |
| Any Complicated  Diarrhea2 1. Uncomplicated diarrhea defined by the absence of symptoms such as cramping,  nausea/vomiting, ≥ grade 2, decreased performance status, pyrexia, sepsis, neutropenia ≥ grade 3,  frank bleeding, and/or dehydration requiring intravenous fluid substitution. 2. Complicated diarrhea defined by the presence of symptoms such as cramping,  nausea/vomiting, ≥ grade 2, decreased performance status, pyrexia, sepsis, neutropenia ≥ grade 3,  frank bleeding, and/or dehydration requiring intravenous fluid substitution. 3. Loperamide should be made available prior to start of study treatment so loperamide  administration can begin at the first signs of diarrhea. 4. Escalation of study treatment to previous dose level is allowed after consultation with the medical  monitor and in the absence of another episode of complicated or severe diarrhea in the 4 weeks  subsequent to dose reduction. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                                                                                                                                                           |                           |  
6.2.11 Dabrafenib +/or Trametinib Dose Modification for **Vision Changes**
Episodes of vision changes have been observed in patients CONFIDENTIAL
This document is confidential. Do not disclose or use except as authorized.  
receiving dabrafenib, trametinib, or the combination of both therapies and can be caused by central serous retinopathy (CSR) with retinal pigment epithelial detachments (RPED), or Retinal Vein Occlusion (RVO). Patients are required to have a standard ophthalmic exam performed by an ophthalmologist at baseline, within 1 week prior to doing on week 9, and any time patients report visual disturbance. For Grade 2 visual changes or greater, this exam must occur within 24 hours of onset (or study team knowledge of the event). The exam will include indirect fundoscopic examination, visual acuity (corrected), visual field examination, tonometry, and direct fundoscopy. Special attention should be given to retinal (*e.g.*, CSR, RPED) or retinal vein abnormalities (*e.g.*, RVO).  
Guidelines regarding event management and dose reduction for visual changes considered to be related to study treatment are provided in the table below.  
| for Vision Changes                                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                              |                |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|----------------|
| CTCAE Grade                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | Management                                                                                                                                                                                                                                                                                                                                                                                                                                        | Action and Dose Modification |                |
| Grade 1 Asymptomatic or  symptomatic but not  limiting ADL; intervention  not indicated.                                                                                                                                                                                                                                                                                                                                                                                              |  Consult ophthalmologist any time  when patient reports visual  disturbance. If there is visual loss  or significant visual changes,  consult ophthalmologist  immediately (within 24 hours of  study team awareness)  Workup to rule out CSR, RPED or  RVO. Consult retinal specialist in  case of CSR or RVO.  Report CSR and RVO as  SAE.  Continue follow up examination(s)  (by retinal specialist if available) for  CSR, RPED and RVO. |                             | Continue study |
| treatmentat the same dose  level until ophthalmologic  examination can be  conducted.*                                                                                                                                                                                                                                                                                                                                                                                                |                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                              |                |
|  If ophthalmologic examination  cannot be performed within 7  days of onset, interrupt study  treatment until CSR and RVO  can be excluded and symptoms  resolve.  If CSR and RVO excluded,  restart study treatment at same  dose level.  If CSR: Interrupt study  treatment until symptoms  resolve and exam by retinal  specialist shows resolution.  May restart study treatment  with one dose level reduction  of trametinib.  If RVO: Permanently  discontinue trametinib. |                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                              |                |  
| for Vision Changes                                                                                                                                                                                                                                                                                                                                               |                                           |                              |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------|------------------------------|
| CTCAE Grade                                                                                                                                                                                                                                                                                                                                                      | Management                                | Action and Dose Modification |
| Grade 2 and Grade 3 Grade 2 defined as:  Symptomatic with  moderate decrease in  visual acuity (20/40 or  better); limiting  instrumental ADL; local or  non-invasive intervention  indicated.                                                                                                                                                                   |                                          | Consult ophthalmologist      |
| immediately (within 24 hours of  study team awareness).                                                                                                                                                                                                                                                                                                          |                                           |                              |
|  Workup to rule out CSR, RPED or  RVO. Consult retinal specialist in  case of CSR or RVO.  Report CSR and RVO as  SAE.  Continue follow up examination(s)  (by retinal specialist if available) for  CSR, RPED and RVO.                                                                                                                                       |                                          | Interrupt study treatment    |
| until signs and symptoms have  resolved to baseline.                                                                                                                                                                                                                                                                                                             |                                           |                              |
|                                                                                                                                                                                                                                                                                                                                                                 | If CSR and RVO                            |                              |
| excluded and symptoms  resolved to baseline, restart  study treatment reduced by one  dose level.                                                                                                                                                                                                                                                                |                                           |                              |
|  If CSR: Interrupt study  treatment until symptoms  resolve and exam by retinal  specialist shows resolution. If  CSR resolves restart study  treatment with trametinib  dose reduced by one dose  level.  If RVO: Permanently  discontinue trametinib.                                                                                                        |                                           |                              |
| Grade 3 defined as:  Symptomatic with marked  decrease in visual acuity  or marked visual field  defect (worse than 20/40  but better than 20/200);  severe pain or medically  significant; operative  intervention indicated. Grade 4 Sight-threatening  consequences; urgent  intervention indicated;  blindness (20/200 or  worse).                           |                                          | Consult ophthalmologist      |
| immediately (within 24 hours of  study team awareness).                                                                                                                                                                                                                                                                                                          |                                           |                              |
|  Workup to rule out CSR, RPED or  RVO. Consult retinal specialist in  case of CSR or RVO.  Report CSR and RVO as  SAE.  Continue follow up examination(s)  (by retinal specialist if available) for  CSR, RPED and RVO.                                                                                                                                       | Permanently discontinue  study treatment. |                              |
| Abbreviations: CSR = central serous retinopathy; RVO = retinal vein occlusion; SAE =  serious adverse event * If visual changes are clearly unrelated to study treatment (e.g., allergic conjunctivitis),  monitor closely but ophthalmic examination is not required. **If ocular toxicities do not resolve within 21 days, permanently discontinue trametinib. |                                           |                              |  
6.2.12 Dabrafenib +/or Trametinib Dose Modification for **Pneumonitis**
Pneumonitis has been observed in patients receiving trametinib. To reduce the risk of pneumonitis, patients will be monitored closely for symptoms, evaluated with imaging and functional tests when appropriate.  
Table 6-11: Dabrafenib + Trametinib Treatment Modification Requirements for  
Pneumonitis  
| Pneumonitis   |                                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                                          |
|---------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| CTCAE Grade   | Management                                                                                                                                                                                                                                                                                                                                          | Action and Dose Modification                                                                                                                                                                                                                                                                                             |
| Grade 1       |  CT scan (high-resolution with lung  windows) recommended.  Clinical evaluation and laboratory  work-up for infection.  Monitoring of oxygenation via pulseoximetry recommended.  Consultation with  pulmonologist recommended.                                                                                                                                                                                                                                                                                                                                                     | Continue study treatment at current  dose.                                                                                                                                                                                                                                                                               |
| Grade 2       |  CT scan (high-resolution with lung  windows).  Clinical evaluation and laboratory  work-up for infection.  Consult pulmonologist.  Pulmonary function tests: If <  normal, repeat every 8 weeks until ≥ normal.  Symptomatic therapy including  corticosteroids if clinically indicated.                                                      |  Interrupt study treatment until  recovery to grade ≤1.  Restart with study treatment  reduced by one dose level.  Escalation to previous dose level  after 4 weeks and consultation  with the overall PI may be  considered.  If no recovery to grade ≤1 within 4  weeks, permanently discontinue  study treatment. |
| Grade 3       |  CT scan (high-resolution with lung  windows).  Clinical evaluation and laboratory  work-up for infection.  Consult pulmonologist.  Pulmonary function tests: If <  normal, repeat every 8 weeks until ≥ normal.  Bronchoscopy with biopsy and/or  BAL if possible.  Symptomatic therapy  including corticosteroids as clinically  indicated. |  Interrupt study treatment until  recovery to grade ≤1.  After consultation with the overall  PI, study treatment may be  restarted reduced by one dose  level.  If no recovery to grade ≤1 within 4 weeks, permanently  discontinue study treatment.                                                                 |
| Grade 4       | Same as Grade 3.                                                                                                                                                                                                                                                                                                                                    | Permanently discontinue study  treatment.                                                                                                                                                                                                                                                                                |  
6.2.13 Dabrafenib +/or Trametinib Dose Modification for Liver Chemistry Changes Table 6-12: Dabrafenib +/or Trametinib Dose Modification Requirements for Liver Chemistry Changes Stopping Criteria and Action
table 6-12 dabrafenib +/or trametinib
6.2.9 dabrafenib +/or trametinib dose

Stopping Criteria and Action Liver chemistry stopping criteria are defined as follows. When any of the liver chemistry stopping criteria are met, **immediately discontinue study treatment**, perform liver event follow-up assessments, and monitor the patient until liver chemistries resolve, stabilize, or return to baseline values.  
ALT ≥3x ULN and bilirubin ≥2x ULN (>35% direct bilirubin) (or ALT ≥3x ULN and international normalized ratio [INR] >1.5, if INR measured). **NOTE:** If serum bilirubin fractionation is not immediately available, study treatment should be discontinued if ALT ≥3x ULN and bilirubin ≥2x ULN. Serum bilirubin fractionation should be performed if testing is available. If testing is unavailable, **record presence of detectable urinary bilirubin on dipstick**, indicating direct bilirubin elevations and suggesting liver injury.  
ALT ≥8x ULN. ALT ≥5x ULN but <8x ULN persists for 2 weeks. ALT ≥3x ULN if associated with the appearance or worsening of symptoms of hepatitis or hypersensitivity such as fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, or eosinophilia.  
ALT ≥5x ULN but <8x ULN and cannot be monitored weekly for >2 weeks.
table 6-12 dabrafenib +/or trametinib
7. adverse events list and

An adverse event is any untoward medical occurrence associated with the use of the study drug(s), whether or not considered drug related. An adverse event (AE) can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding, for example), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the study drug(s).
table 6-12 dabrafenib +/or trametinib
7.1 expedited adverse event reporting

Investigators **must** report to the DF/HCC Overall Principal Investigator (PI) any serious adverse event (SAE) that occurs from the time of the first dose of study treatment up until 90 days after the last dose of study treatment. The report should be submitted on the MedWatch 3500A form. A serious adverse event is any adverse event that is any of the following:
Fatal (i.e., the adverse event actually causes or leads to death)
Life threatening (i.e., the adverse event, in the view of the investigator, places the patient at immediate risk of death)
Requires or prolongs inpatient hospitalization Results in persistent or significant disability/incapacity (i.e., the adverse event results in substantial disruption of the patient's ability to conduct normal life functions)
A congenital anomaly/birth defect in a neonate/infant born to a mother exposed to the investigational product Considered a significant medical event by the investigator (e.g., may jeopardize the patient or may require medical/surgical intervention to prevent one of the outcomes listed above)
Pyrexia accompanied by hypotension, dehydration, renal insufficiency and/or severe (grade 3) rigors/chills in the absence of an obvious infectious cause should be reported as an SAE.  
Occurrence of Cutaneous squamous cell carcinoma (SCC) must be reported as an SAE.  
Central serous retinopathy (CSR) and Retinal Vein Occlusion (RVO) must be reported as an SAE.  
All adverse events that do not meet any of the criteria for serious should be regarded as **nonserious adverse events**
Investigators must report each serious adverse event to the Overall PI within 24 hours of learning of the occurrence. In the event that the participating investigator does not become aware of the serious adverse event immediately (e.g., participant sought treatment elsewhere), the participating investigator is to report the event within 24 hours after learning of it and document the time of his or her first awareness of the adverse event. Report serious adverse events by telephone, email or facsimile using the MedWatch 3500A form to:
Ryan J. Sullivan, MD Phone: 617-724-4000 Email: rsullivan7@mgh.harvard.edu Fax: 617-726-1949 The participating investigator must provide follow-up information on the serious adverse event. Follow-up information should describe whether the event has resolved or continues, if and how the event was treated, and whether the participant will continue or discontinue study participation.  
In addition, the participating investigator will be responsible for reporting these serious adverse events to Merck via the MedWatch 3500A form. The contact for Merck is listed below:
Alicia M. Schneider Scientific Leadership & Research Manager - Oncology Merck Investigator Studies Program/Scientific Engagements Global Center for Scientific Affairs| Merck Research Laboratories Phone: 267-305-0191 Email: Alicia.Schneider@merck.com Fax: 267-305-6534 7.2 Expedited Reporting to the Food and Drug Administration (FDA)
The Overall PI, as study sponsor, will be responsible for all communications with the FDA. The Overall PI will report to the FDA, regardless of the site of occurrence, any serious adverse event that meets the FDA's criteria for expedited reporting following the reporting requirements and timelines set by the FDA. The Overall PI must report to the FDA any suspected adverse reaction to study treatment (i.e., including active comparators) that is both serious and unexpected.
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7.3 expedited reporting to hospital

Participating investigators will report to their local Risk Management office any participant safety reports or sentinel events that require reporting according to institutional policy.
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7.4 routine adverse event reporting

All Adverse Events **must** be reported in routine study data submissions to the Overall PI on the toxicity case report forms. AEs reported through expedited processes (e.g., reported to the IRB, FDA, etc.) must also be reported in routine study data submissions.
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7.5 monitoring of adverse events

All adverse events, both serious and non-serious, and deaths that are experienced by participants will be collected from the time of the first dose of study treatment up until 90 days after the last dose study treatment, or until death if death occurs first. The presence and resolution of AEs and SAEs (with dates) should be documented on the appropriate case report form and recorded in the participant's medical record to facilitate source data verification. For some SAEs, the Overall PI or designee may follow-up by telephone, fax, and/or monitoring visit to obtain additional case details deemed necessary to appropriately evaluate the SAE report (e.g., hospital discharge summary, consultant report, or autopsy report).
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8. pharmaceutical information

A list of the adverse events and potential risks associated with the agents administered in this study can be found in the investigative drug brochure.
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8.1 pembrolizumab 8.1.1 description

Pembrolizumab (MK-3475) is a humanized anti-PD-1 mAb of the IgG4/kappa isotype with a stabilizing S228P sequence alteration in the fragment crystallizable (Fc) region. MK-3475 binds to human PD-1 and blocks the interaction between PD-1 and its ligands. The theoretical molecular weight of the polypeptide is 146,288 Da and its theoretical pI is 7.5. The parental murine anti-human PD-1 antibody (hPD-1.09A) was produced by immunizing mice with hPD-1 DNA. The MK-3475 antibody was generated by humanization of the parental antibody by the Medical Research Council (Cambridge, UK) using complementarilydetermining region (CDR) grafting technology (U.S. Patent No. 5,225,539). The gene segments encoding the variable heavy and light chains of MK-3475, as well as human IgG4, were codon-optimized, synthesized, and ligated into a vector. A single expression plasmid, pAPD11V1-GA was constructed for the expression of both the heavy and light antibody chains of MK-3475. The nucleotide sequences encoding the heavy and light chains, along with their respective promoters and poly A signal sequences have been confirmed by DNA sequence analysis. The pAPD11V1-GA expression vector was subsequently used to transfect CHO-DXB-11 cells for the development of the MK-3475-producing cell line. The theoretical molecular weight of the polypeptide is 146,288 Da and its theoretical pI is 7.5. Pembrolizumab exhibits linear pharmacokinetics at dose levels of clinical relevance (1-10 mg/kg). It exhibits low clearance and limited volume of distribution that is typical for therapeutic antibodies. Mean estimated t1/2 values are 14.1-21.6 days. The nomenclature of pembrolizumab is provided in the table below:
The nomenclature of pembrolizumab is provided in the table below:  
| Code Name       | MK 3475 (Anti-PD-1)                                                                                                                                                                |
|-----------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Other Code Name | SCH 900475 (Anti-PD-1)                                                                                                                                                             |
| Chemical Name   | Humanized X PD-1_mAb (H409A11) IgG4                                                                                                                                                |
| CAS Number      | 1374853-91-4                                                                                                                                                                       |
| CAS Name        | Anti-(human protein PDCD1 (programmed cell death 1)) immunoglobulin G4 (human-Mus musculus monoclonal heavy chain) disulfide with human-Mus musculus monoclonal light chain, dimer |
| Generic Name    | Not available                                                                                                                                                                      |
| Commercial Name | Pembrolizumab                                                                                                                                                                      |
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8.1.2 form

The investigator shall take responsibility for and shall take all steps to maintain appropriate records and ensure appropriate supply, storage, handling, distribution and usage of investigational product in accordance with the protocol and any applicable laws and regulations. Pembrolizumab is supplied as a clear to opalescent solution that is essentially free of extraneous particles and may contain proteinaceous particulates. One dosage form of pembrolizumab will be provided by Merck in Type I glass vials intended for single use only as summarized in the following table:  
| Product Name & Potency    | Dosage Form            |
|---------------------------|------------------------|
| Pembrolizumab 100 mg/ 4mL | Solution for Injection |  
Pembrolizumab solution for infusion is a sterile, non-pyrogenic, aqueous, preservative-free solution. Pembrolizumab solution for infusion contains an excess fill of 6.25 mg (equivalent to 0.25 mL solution) to ensure the recovery of label claim of 100 mg pembrolizumab per vial (equivalent to
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8.1.3 storage and stability

As specified in the Pharmacy Manual for pembrolizumab as provided by the Overall PI. Clinical supplies must be stored in a secure, limited-access location under the storage conditions specified on the label.
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8.1.4 compatibility

The following infusion set materials are compatible with Pembrolizumab  
PVC infusion set that is plasticized using Di-2-ethylhexyl Terephthalate DEHP
PVC and tri-(2-ethylhexyl) trimellitate (TOTM) infusion set
Polyethylene lined PVC infusion set  
Polyrethane Plybutadiene A sterile, non-pyrogenic, low-protein binding 0.2 to 5 μm in-line filter made of polyethersulfone (PES) or polysulfone must be used during administration to remove any adventitious particles. If the infusion set does not contain a 0.2 to 5 μm in-line filter, it is recommended to use a 0.2 to 5 μm add-on filter which may contain an extension line (the materials of the extension line and filter should be as mentioned above).
A sterile, non-pyrogenic, low-protein binding 0.2 to 5 μm in-line filter made of polyethersulfone (PES) or polysulfone must be used during administration to remove any adventitious particles. If the infusion set does not contain a 0.2 to 5 μm in-line filter, it is recommended to use a 0.2 to 5 μm add-on filter which may contain an extension line (the materials of the extension line and filter should be as mentioned above).
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8.1.5 handling

Receipt and dispensing of trial medication must be recorded by an authorized person at the trial site. Clinical supplies may not be used for any purpose other than that stated in the protocol.
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8.1.7 administration

Pembrolizumab will be administered as a 30 minute IV infusion using an infusion pump (treatment cycle intervals may be increased due to toxicity as described in Section 6). Sites should make every effort to target infusion timing to be as close to 30 minutes as possible. However, given the variability of infusion pumps from site to site, a window of -5 minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). Attach the infusion line to the pump and prime the line, either with normal saline (at least 25 mL) or with infusion solution as per local SOP, before starting the infusion.  
Maximum infusion rate should not exceed 6.7 ml/min through a peripheral or indwelling catheter. Use 30 mL normal saline to flush the infusion line at the end of the infusion if institutional guidelines allow. Unused infusion solution should not be used for another infusion of the same participant or different participant. DO NOT administer the product as an intravenous push or bolus. DO NOT combine, dilute or administer it as an infusion with other medicinal products. A central line is not required for Pembrolizumab administration, but may be used if available.
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8.1.8 ordering

Participating Institutions will order their own investigational agent (Pembrolizumab) directly from Merck using the Drug Supply Request Form. Please allow for 3 weeks for drug to arrive after the order is submitted. The Participating Institution will ensure that the pharmacy will be able to receive and store the agent according to state and federal guidelines. The local IRB should be kept informed of who will supply the agent (i.e., Merck pharmaceuticals Inc.) so that any regulatory responsibilities can be met in a timely fashion.
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8.1.9 accountability

The investigator is responsible for keeping accurate records of the clinical supplies received from Merck or designee, the amount dispensed to and returned by the subjects and the amount remaining at the conclusion of the trial.
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8.1.10 destruction and return

Upon completion or termination of the study, all unused and/or partially used investigational product will be destroyed at the site per institutional policy. It is the Investigator's responsibility to arrange for disposal of all empty containers, provided that procedures for proper disposal have been established according to applicable federal, state, local and institutional guidelines and procedures, and provided that appropriate records of disposal are kept.
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8.2 dabrafenib mesylate (gsk2118436b) (nsc

8.2.1 **Chemical Name:** N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzene sulfonamide, methanesulfonate salt
8.2.2 **Other Names:** GSK2118436, GSK2118436A (free base), Tafinlar 8.2.3 **Classification:** BRAF inhibitor 8.2.4 CAS Registry Number: 1195768-06-9 8.2.5 **Molecular Formula:** C23H20F3N5O2S2 - CH4O3S
8.2.6 **M.W.:** 615.68 (mesylate salt) 8.2.7 **Mode of Action:** Dabrafenib mesylate (GSK2118436B) is a potent and selective BRAF kinase inhibitor. This inhibition suppresses downstream activity of pERK, a biomarker, and has antiproliferative activity against BRAF mutant tumors. The mode of action is consistent with ATP-competitive inhibition.  
8.2.8 **How Supplied:** Dabrafenib mesylate (GSK2118436B) capsules will be supplied commercially.  
- 50 mg capsule is Swedish orange, size 2 with markings of four black bars.  
- 75 mg capsule is pink, size 1 with markings of four black bars.  
Capsule excipients include microcrystalline cellulose, magnesium stearate
(vegetable source), colloidal silicon dioxide. Shell composition consists of an opaque hypromellose capsule, composed of red iron oxide (E172), titanium dioxide (E171), and hypromellose (E464). Four black bars are printed on the hypromellose capsules using black ink. The black ink contains black iron oxide (E172), shellac, propylene glycol, and ammonium hydroxide.  
8.2.9 **Storage:** Store between 15°C to 30°C (59°F to 86°F). 8.2.10 **Stability:** Shelf-life studies of dabrafenib mesylate (GSK2118436B) are ongoing. 8.2.11 **Route of Administration:** Oral administration. Take dabrafenib mesylate
(GSK2118436B ) 1 hour prior or 2 hours after a meal. If a dose is missed, it should not be taken if it is less than 6 hours until the next dose.  
8.2.12 **Potential Drug Interactions:** *In vitro* studies show that dabrafenib mesylate
(GSK2118436B) induces CYP3A4, 2B6, 2C9,8, 2C9, and 2C19 enzymes. Use caution in patients who are taking substrates that are metabolized in these enzyme pathways.  
Dabrafenib mesylate (GSK2118436B) metabolism appears to be mediated by CYP3A4 and CYP2C8. Use caution if strong inducers or inhibitors of CYP2C8 or 3A4 are co-administered with dabrafenib.  
Patients should avoid ingesting the fruit or juice of Seville oranges, grapefruit, pummelos, exotic citrus fruits, or grapefruit hybrids for at least 24 hours prior to the start of dosing because of inhibition of intestinal CYP3A4.  
Dabrafenib solubility is pH-dependent and experiences decreased solubility at higher pH. Use caution in patients who are taking drugs that elevate gastric pH due to the theoretical risk of decreasing oral bioavailability of dabrafenib.
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8.2.13 availability

Dabrafenib mesylate (GSK2118436B) is FDA-approved for the treatment of unresectable or metastatic BRAF-mutant melanoma. It will be used at its standard dose and schedule for its indicated purpose. Commercial dabrafenib will be used throughout the study and obtained from specialty pharmacies and with coverage provided by participant's individual health insurance.
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8.3 trametinib dimethyl sulfoxide (gsk1120212b)

8.3.1 Chemical Name **(IUPAC):** equimolecular combination of N-(3-{3-cyclopropyl-5-
[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide with (methylsulfinyl)methane  
8.3.2 **Other Names:** trametinib, GSK1120212, JTP-74057, JTP-78296, JTP-75303, Mekinist
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8.3.3 cas registry number 1187431-43-1

8.3.4 **Classification:** MEK inhibitor 8.3.5 **Molecular Formula:** C26H23FIN5O4 - C2H6OS
8.3.6 **M.W.:** 693.54 (dimethyl sulfoxide solvate), 615.41 (anhydrous parent) 8.3.7 **Approximate Solubility:** Trametinib dimethyl sulfoxide is almost insoluble in water (<0.0001 mg/mL at 25° C)
8.3.8 **Mode of Action:** Trametinib dimethyl sulfoxide is a reversible, highly selective, allosteric inhibitor of mitogen-activated extracellular signal regulated kinase 1
(MEK1) and MEK2. Tumor cells commonly have hyperactivated extracellular signal-related kinase (ERK) pathways in which MEK is a critical component. Trametinib dimethyl sulfoxide inhibits activation of MEK by RAF kinases and MEK kinases.  
8.3.9 **Description:** Trametinib dimethyl sulfoxide is a white to almost white powder.
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8.3.10 how supplied tablets will

The tablet core contains mannitol, microcrystalline cellulose, hypromellose, croscarmellose sodium, magnesium stearate (non-animal), colloidal silicon dioxide, and sodium lauryl sulfate.  
- 0.5 mg tablets are white or yellow, modified oval, biconvex and filmcoated. Aqueous film coating consists of Opadry Yellow 03B120006
(hypromellose, titanium dioxide, polyethylene glycol, iron oxide yellow).  
- 2 mg tablets are pink, round, biconvex and film-coated. Aqueous film coating consists of Opadry Pink YS-1-14762-A, hypromellose (titanium dioxide, polyethylene glycol, polysorbate 80, iron oxide red).  
8.3.11 **Storage:** Store tablets at 2°C - 8°C (36°F - 46°F) in the original bottle. Bottles should be stored in the manufacturer's package carton for light protection. Protect from moisture. For additional storage instructions, please refer to package insert.  
8.3.12 **Stability:** Shelf life studies of trametinib dimethyl sulfoxide are ongoing. 8.3.13 **Route of Administration:** Oral. Take by mouth on an empty stomach, either 1 hour before or 2 hours after a meal. If a dose is missed, it should not be taken if it is less than 12 hours until the next dose.
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8.3.14 potential drug interactions

In vitro studies suggest that trametinib dimethyl sulfoxide is not a substrate of CYP enzymes or of human Pgp, BCRP, OATP1B1 or OATP1B3 transporters. Trametinib dimethyl sulfoxide is a weak CYP2C8 inhibitor and weak CYP3A4 inducer. Drug-drug interactions with sensitive substrates of 2C8 and 3A4 are not anticipated.  
8.3.15 **Availability**
Trametinib dimethyl sulfoxide (GSK1120212B) is FDA-approved for the treatment of unresectable or metastatic BRAF-mutant melanoma. It will be used at its standard dose and schedule for its indicated purpose.  
Commercial trametinib will be used throughout the study and obtained from specialty pharmacies and with coverage provided by participant's individual health insurance.
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9. biomarker correlative and special

Tumor biopsies are required be performed in all patients for both cohorts at four time points (as all patients are enrolled onto the first Stage of the Simon 2stage design):
1. Prior to commencing MTT 2. Following 2-week MTT lead-in 3. Following 6-weeks of combined pembrolizumab with MTT (if feasible) 4. A biopsy at the time of disease progression will also be performed (this is optional)
Multiple cores will be obtained and/or sufficient tumor excised at each biopsy time-point, assuming that it is safe to do so (e.g. patients with rapid responses may have very little tumor to biopsy safely at the latter time points). Core biopsies or portions of the excised tumor will be processed with the following hierarchy in mind:  
| 1. DNA/RNA/protein extraction         | (Sullivan)   |
|---------------------------------------|--------------|
| 2. Disaggregation and flow cytometry  | (Sharpe)     |
| 3. FFPE                               | (Sullivan)   |
| 4. Single-cell RNA seq of T cells     | (Hacohen)    |
| 5. Single-cell RNA seq of tumor cells | (Sharpe)     |  
Analysis will then be carried out to compare changes seen longitudinally and to determine if those changes are associated with improved outcomes to therapy with study treatment. Most of this work will exploratory in nature, as analysis CONFIDENTIAL
This document is confidential. Do not disclose or use except as authorized.  
such as the technology for single-cell sequencing of tumor and T-cells is emerging and no data is presently available to inform how these analyses might be used to help predict which patients are most likely to benefit from singleagent or combination therapy. However, based on previous work, there are two assays that will be incorporated into a predictive model, PD-L1 expression by immunohistochemistry (IHC) and immuno-sequencing (immunoSEQ) using the Adaptive Biotechnologies platform.
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9.1.1 handling of specimens(s)

As described above, four cores will be obtained and fixed. The SOP for collection, handling, and processing is attached (Appendix C). Samples will either be stored at the individual sites and sent in batch at the time of planned correlative study analysis or sent to and stored at the Sullivan Laboratory at MGH. FFPE samples will be stored at room temperature. Frozen samples will be stored in a -80 degree freezer.
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9.1.2 shipping of specimen(s)

Samples may be sent at the time of collection or in batches via overnight shipping. Frozen samples should be sent on dry ice; paraffin blocks may be sent at room temperature conditions. Please email laboratory notification of shipment prior to sending. The address:
Sullivan/Flaherty Laboratory c/o Michal Barzily, Ph.D.  
55 Fruit Street; Jackson 9th Floor Boston, MA 02114 Phone: 617-643-3614 Email: RSULLIVAN7@mgh.harvard.edu Site(s) Performing Correlative Study All sites are expected to participate in these correlative studies.
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9.2 pd-l1 immunohistochemistry

Immunohistochemistry for PD-L1 and CD8 will be performed in Dr. Jeffrey Engelman's laboratory using a monoclonal anti-PD-L1 antibody (Clone E1L3N, Cell Signaling Technologies; Danvers, MA) and a CD8 monoclonal antibody (4B11, RTU, Leica Biosystems, Buffalo Grove, IL), respectively, with an automated stainer (Bond Rx, Leica Microsystems, Buffalo Grove, IL). As there is no recognized standard PD-L1 scoring protocol, we will define PD-L1 positivity as membranous +/- cytoplasmic staining of tumor cells of any intensity using two different cut-offs (≥1% and ≥5 tumor cells) since both have been associated with clinical benefit to PD-1 pathway blockade in melanoma.
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9.3 adaptive biotechnologies immunoseq

FFPE specimens (or isolated tumor DNA) and frozen PBMC samples will be shipped to Adaptive Biotechnologies. Samples sent to Adaptive will be handled in accordance with Adaptive SOPs. All arriving packages will be opened the same day they arrive and inspected thoroughly for correct labeling and packaging integrity. Adaptive will perform "survey level" sequencing on each tissue sample and "deep level" sequencing on each of the PBMC derived gDNA samples provided. As part of these analyses, Adaptive uses a proprietary assay for the amplification and massive parallel sequencing of millions of CDR3 regions in the T cell receptor beta gene (TCRB) using a multiplex PCR amplification across the VDJ junction of rearranged TCRB.  
Using Adaptive's unique capacity to perform next generation sequencing of Tcell receptors for this study, a large amount of data will be generated using tissue specimens and PBMC samples from all collection time points. In addition to the immunoSEQ Analyzer software, which is available to all of Adaptive clients to aid in data analysis and visualization, Adaptive can directly involve its computational biology and biostatistics personnel in the data analysis.
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9.4 tumor microenvironment assessment

Tumor microenvironment assessment will be performed in collaboration with Dr. Arlene Sharpe. At least two biopsies will be earmarked for this work and will be sent to Dr. Sharpe's laboratory. To determine how the tumor cells are modulated during MTT alone, anti-PD1 plus MTT and anti-PD1 monotherapy combination, we will use single-cell and bulk RNA-seq, whole exome, etc. to assess the evolution of tumor cells during therapy. Sequential biopsies will provide a means to analyze evolution of tumor cell responsiveness and resistance to therapy To determine how immune cells in the tumor microenvironment are modulated during MTT alone, anti-PD1 plus MTT and anti-PD1 monotherapy combination, we will use single-cell and bulk RNA-seq, and assays to compare immune cell function (e.g., T cell activation vs. exhaustion using cellular immunologic assays, as well as analysis of phosphoproteins and ubiquitination signaling) in temporal biopsies. These studies should reveal how anti-tumor immunity is modulated during the lead-in phase of the clinical trial with MTT, during MTT and anti-PD1 combination therapy and during subsequent anti-PD1 monotherapy.
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9.5.1.2 blood-based braf assay

To explore the value of pretreatment BRAF level in predicting response to dabrafenib, trametinib, and Pembrolizumab, we will test isolated PBL and serum samples obtained prior to commencement of therapy. RNA will then be isolated from PBL and serum and BRAFV600 analysis will be performed as described in Figure 2.4.1. Quantitative analysis will be performed and levels will be reported in ng/uL. Two categories of levels will be analyzed, based on our previous data, those with values >900 ng/uL and < 900 ng/uL. To explore the pharmacodynamic effects of dabrafenib, trametinib, and Pembrolizumab therapy on peripheral blood BRAFV600 mutation detection, we will serial test isolated PBL and serum samples. RNA will then be isolated from PBL and serum and BRAFV600 analysis will be performed as described in Figure 2.4.1. Patients will have quantitative analysis before and baseline values will be in ng/uL. Peripheral blood will be drawn pretreatment, on Day 15 of the first cycle of treatment, and then on Day 1 of every subsequent cycle of therapy. Pre- and on-treatment BRAFV600 levels will be compared, and a blood-based response will be defined as a 50% or more decrease in signal based on acrylamide gel analysis. A blood-based progression will be defined as a doubling in signal based compared with nadir level on acrylamide gel analysis. A comparison of blood-based response or progression (>50% improvement vs. < 50% improvement; or 100% increase vs. < 100% increase) with clinical response (CR/PR by RECIST1.1) and clinical progression (PD) will then be performed to assess concordance between changes over time with our assay and with imaging.
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10.study calendar

Baseline evaluations are to be conducted within 2-weeks prior to start of protocol therapy. Scans must be done <4 weeks prior to the start of therapy. All assessments must be performed prior to administration of any study medication. On days when TSH is required, TSH is not required to be resulted prior to administration of study medication. All study assessments and medications should be administered within + 3 days of the protocol-specified date, unless otherwise noted. Laboratory values do not need to re-meet eligibility criteria on Week 1, Day 1.  
| Targeted  Therapy  Lead-In                                                                                                     | Combined Targeted  Therapy plus Immune  Therapy                 | Single-agent pembrolizumab                    | OffStudym                 | Follow   |       |       |       |       |    |           |    |    |    |
|--------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------|-----------------------------------------------|-----------------|----------|-------|-------|-------|-------|----|-----------|----|----|----|
| Wk 1                                                                                                                           | Wk 2                                                            | Wk 3                                          | Wk 6            | Wk 9     | Wk 12 | Wk 15 | Wk 18 | Wk 21 | Wk | Wk 27-107 | Up |    |    |
| 24                                                                                                                             |                                                                 |                                               |                 |          |       |       |       |       |    |           |    |    |    |
| Pre- Study  -14 to -1  days  X------------------------------------------------------------X                                    | - - - - - - - - - - - - - - - - - - - - - - - -                 | - - - - - - - -                               |                 |          |       |       |       |       |    |           |    |    |    |
| -                                                                                                                              |                                                                 |                                               |                 |          |       |       |       |       |    |           |    |    |    |
| Dabrafeniba                                                                                                                    | X-------------------------------------------------------------X | - - - - - - - - - - - - - - - - - - - - - - - | - - - - - - - - |          |       |       |       |       |    |           |    |    |    |
| Trametiniba                                                                                                                    | -                                                               |                                               |                 |          |       |       |       |       |    |           |    |    |    |
| Pembro                                                                                                                         | X                                                               | X                                             | X               | X        | X     | X     | X     | X     | Xl |           |    |    |    |
| Informed consent                                                                                                               | X                                                               |                                               |                 |          |       |       |       |       |    |           |    |    |    |
| Historyb                                                                                                                       | X                                                               | X                                             | X               | X        | X     | X     | X     | X     | X  | X         | X  |    |    |
| Dermatology Examc                                                                                                              | X                                                               | X                                             |                 |          |       |       |       |       |    |           |    |    |    |
| Ophthalmology Examd                                                                                                            | X                                                               | X                                             |                 |          |       |       |       |       |    |           |    |    |    |
| X----------------------------------------------------------------------------------------------------------------------------X | X                                                               |                                               |                 |          |       |       |       |       |    |           |    |    |    |
| Concurrent meds                                                                                                                | X                                                               |                                               |                 |          |       |       |       |       |    |           |    |    |    |
| Physical exam (Wt, BSAn ,  VSo )                                                                                               | X                                                               | X                                             | X               | X        | X     | X     | X     | X     | X  | X         |    | X  | X  |
| Height                                                                                                                         | X                                                               |                                               |                 |          |       |       |       |       |    |           |    |    |    |
| ECGe                                                                                                                           | X                                                               | X                                             | X               |          |       |       |       |       |    |           |    |    |    |
| Performance Status                                                                                                             | X                                                               | X                                             | X               | X        | X     | X     | X     | X     | X  | X         | X  | X  |    |
| CBC w/diff, plts                                                                                                               | X                                                               | X                                             | X               | X        | X     | X     | X     | X     | X  | X         | X  | X  |    |
| Serum chemistryf                                                                                                               | X                                                               | X                                             | X               | X        | X     | X     | X     | X     | X  | X         | X  | X  |    |
| TSH                                                                                                                            | X                                                               | X                                             | X               | X        | X     | X f   |       |       |    |           |    |    |    |
| X----------------------------------------------------------------------------------------------------------------------------X | X                                                               | XP                                            | XP              |          |       |       |       |       |    |           |    |    |    |
| Adverse event evaluationP                                                                                                      | X                                                               | X                                             | X               | X        | X     | Xr    |       |       |    |           |    |    |    |
| Radiologic evaluationg B-HCGh                                                                                                  | X                                                               | X                                             | X               | X        | X     | X     | X     |       |    |           |    |    |    |
| Echo/MUGAi                                                                                                                     | X                                                               | X                                             |                 |          |       |       |       |       |    |           |    |    |    |  
| BRAFj             | X    | X    | X    | X    | X   | X   | X   | X   | X   | X j   |
|-------------------|------|------|------|------|-----|-----|-----|-----|-----|-------|
| PBMCk             | X    | X    | X    | (X)  |     |     |     |     |     |       |
| Tumor Biopsiesk   | X    | X    | X    | (X)  |     |     |     |     |     |       |
| Rash Prophylaxisq | X  q | X  q | X  q | X  q |     |     |     |     |     |       |  
a. MAPK targeted therapy (MTT): In cohort 1, participants will be treated with the combination of dabrafenib and trametinib. In cohort 2, participants will be treated with trametinib. MTT will commence week 1, day 1 and be given continuously (except in the setting of toxicity) for two weeks in the lead-in phase and then 6-weeks in combination with pembrolizumab. Only if patients develop symptomatic or radiographic progression at anytime AFTER initial MTT-phase completion, then MTT may be resumed at a previously tolerated dose in combination with ongoing pembrolizumab.  
b. Participants will be seen on day 1 (+/-3 days) of week 1, week 3, and then every three weeks while remaining on therapy. If treatment is discontinued for a reason other than disease progression, study discontinuation, or withdrawal of consent, patients will be seen every 12 weeks (+/- 2 weeks).  
c. Dermatological examination done at screening, and within 1 week prior to dosing on week 9. A full-body skin examination and removal of pre-existing calluses and keratotic skin is recommended prior to initiation of study treatment (See Table 6-4).  
d. Ophthalmological examination done at screening and within 1 week prior to treatment on week 9. For Grade 2 visual changes or greater, an exam must occur within 24 hours of onset (or study team knowledge of the event). Consult ophthalmologist any time when patient reports visual disturbance. If there is visual loss or significant visual changes, consult ophthalmologist immediately (within 24 hours)
e. Single EKG will be collected at screening and repeated on day 1 of weeks 3 and 6 in cohort 1. f. Albumin, alkaline phosphatase, total bilirubin, BUN, creatinine, calcium, chloride, glucose, HCO3, LDH, phosphorus, potassium, total protein, SGOT [AST], SGPT [ALT], sodium. TSH will be performed at baseline, week 3 and then every 6 weeks.  
g. Radiographic imaging will be performed within 4 weeks of lead-in therapy commencement, within 1 week prior to Week 9, every six weeks thereafter (within 1 week prior to W15, W21, W27) until week 27, and then every 12 weeks (+/- 2 weeks) through 48 weeks of pembrolizumab therapy. Additionally, following initial disease progression, if a participant is treated beyond progression, tumor assessments must be repeated > 4 weeks after initial disease progression in order to confirm disease progression. The tumor assessment must not be repeated any sooner than 4 weeks after initial disease progression.  
h. Serum pregnancy test (women of childbearing potential). i. Ejection fraction measurement done at screening and within 1 week prior to dosing on week 9 . The same modality (ECHO or MUGA) should be used at baseline and at follow-up; should be performed with increased frequency if clinically indicated.  
j. Correlative blood analysis will be performed prior to MTT, prior to pembrolizumab therapy initiation, prior to every pembrolizumab dose through week 27, prior to every other pembrolizumab dose after week 27 (week 33, week 39, etc.), and at time of disease progression. BRAF samples will be collected in 1-10 mL green sodium heparin and 1-10 mL red top serum tubes k. Tumor biopsies and PBMC blood analysiswill be performed pretreatment, prior to pembrolizumab therapy initiation (up to 5 days prior), at the conclusion of combined targeted therapy plus pembrolizumab therapy (up to 5 days prior), and then at the time of disease progression. PBMC samples will be collected in 2-8 mL blue tiger top CPT tubes.  
l. Pembrolizumab will be administered at a dose of 200 mg IV every three weeks. m. Off-study evaluation visit is completed within 30 days after the last dose of study medication. If treatment is discontinued for a reason other than completion of two years of pembrolizumab, disease progression or withdrawal of consent, patients will be seen every 12 weeks (+/- 2 weeks) with imaging performed prior to these evaluations. If patients have an ongoing response or stable disease 18 months (+/- 2 months) after their last dose of pembrolizumab, patients will be followed every three-twelve months, based on investigator discretion, with imaging performed every 3-12 months.  
n. BSA should be calculated using the baseline height.  
o. Vitals signs include pulse, body temperature, blood pressure (BP), and respiratory rate. In subjects with an initial BP reading within the hypertensive range, a second reading should be taken at least 1 minute later per Section 6.2.8BRAD
p. Adverse Event Reporting: All adverse events, both serious and non-serious, will be collected from the time of the first dose of study treatment up until 90 days after the last dose of study treatment, or until death if death occurs first. The adverse events are followed to their resolution, or until the participating investigator assesses them as stable, or the participating investigator determines the event to be irreversible, or the participant is lost to follow-up. If the subject is not seen in clinic after the off-study visit, required Adverse Event details must be collected over the phone.  
q. Rash prophylaxis is recommended for the first 6 weeks of study treatment. See Table 6-3. r. If treatment is discontinued for a reason other than disease progression, study discontinuation, or withdrawal of consent, participants will be seen every 12 weeks (+/- 2 weeks) for imaging until progressive disease or death, whichever comes first.
table 6-12 dabrafenib +/or trametinib
11.measurement of effect

Although response is not the primary endpoint of this trial, participants with measurable and/or non-measurable disease will be assessed by RECIST 1.1.
table 6-12 dabrafenib +/or trametinib
11.1 antitumor effect - solid

Radiographic imaging will be performed within 4 weeks of lead-in therapy commencement, within 1 week prior to Week 9, every six weeks thereafter (within 1 week prior to W15, W21, W27) until week 27, and then every 12 weeks (+/- 2 weeks) through 48 weeks of pembrolizumab therapy. In addition to a baseline scan, confirmatory scans should also be obtained not less than 4 weeks following initial documentation of objective response. If treatment is discontinued for completion of two years of pembrolizumab, or for a reason other than disease progression or withdrawal of consent, patients will be seen every 12 weeks (+/- 2 weeks) with imaging performed within 2 weeks prior to these evaluations. Response and progression will be evaluated in this study using the new international criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guideline. *(Eisenhauer et al., 2009).* Changes in the diameter (unidimensional measurement) of the tumor lesions are used in the RECIST criteria.
table 6-12 dabrafenib +/or trametinib
11.1.1 definitions

Evaluable for Target Disease response. Only those participants who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for target disease response. These participants will have their response classified according to the definitions stated below. (Note: Participants who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) Evaluable Non-Target Disease Response. Participants who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for non-target disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions.
table 6-12 dabrafenib +/or trametinib
11.1.2 disease parameters

Measurable disease. Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as ≥ 20 mm by chest x-ray or ≥10 mm with CT scan, MRI, or calipers by clinical exam. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Note: Tumor lesions that are situated in a previously irradiated area might or might not be considered measurable. Malignant lymph nodes. To be considered pathologically enlarged and measurable, a lymph node must be ≥15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed.  
Non-measurable disease. All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, abdominal masses (not followed by CT or MRI), and cystic lesions are all considered non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same participant, these are preferred for selection as target lesions. Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Non-target lesions. All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow up.
table 6-12 dabrafenib +/or trametinib
11.1.3 methods for evaluation of

All measurements should be taken and recorded in metric notation using a ruler, calipers, or a digital measurement tool. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up.  
Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam. Clinical lesions. Clinical lesions will only be considered measurable when they are superficial (*e.g.*, skin nodules and palpable lymph nodes) and ≥10 mm in diameter as assessed using calipers (*e.g.*, skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended. Chest x-ray. Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung; however, CT is preferable. Conventional CT and MRI. This guideline has defined measurability of lesions on CT scan based on the assumption that CT thickness is 5mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size of a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (*e.g.* for body scans).  
Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if FDG-PET. While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment of progression (particularly possible 'new' disease). New lesions on the basis of FDG-PET imaging can be identified according to the following algorithm:
(a) Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD based on a new lesion. (b) No FDG-PET at baseline and a positive FDG-PET at follow-up: If the positive FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD. If the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, additional follow-up CT scans are needed to determine if there is truly progression occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-PET scan). If the positive FDG-PET at follow-up corresponds to a pre-existing site of disease on CT that is not progressing on the basis of the anatomic images, this is not PD. (c) FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in cases where a residual radiographic abnormality is thought to represent fibrosis or scarring. The use of FDG-PET in this circumstance should be prospectively described in the protocol and supported by disease-specific medical literature for the indication. However, it must be acknowledged that both approaches may lead to false positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity.  
Note: A 'positive' FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of the surrounding tissue on the attenuation corrected image. PET-CT. At present, the low dose or attenuation correction CT portion of a combined PET-CT is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed. Ultrasound. Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Ultrasound examinations cannot be reproduced in their entirety for independent review at a later data and, because they are operator dependent, it cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next. If new lesions are identified by ultrasound in the course of the study, confirmation by CT or MRI is advised. If there is concern about radiation exposure from CT, MRI may be used instead of CT in selected instances. Endoscopy, Laparoscopy. The utilization of these techniques for objective tumor evaluation is not advised. However, such techniques may be useful to confirm complete pathological response when biopsies are obtained or to determine relapse in trials where recurrence following complete response (CR) or surgical resection is an endpoint. Tumor markers. Tumor markers alone cannot be used to assess response. If markers are initially above the upper normal limit, they must normalize for a participant to be considered in complete clinical response. Specific guidelines for both CA-125 response (in recurrent ovarian cancer) and PSA response (in recurrent prostate cancer) have been published [*JNCI* 96:487-488, 2004; *J Clin* Oncol 17, 3461-3467, 1999; *J Clin Oncol* 26:1148-1159, 2008]. In addition, the Gynecologic Cancer Intergroup has developed CA-125 progression criteria which are to be integrated with objective tumor assessment for use in first-line trials in ovarian cancer [*JNCI* 92:1534-1535, 2000]. Cytology, Histology. These techniques can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (*e.g.*, residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease.
table 6-12 dabrafenib +/or trametinib
11.1.4 response criteria

11.1.4.1 Evaluation of Target Lesions Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.  
11.1.4.2 Evaluation of Non-Target Lesions
Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be nonpathological in size (<10 mm short axis).  
Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response.  
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator).
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11.1.4.3 evaluation of new lesions

The finding of a new lesion should be unequivocal (i.e. not due to difference in scanning technique, imaging modality, or findings thought to represent something other than tumor (for example, some 'new' bone lesions may be simply healing or flare of pre-existing lesions). However, a lesion identified on a follow-up scan in an anatomical location that was not scanned at baseline is considered new and will indicate PD. If a new lesion is equivocal (because of small size etc.), follow-up evaluation will clarify if it truly represents new disease and if PD is confirmed, progression should be declared using the date of the initial scan on which the lesion was discovered.  
11.1.4.4 Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| Target                                   | Non-Target                                                                          | New       | Overall   | Best Overall Response                            |
|------------------------------------------|-------------------------------------------------------------------------------------|-----------|-----------|--------------------------------------------------|
| Lesions                                  | Lesions                                                                             | Lesions   | Response  | when Confirmation is  Required*                  |
| CR                                       | CR                                                                                  | No        | CR        | >4 wks Confirmation**                            |
| CR                                       | Non-CR/NonPD                                                                                     | No        | PR        |                                                  |
| CR                                       | Not evaluated                                                                       | No        | PR        |                                                  |
| PR                                       | Non-CR/NonPD/not  evaluated                                                                                     | No        | PR        | >4 wks Confirmation**                            |
| SD                                       | Non-CR/NonPD/not  evaluated                                                                                     | No        | SD        | Documented at least once  >4 wks from baseline** |
| PD                                       | Any                                                                                 | Yes or No | PD        |                                                  |
| Any                                      | PD***                                                                               | Yes or No | PD        | no prior SD, PR or CR                            |
| Any                                      | Any                                                                                 | Yes       | PD        |                                                  |
|                                         | See RECIST 1.1 manuscript for further details on what is evidence of a new  lesion. |           |           |                                                  |
| **                                       | Only for non-randomized trials with response as primary endpoint.                   |           |           |                                                  |
| ***                                      | In exceptional circumstances, unequivocal progression in non-target lesions         |           |           |                                                  |
| may be  accepted as disease progression. |                                                                                     |           |           |                                                  |  
For Participants with Measurable Disease (*i.e.***, Target Disease)**
Note: Participants with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be reported as "*symptomatic deterioration."* Every effort should be made to document the objective progression even after discontinuation of treatment.  
For Participants with Non-Measurable Disease (*i.e.*, Non-Target Disease)  
| Non-Target Lesions   | New Lesions                                                                                                                                                                                                                                  | Overall Response   |
|----------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------|
| CR                   | No                                                                                                                                                                                                                                           | CR                 |
| Non-CR/non-PD        | No                                                                                                                                                                                                                                           | Non-CR/non-PD*     |
| Not all evaluated    | No                                                                                                                                                                                                                                           | not evaluated      |
| Unequivocal PD       | Yes or No                                                                                                                                                                                                                                    | PD                 |
| Any                  | Yes                                                                                                                                                                                                                                          | PD                 |
|                     | 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease since  SD is increasingly used as an endpoint for assessment of efficacy in some trials  so to assign this category when no lesions can be measured is not advised |                    |  
11.1.5 Duration of Response Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started, or death due to any cause. Participants without events reported are censored at the last disease evaluation). Duration of overall complete response: The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented, or death due to any cause. Participants without events reported are censored at the last disease evaluation.  
Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.
table 6-12 dabrafenib +/or trametinib
11.1.6 progression-free survival

Overall Survival: Overall Survival (OS) is defined as the time from randomization (or registration) to death due to any cause, or censored at date last known alive. Progression-Free Survival: Progression-Free Survival (PFS) is defined as the time from randomization (or registration) to the earlier of progression or death due to any cause. Participants alive without disease progression are censored at date of last disease evaluation. Time to Progression: Time to Progression (TTP) is defined as the time from randomization (or registration) to progression, or censored at date of last disease evaluation for those without progression reported.
table 6-12 dabrafenib +/or trametinib
11.1.7 response review

Independent, central review of the radiology assessment will be performed by through the Dana Farber Harvard Cancer Center Tumor Imaging Metrics Core (TIMC).
table 6-12 dabrafenib +/or trametinib
12.data reporting / regulatory requirements

Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7 (Adverse Events: List and Reporting Requirements).
table 6-12 dabrafenib +/or trametinib
12.1 data reporting 12.1.1 method

The ODQ will collect, manage, and perform quality checks on the data for this study. Note: If your study has been assigned to CDUS-Complete reporting, all adverse events (both routine and expedited) that have occurred on the study and meet the mandatory CDUS reporting guidelines must be reported via the monitoring method identified above. If your study has been assigned to CDUS-Abbreviated reporting, no adverse event reporting (routine or expedited) is required to be reported via CDUS.
table 6-12 dabrafenib +/or trametinib
12.1.2 responsibility for data submission

Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or data forms to the ODQ according to the schedule set by the ODQ.
table 6-12 dabrafenib +/or trametinib
for cdus and cmts submissions

Participant institutions are responsible for submitting CDUS data and/or data forms to either the Coordinating Center or to the Lead Organization on the study quarterly. The date for submission to the Coordinating Center or to the Lead Organization will be set by them. CDUS does not accept data submissions from the participant institutions on the study. When setting the dates, allow time for Coordinating Center compilation, Overall PI review, and timely submission to CTEP by the quarterly deadlines. For trials monitored by CTMS, a quarterly report of data will be provided by Theradex to the Coordinating Center. Either the Coordinating Center or the Lead Organization is responsible for compiling and submitting CDUS data to CTEP for all participant institutions and for providing the data to the Overall PI for review.
table 6-12 dabrafenib +/or trametinib
12.2 data safety monitoring

The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity and accrual data from this study. The committee is composed of clinical specialists with experience in oncology and who have no direct relationship with the study. Information that raises any questions about participant safety will be addressed with the Overall PI and study team. The DSMC will review each protocol up to four times a year or more often if required to review toxicity and accrual data. Information to be provided to the committee may include: up-to-date participant accrual; current dose level information; DLT information; all grade 2 or higher unexpected adverse events that have been reported; summary of all deaths occurring with 30 days of intervention for Phase I or II protocols; for gene therapy protocols, summary of all deaths while being treated and during active followup; any response information; audit results, and a summary provided by the study team.  
Other information (e.g. scans, laboratory values) will be provided upon request.
table 6-12 dabrafenib +/or trametinib
12.3 regulatory considerations 12.3.1 protocol

This protocol, the proposed informed consent and all forms of participant information related to the study (e.g., advertisements used to recruit participants) and any other necessary documents must be submitted, reviewed and approved by a properly constituted IRB governing each study location. Any changes made to the protocol must be agreed upon by the DFHCC Overall Principal Investigator, shall be submitted as amendments, and must be approved by the IRB prior to implementation. Any changes in study conduct must be reported to the IRB. The DF/HCC Overall Principal Investigator (or Protocol Chair) will disseminate protocol amendment information to all participating investigators. All decisions of the IRB concerning the conduct of the study must be made in writing.
table 6-12 dabrafenib +/or trametinib
12.3.2 informed consent

All participants must be provided a consent form describing this study and providing sufficient information for participants to make an informed decision about their participation in this study. The formal consent of a participant, using the IRB approved consent form, must be obtained before the participant is involved in any study-related procedure. The consent form must be signed and dated by the participant or the participant's legally authorized representative, and by the person obtaining the consent. The participant must be given a copy of the signed and dated consent document. The original signed copy of the consent document must be retained in the medical record or research file.  
12.3.3 Ethics and Good Clinical Practice (GCP)
This study is to be conducted according to the following considerations, which represent good and sound research practice:
E6 Good Clinical Practice: Consolidated Guidance www.fda.gov/downloads/RegulatoryInformation/Guidances/UCM12951 5.pdf US Code of Federal Regulations (CFR) governing clinical study conduct and ethical principles that have their origin in the Declaration of Helsinki  
o Title 21 Part 11 - Electronic Records; Electronic Signatures www.access.gpo.gov/nara/cfr/waisidx_02/21cfr11_02.html o Title 21 Part 50 - Protection of Human Subjects www.access.gpo.gov/nara/cfr/waisidx_02/21cfr50_02.html o Title 21 Part 54 - Financial Disclosure by Clinical Investigators www.access.gpo.gov/nara/cfr/waisidx_02/21cfr54_02.html o Title 21 Part 56 - Institutional Review Boards www.access.gpo.gov/nara/cfr/waisidx_02/21cfr56_02.html o Title 21 Part 312 - Investigational New Drug Application www.access.gpo.gov/nara/cfr/waisidx_02/21cfr312_02.html State laws
DF/HCC research policies and procedures http://www.dfhcc.harvard.edu/clinical-research-support/clinicalresearch-unit-cru/policies-and-procedures/
It is understood that deviations from the protocol should be avoided, except when necessary to eliminate an immediate hazard to a research participant. In such case, the deviation must be reported to the IRB according to the local reporting policy.
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12.4 study documentation

The investigator must prepare and maintain adequate and accurate case histories designed to record all observations and other data pertinent to the study for each research participant. This information enables the study to be fully documented and the study data to be subsequently verified. Original source documents supporting entries in the case report forms include but are not limited to hospital records, clinical charts, laboratory and pharmacy records, recorded data from automated instruments, microfiches, photographic negatives, microfilm or magnetic media, and/or x-rays.
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12.5 records retention

All study-related documents must be retained for the maximum period required by applicable federal regulations and guidelines or institutional policies.
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13.statistical considerations

This is a phase II trial to assess the effect of adding an abbreviated MAPK-targeted therapy to pembrolizumab in patients with unresectable or metastatic melanoma. Two cohorts of patients will be enrolled and analyzed separately: Patients with BRAFV600 mutant melanoma, and patients with BRAFV600 wild-type melanoma. The trial will be based on parallel Simon two-stage designs according to disease cohort and will provide preliminary estimates of toxicity and efficacy.
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13.1 study design/endpoints

Primary Endpoint: The rate of clinical benefit (CBR), defined as the proportion of patients who have CR/PR/SD at the week-24 scan while remaining off MAPK-targeted therapy (MTT) after induction. To be evaluable for the primary endpoint, patients must receive at least one dose of pembrolizumab, any patient who is unevaluable for response will be classified as a non-responder.
table 6-12 dabrafenib +/or trametinib
secondary endpoints

13.1.1 Overall Survival (OS): The time between the first dose of targeted therapy and death from any cause. For patients who are lost to follow-up or who have no documentation of death at the time of final analysis, follow-up will be censored at the date of last assessment of vital status.  
13.1.2 Safety and tolerability: Safety and toxicity will be monitored throughout the study.  
An early, comprehensive safety review will be conducted at the end of the firststage of the Simon two-stage design to review the data for unexpected severe toxicities due to the drug combination.  
13.1.3 Describe temporal changes in the tumor microenvironment as a function of therapy.  
Design: For each disease cohort, the Simon two-stage design is based on a type-I error rate of 0.1, at least 85% power, and a total sample size of 25 subjects. A complete safety assessment is planned to coincide with the review of clinical benefit at the end of the first stage. Within cohort, the null hypothesis of a true CBR of 0.50 will be tested against a onesided alternative CBR of 0.75. The benchmark CBR rate of 0.50 is based on the results of the Keynote 001 trial in patients with refractory or first-line malignant melanoma treated with pembrolizumab (Robert, ASCO 2016) that reported a CBR of 0.51 regardless of BRAF mutation status. A short course of MAPK-targeted therapy in addition to pembrolizumab would be considered worthy of further investigation if there is evidence of an improvement in CBR to 0.75. Fourteen (14) subjects that are evaluable for the primary endpoint (defined as receiving at least 1 dose of dabrafenib/trametinib/pembrolizumab combination therapy prior to treatment discontinuation for any reason.) will be enrolled in the first stage. If 8 or fewer subjects with clinical benefit are observed, enrollment will stop. If there are 9 or more subjects with clinical benefit, then an additional 11 subjects will be enrolled for a total of 25. The null hypothesis will be rejected if 16 or more subjects with clinical benefit are observed in 25 subjects. This design yields power of 86% (target type II error of 0.15) when the CBR rate is 0.75. If the null hypothesis is true, the probability is 0.79 that enrollment to a cohort will stop at the end of the first stage. This high probability of stopping under the null was chosen to require strong evidence of benefit before opening the second stage.
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13.2 sample size accrual rate

The total sample size for this study is 50 patients (25 patients per cohort). At an anticipated accrual rate of 2 patients per month, enrollment is expected to be completed in approximately two years.
table 6-12 dabrafenib +/or trametinib
13.3 analysis of primary endpoints

Clinical benefit rates will be presented for each cohort with 90% confidence intervals based on the method of Atkinson and Brown, which allows for the two-stage design. For samples of size 25, each confidence interval will be no wider than 0.35.
table 6-12 dabrafenib +/or trametinib
13.4 analysis of secondary endpoints

PFS and OS: The distributions of secondary endpoints of PFS and OS will be based on the method of Kaplan-Meier. Medians of the distributions of PFS and OS will be estimated and presented with 90% confidence intervals based on log(-log(endpoint)) methodology. One-year estimates of OS will also be presented using the same methods. Early Safety Review: An early, comprehensive safety review will occur at the end of the first stage of the Simon two-stage design and will take place when one cohort has reached the end of its respective first stage according to the Simon design. Since we do not anticipate that adverse events will differ according to BRAF mutation status, all available safety data will be combined from the two cohorts, resulting in an early safety review for up to 28 patients. All reported AEs will be summarized. For a sample of 28 patients, there is high probability of observing at least one event if a toxicity has an incidence of at least 5%. If the true incidence of an unexpected or severe toxicity is 5% or greater, the probability is at least 0.75 that one or more patients out of 28 will experience the toxicity during the early safety monitoring period. If the true incidence of unexpected or severe toxicity is 1% or less, the probability is 0.25 or less that at least one patient of 28 will experience the toxicity during the early safety monitoring period. 0 summarizes the probabilities of observing at least one patient with a severe or unexpected toxicity during the initial safety review for a range of true incidence rates.  
| True Incidence of Unexpected or Severe   | Probability of Observing One or More   |
|------------------------------------------|----------------------------------------|
| Toxicity                                 | Patients with Toxicity among First 28  |
| 1%                                       | 0.25                                   |
| 3%                                       | 0.57                                   |
| 5%                                       | 0.76                                   |
| 8%                                       | 0.90                                   |
| 9%                                       | 0.93                                   |
| 10%                                      | 0.95                                   |
| 15%                                      | 0.99                                   |
table 6-12 dabrafenib +/or trametinib
13.5 analysis of correlative measures

Analysis of MTT induced changes on T-cell clonality / PDL1 expression and correlation with clinical benefit. To investigate whether MTT-associated changes following a brief course of MTT can predict who will benefit from the addition of anti-PD1 therapy, assessments will be based on: (a) clonality of tumor-infiltration lymphocytes (TILs), and (b) PD-L1 IHC. TIL clonality is a term used to quantify the diversity of clones and frequency of any given clone. It is calculated by ImmunoSeq Adaptive Biotechnologies software using a clonality score, defined as [1-(entropy)/log2(\# of productive unique sequences)], where the entropy term takes into account the varying clone frequencies of TILs. A maximally diverse population, where every sequence is represented one time, is associated with a clonality score of zero, and a perfectly monoclonal population with a clonality score of one. Assessment times will be pre-treatment and after the 2-week MTT lead-in. Each cohort will be investigated separately. To investigate the hypothesis that there will be an increase in TIL clonality after MTT lead-in, the overall changes in clonality of TILs after MTT will be expressed as a fold-change (2-week/pre) and compared with unity using a Wilcoxon signedrank test with 80% power, and a 0.1, two-sided significance level. For a range of standard deviations of the fold-change between 0.8 and 1.2, we will be able to detect effect sizes between 1.4 and 1.65 for this comparison. We will further refine this question to address whether increases in clonality are related to response. Clones that comprise the largest percentage of the total T-cell population after MTT lead-in will be identified and sorted by frequency. The percentage of the overall T-cell population included in the top 5% of clones will be compared according to response (achieving/not achieving clinical benefit) using Wilcoxon rank-sum tests. If the observed CBR is 0.75, resulting in 19 patients with clinical benefit and 6 without, a Wilcoxon rank-sum test with 0.1, two-sided significance level will have 80% power to detect a difference in percentage T-cells that is approximately 1.3 times the common standard deviation. Similar analyses will be performed for the top 2.5% or 1% of clones. To assess if changes in clonality after MTT lead-in are associated with infiltration by new TIL clones, we will compare pre-treatment and post-MTT biopsies to identify clones that were absent from the pre-treatment biopsies, and summarize the percentage of individual clones that were identified to be new. This will be assessed for all patients in a cohort using Wilcoxon signed-rank tests, and according to clinical response using Wilcoxon rank-sum tests. In additional analyses, the percentage of pre-existing clones, rather than new clones, will be compared according to response to see if patients with high proportions of preexisting clones after MTT lead-in exhibit a better response than patients with low proportions. For PD-L1 expression, patients will be classified, retrospectively, into two subgroups defined by clinical benefit at 24 weeks. Fold-changes in PD-L1 IHC (2-week/pre) will be compared for patients achieving/not achieving clinical benefit using Wilcoxon rank-sum tests. If the observed CBR is 0.75, resulting in group sizes of 19 and 6, a Wilcoxon rank-sum test with 0.1, two-sided significance level will have 80% power to detect a difference in fold-changes that is approximately 1.3 times the common standard deviation.
table 6-12 dabrafenib +/or trametinib
13.6 analysis of safety and

Analysis set and grouping for the analyses: All patients who receive one or more doses of the drug combination will be included in the safety analysis. All listings and tables will be presented by treatment. Adverse events (AEs): All adverse events recorded during the study will be summarized. The incidence of treatment-emergent adverse events will be summarized by treatment according to primary system organ class, severity based on the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, type of adverse event, and relationship to treatment. Deaths reportable as SAEs and non-fatal serious adverse events will be listed by patient and tabulated by primary system organ class, type of adverse event, and dose level. Laboratory abnormalities: All laboratory values will be converted into SI units, as appropriate, and the severity grade calculated using CTCAE, version 4.0. Parameters for which a grading does not exist will be classified into low/normal/high group by means of laboratory normal ranges. For each laboratory test (e.g., hematology, biochemistry) a listing of laboratory values will be provided by laboratory parameter, patient and treatment group. The frequency of notable lab abnormalities (i.e., newly occurring CTCAE grade-3 or -4 laboratory toxicities), will be displayed by parameter, cycle, and dose level. Similarly, the frequency of all laboratory abnormalities will be displayed by parameter, worst CTCAE version 4.0 grade experienced and dose level. A separate listing will display notable laboratory abnormalities (i.e., newly occurring CTCAE grade-3 or -4 laboratory toxicities). Laboratory data will be summarized by presenting grade shift tables for those parameters for which CTCAE version 4.0 allows classification. All remaining data will be summarized by presenting shift tables based on normal ranges. Laboratory data will be also be displayed by presenting summary statistics of raw data and change from baseline values (means, medians, standard deviations, ranges).
table 6-12 dabrafenib +/or trametinib
13.7 reporting and exclusions 13.7.1

Evaluation of toxicity. *Define "evaluable."* For example: All participants will be evaluable for toxicity from the time of their first treatment.
table 6-12 dabrafenib +/or trametinib
13.7.2 evaluation of the primary

Evaluation of response. All participants included in the study must be assessed for response to treatment, even if there are major protocol treatment deviations or if they are ineligible. Each participant should be assigned one of the following categories: 1) complete response, 2) partial response, 3) stable disease, 4) progressive disease, 5) early death from malignant disease, 6) early death from toxicity, 7) early death because of other cause, or 9) unknown (not assessable, insufficient data). By arbitrary convention, category 9 usually designates the "unknown" status of any type of data in a clinical database.  
All of the participants who met the eligibility criteria (with the possible exception of those who did not receive triplet therapy) should be included in the main analysis of the response rate. Participants in response categories 4-9 should be considered to have a treatment failure (disease progression). Thus, an incorrect treatment schedule or drug administration does not result in exclusion from the analysis of the response rate. Precise definitions for categories 4-9, and sometimes category 3, will be protocol specific. All conclusions should be based on all eligible participants. Subanalyses may then be performed on the basis of a subset of participants, excluding those for whom major protocol deviations have been identified (e.g., early death due to other reasons, early discontinuation of treatment, major protocol violations, etc.). However, these subanalyses may not serve as the basis for drawing conclusions concerning treatment efficacy, and the reasons for excluding participants from the analysis should be clearly reported. The 95% confidence intervals should also be provided.
table 6-12 dabrafenib +/or trametinib
14.publication plan

The results should be made public within 24 months of reaching the end of the study. The end of the study is the time point at which the last data items are to be reported, or after the outcome data are sufficiently mature for analysis, as defined in the section on Sample Size, Accrual Rate and Study Duration. If a report is planned to be published in a peer-reviewed journal, then that initial release may be an abstract that meets the requirements of the International Committee of Medical Journal Editors. A full report of the outcomes should be made public no later than three (3) years after the end of the study.
table 6-12 dabrafenib +/or trametinib
references

Divito, K. A., Charette, L. A., Rimm, D. L., and Camp, R. L. (2004). Long-term preservation of antigenicity on tissue microarrays. Lab Invest 84, 1071-8.  
Fergenbaum, J. H., Garcia-Closas, M., Hewitt, S. M., Lissowska, J., Sakoda, L. C., and Sherman, M. E. (2004). Loss of antigenicity in stored sections of breast cancer tissue microarrays. Cancer Epidemiol Biomarkers Prev 13, 667-72.  
Hewitt, S. M., Lewis, F. A., Cao, Y., Conrad, R. C., Cronin, M., Danenberg, K. D.,
Goralski, T. J., Langmore, J. P., Raja, R. G., Williams, P. M., et al. (2008). Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 132, 1929-35.  
Research, N. C. I. O. O. B. a. B. (2007). National Cancer Institute Best Practices for Biospecimen Resources Riss, J., Khanna, C., Koo, S., Chandramouli, G. V., Yang, H. H., Hu, Y., Kleiner, D. E.,
Rosenwald, A., Schaefer, C. F., Ben-Sasson, S. A., et al. (2006). Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma. Cancer Res 66, 7216-24.  
Singh, B. (2007). Initial pathology assessment prior to preoperative therapy. In Preoperative Therapy in Invasive Breast Cancer: Reviewing the State of the Science and Exploring New Research Directions, (Bethesda, Maryland).
table 6-12 dabrafenib +/or trametinib
appendix a performance status criteria

| ECOG Performance Status Scale                                                                                                                                                               | Karnofsky Performance Scale                                                  |                                                                  |                                                 |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|------------------------------------------------------------------|-------------------------------------------------|
| Grade                                                                                                                                                                                       | Descriptions                                                                 | Percent                                                          | Description                                     |
| 100                                                                                                                                                                                         | Normal, no complaints, no                                                    |                                                                  |                                                 |
| Normal activity. Fully active,                                                                                                                                                              | evidence of disease.                                                         |                                                                  |                                                 |
| 0                                                                                                                                                                                           | able to carry on all pre-disease                                             | Able to carry on normal activity;                                |                                                 |
| performance without restriction.                                                                                                                                                            | 90                                                                           | minor signs or symptoms of  disease.                             |                                                 |
| Symptoms, but ambulatory.  Restricted in physically  strenuous activity, but  ambulatory and able to carry  out work of a light or sedentary  nature (e.g., light housework,  office work). | 80                                                                           | Normal activity with effort; some  signs or symptoms of disease. |                                                 |
| 1                                                                                                                                                                                           | Cares for self, unable to carry on                                           |                                                                  |                                                 |
| 70                                                                                                                                                                                          | normal activity or to do active  work.                                       |                                                                  |                                                 |
| In bed <50% of the time.                                                                                                                                                                    | Requires occasional assistance,                                              |                                                                  |                                                 |
| Ambulatory and capable of all                                                                                                                                                               | 60                                                                           | but is able to care for most of  his/her needs.                  |                                                 |
| 2                                                                                                                                                                                           | self-care, but unable to carry  out any work activities. Up and              | Requires considerable                                            |                                                 |
| about more than 50% of waking                                                                                                                                                               | 50                                                                           | assistance and frequent medical                                  |                                                 |
| hours.                                                                                                                                                                                      | care.                                                                        |                                                                  |                                                 |
| 40                                                                                                                                                                                          | Disabled, requires special care  and assistance.                             |                                                                  |                                                 |
| 3                                                                                                                                                                                           | In bed >50% of the time.  Capable of only limited selfcare, confined to bed or chair                                                                              | Severely disabled, hospitalization                               |                                                 |
| more than 50% of waking                                                                                                                                                                     | 30                                                                           | indicated. Death not imminent.                                   |                                                 |
| hours. 100% bedridden. Completely                                                                                                                                                           | 20                                                                           | Very sick, hospitalization  indicated. Death not imminent.       |                                                 |
| 4                                                                                                                                                                                           | disabled. Cannot carry on any  self-care. Totally confined to  bed or chair. | 10                                                               | Moribund, fatal processes  progressing rapidly. |
| 5                                                                                                                                                                                           | Dead.                                                                        | 0                                                                | Dead.                                           |
appendix b information on possible
appendix a performance status criteria

Information on Possible Interactions with Other Agents for Patients and Their Caregivers and Non-Study Healthcare Team The patient ____________________________ is enrolled on a clinical trial using the experimental agents, Dabrafenib plus Trametinib dimethyl sulfoxide or trametinib dimethyl sulfoxide. This clinical trial is sponsored by the Dana Farber Harvard Cancer Center. This form is addressed to the patient, but includes important information for others who care for this patient.  
Dabrafenib mesylate and trametinib dimethyl sulfoxide interact with many drugs that are processed by your liver. Because of this, it is very important to tell your study doctors about all of your medicine before you start this study. It is also very important to tell them if you stop taking any regular medicine, or if you start taking a new medicine while you take part in this study. When you talk about your medicine with your study doctor, include medicine you buy without a prescription at the drug store (over-the-counter remedy), or herbal supplements such as St. John's wort. Many health care prescribers can write prescriptions. You must also tell your other prescribers (doctors, physicians' assistants or nurse practitioners) that you are taking part in a clinical trial. Bring this paper with you and keep the attached information card in your wallet. These are the things that you and they need to know: Dabrafenib mesylate interacts with certain specific enzymes in your liver.  
The enzymes in question are *CYP450 3A4, 2C8, 2C9, 2C19, 2B6.* Dabrafenib mesylate levels are affected by some of these enzymes and can lower the levels of other medicines you take.  
Dabrafenib mesylate must be used very carefully with other medicines that need these liver enzymes to be effective or to be cleared from your system.  
Other medicines may also affect the activity of the enzyme.  
o Substances that increase the enzyme's activity ("inducers") could reduce the effectiveness of the drug, while substances that decrease the enzyme's activity ("inhibitors") could result in high levels of the active drug, increasing the chance of harmful side effects. Dabrafenib mesylate should not be taken with any other drugs that are strong inducers or inhibitors of CYP 3A4 or 2C8. Prohibited medications include azole antifungals, some antiepileptic drugs, some antibiotics and some immunosuppressants. Please check with the study investigator before prescribing or dispensing strong inhibitors/inducers of CYP 3A4 or 2C8. Mild/moderate inhibitors/inducers should be used with caution.  
o Dabrafenib mesylate is considered an inducer of CYP 3A4, CYP2C8/9, 2B6 and possibly 2C19 , meaning that it can decrease the levels of other drugs that are processed by these enzymes. This can lead to harmful side effects and/or reduce the effectiveness of those medications.  
You and healthcare providers who prescribe drugs for you must be careful about adding or removing any drug in this category.  
Before you start the study, your study doctor will work with your regular prescriber to switch any prohibited medicines that are considered "strong inducers/inhibitors or substrates of *CYP 3A4 and 2C8*."
Your prescribers should look at this web site http://medicine.iupui.edu/clinpharm/ddis/table.aspx or consult a medical reference to see if any medicine they want to prescribe is on a list of drugs to avoid.  
Please be very careful! Over-the-counter drugs have a brand name on the labelit's usually big and catches your eye. They also have a generic name—it's usually small and located above or below the brand name, and printed in the ingredient list. Find the generic name and determine, with the pharmacist's help, whether there could be an adverse interaction.  
Be careful:
o If you take acetaminophen regularly: You should not take more than 3 grams a day if you are an adult or 2.4 grams a day if you are older than 65 years of age. Read labels carefully! Acetaminophen is an ingredient in many medicines for pain, flu, and cold.  
o If you take herbal medicine regularly: You should not take St. John's wort while you are taking dabrafenib mesylate Other medicines can be a problem with your study drugs.  
You should check with your doctor or pharmacist whenever you need to use an over-the-counter medicine or herbal supplement.  
Your regular prescriber should check a medical reference or call your study doctor before prescribing any new medicine for you.
appendix b information on possible
your study doctor's name is

INFORMATION ON POSSIBLE DRUG INTERACTIONS  
You are enrolled on a clinical trial using the experimental agent __________________. This clinical trial is sponsored by the NCI. __________________ interacts with drugs that are processed by your liver. Because of this, it is very important to:  
Tell your doctors if you stop taking regular medicine or if you start  
taking a new medicine.  
Tell all of your prescribers (doctor, physicians' assistant, nurse  
practitioner, and pharmacist) that you are taking part in a clinical trial.  
Check with your doctor or pharmacist whenever you need to use  
an over-the-counter medicine or herbal supplement.  
__________________ interacts with a specific liver enzyme called CYP______, and must be used very carefully with other medicines that interact with this enzyme.  
Before you start the study, your study doctor will work with your regular prescriber to switch any medicines that are considered "strong inducers/inhibitors or substrates of **CYP______**."
Before prescribing new medicines, your regular prescribers should go to http://medicine.iupui.edu/clinpharm/ddis/table.aspx for a list of drugs to avoid, or contact your study doctor.  
Your study doctor's name is **_____________________________**  
|  Your study doctor's name is _____________________________ and can be contacted at _________________________________.   |
|--------------------------------------------------------------------------------------------------------------------------|
appendix b information on possible
appendix c bioassay

PROCESSING OF TISSUE SPECIMENS Adapted from: 1. Leyland-Jones, B.R. et al. Recommendations for collection and handling of specimens from group breast cancer clinical trials. J Clinical Oncology, 26 (34): 56385644, 2008.  
-Full guidelines from that manuscript are referenced on the World Wide Web at http://ctep.cancer.gov/guidelines/spec_bc_grptrials.html 2. University of Texas MD Anderson Cancer Center Institutional Tissue Bank Standard Operating Procedures, Version 7.0
appendix b information on possible
materials/equipment

| Liquid Nitrogen in approved LN2 transport carrier   | Surgical mask/eye protection   |
|-----------------------------------------------------|--------------------------------|
| Safety glasses or face shield                       | Clean Laboratory coat          |
| Freezer gloves                                      | Clean protective shoes         |
| Disposable latex gloves                             | Cryovials                      |
| Disposable scalpels or scalpel blades (or single    | Racks for cryovials            |
| edge razor blade) Forceps                           | Petri dish                     |
| Histoprep Marker                                    | OCT cyro-compound              |
| TissueTek cryomold                                  | Kimwipes                       |
| 10% Neutral Buffered formalin                       | 100% Isopropanol               |
| 95% Ethanol (alcohol)                               |                                |
appendix b information on possible
hazardous materials

1. 10% Neutral Buffered Formalin Formaldehyde : severe eye and skin irritant. Sensitizes by skin and respiratory contact. Toxic by ingestion and inhalation. Target organs effects on respiratory system. Corrosive. Carcinogen.  
| Emergency First Aid Procedures:   | Eye: Irrigate immediately with large quantity of  water for a least 15 minutes. Get medical  attention immediately Skin: Flush with water for at least 15 minutes Ingestion: Dilute immediately with water or  milk. Induce vomiting. Call physician.   |
|-----------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Inhalation: Remove to fresh air. Give artificial  respiration if necessary.   |                                                                                                                               |
|-------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|
| If released or spilled:                                                       | Use formaldehyde spill kit for small spills of  1000ml; For larger spills contact Environmental  Health & Safety              |
| Waste Disposal Method                                                         | Whatever cannot be saved for recovery or  recycled should be disposed of according to  local, state, and federal regulations. |
| Respiratory Protection                                                        | If the exposure level is exceeded, wear a full  facepiece respirator equipped with a  formaldehyde cartridge.                 |
| Ventilation                                                                   | Work in well ventilated areas                                                                                                 |
| Precautionary Labeling                                                        | Label with formaldehyde label                                                                                                 |
| Handling and Storage                                                          | Wash hands thoroughly after handling. Avoid                                                                                   |
| Considerations                                                                | eye contact. Protect from freezing and physical  damage. Store at controlled room  temperature, 15-30 C.                     |  
| 2.                                                                                                                                | Liquid Nitrogen                                       |
|-----------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|
| Emergency First Aid                                                                                                               | For Cold Liquid Frostbite:                            |
| Procedures:                                                                                                                       | If any liquefied atmosphere gas contacts the skin or  eyes, remove any clothing that may constrict blood  circulation to the frozen area. Immediately flood or  submerge the affected body area with large quantities of  clean, unheated water and then apply cold compresses.  A source of water should be nearby and easily  accessible wherever liquid nitrogen activities are being  conducted. If the skin is blistered, or there is any chance  that the eyes have been affected, get the patient to a  physician immediately for treatment. For Grogginess or Unconsciousness While Handling  Liquid Nitrogen: If a person seems to become groggy or loses  consciousness while working with liquid nitrogen, get  him to a well-ventilated area immediately. Use a selfcontained breathing apparatus if necessary. If breathing  has stopped, apply artificial respiration. Whenever a  person loses consciousness, call 911 immediately.                                                       |
| If released or spilled:  Waste Disposal Method  Respiratory Protection  Ventilation  Precautionary Labeling  Handling and Storage | Use only containers specifically designed for holding |
appendix b information on possible
fresh/frozen tissue processing processing options

OCT is especially useful for preserving histology, and may improve RNA
recovery, and thus is recommended. A frozen section resulting from an OCTembedded specimen will provide important information on the presence and quantity of tumor. Moreover, OCT prevents the tissue from desiccation and crumbling and also acts as an insulator from thermal change and limits ice crystal formation. If tissue samples are obtained by core (punch or needle) biopsies, each core should be separately embedded in the OCT.  
If OCT processing is not possible, controlled snap-freezing in cooled isopentane or with a heat extractor is recommended over simple dry-ice freezing. Snapfrozen specimens should be placed in an appropriate container (e.g., cryovial or cassette) and transferred to -80°C or colder for storage
appendix b information on possible
some guiding principles for fresh/frozen

Do not slow-freeze. Samples should be snap-frozen. Slow freezing promotes the formation of ice crystals, which damage the nucleic acids (e.g., RNA) in the specimen. The slower a sample freezes, the larger the ice crystals. Older models of cryostats (bath/dewar or vacuum type) that require > one minute to freeze a specimen should be avoided.  
Do not place the specimen directly in liquid nitrogen. Instead, place the OCTfilled cryomold onto a stable structure (i.e. Petri dish) that is on the surface of the liquid nitrogen.  
If only dry ice is available, adding alcohol (e.g., isopropanol or ethanol) to the dry ice can make a slurry that will help freeze the specimen more effectively (the alcohol will increase the thermal conductivity of the dry ice). This, however, is not a preferred over liquid nitrogen for snap-freezing.  
Do not add serum to the specimen
Do not touch the biopsy without sterile gloves Sterile or disposable equipment should be used, including for dissection and for snap-freezing.  
Instruments should be changed or cleaned between dissecting normal and tumor tissue.  
If a pen is used to label cryovials or other receptacles that will be stored in freezing conditions, ensure that the pen is waterproof/solvent-proof and can withstand long-term freezing conditions.  
Copies of any relevant pathology reports and material submission forms should be sent along with the specimens to the central bank. Reports should be coded in a way they can be matched to the specimen(s) while also protecting patient confidentiality requirements.  
If possible, representative adjacent normal tissue should be provided in addition to the tumor tissue. Normal tissue should be maximally distant from the tumor
(minimum 2 cm). Collection of germline DNA (i.e. from PBMCs) should be considered in all protocols that include the collection of tumor tissue for DNA isolation.
appendix b information on possible
size/number of fresh/frozen biopsies

Punch biopsies (4-6 mm preferred, 2 mm minimum) of cutaneous and subcutaneous lesions are preferable to core needle biopsies. A single biopsy is generally sufficient for research purposes.  
Core needle biopsies: It should be stressed that core biopsy - whether collected as fresh/frozen or FFPE - is superior to fine needle aspiration (FNA) (Singh, 2007). Cores are more representative than FNA, providing more accurate diagnostic information - including invasive versus in situ, grade, percent tumor, and other important histopathologic information. The minimum size for fresh/frozen tissue collection is generally 0.25 cubic cm, which is generally achieved with approximately four passes of a 14-gauge needle. In the diagnostic setting, the collection of 2-4 fresh/frozen cores for research is recommended, in addition to 2 cores for diagnosis. The fraction of cores that will contain viable tumor will decrease if/as the tumor shrinks in response to therapy. Therefore, fewer cores may be obtainable as lesions grow smaller. If a previous biopsy site is noted in the specimen to be sampled, biopsies should not be taken from near that site. Core biopsies alter the biology of tissue - e.g., they introduce inflammatory material from wound reaction and biomolecules involved in wound healing - which can be problematic if a subsequent core taken from that tissue is used for assay development. Notably, genes involved in wound healing are very similar to those involved in cancer progression (Riss et al., 2006).
appendix b information on possible
fresh/frozen tissue collection in the

During surgery, fresh/frozen tissue may be acquired by the transfer of the surgical specimen from the operating room to the pathology department in a tumor container without fixatives. Once it arrives in the pathology department, the sample may be sectioned, and then cores taken with a punch biopsy instrument and snap-frozen. This method is preferred if ischemia time can be limited to 30 minutes or less. *Note:* use a sterile RNAse free container if RNA work will be done with any of the tissue.  
*Alternative*: Taking cores and immediately freezing them in the operating room -
i.e., a core biopsy is taken from the specimen immediately after the specimen is removed from the patient. Operating room acquisition of cores from the specimen right after excision helps keep ischemia time under 10 minutes, especially in situations where pathologists are some distance from the operating room.  
Time to Freezing and Storage Temperature for Fresh/Frozen Samples Fresh tissue samples should be frozen as soon as possible. If they cannot be frozen immediately, they should be frozen within 30 minutes.  
Once snap-frozen, samples should be immediately transferred either to liquid nitrogen (preferred) or to a -80°C freezer. Specimens should be carried to such freezers on dry ice.  
Frozen samples should be stored long-term either in liquid nitrogen or in a locked freezer with a temperature of -80°C or colder. The freezer should be on electrical emergency power line and alarmed. If future uses of the tissue are unknown, storing the tissue in the vapor phase of liquid nitrogen will help to ensure longterm viability.  
Storage equipment may include small (2x3 inch) plastic, zip-top bags; megacassettes (for example, with each tissue or cryomold wrapped in aluminum foil);
and cryoboxes and plastic racks (cryovial storage).
appendix b information on possible
quality assurance for fresh/frozen samples

A frozen section should be cut from the OCT block and stained with H&E to confirm tumor presence, percent of tumor cells, preservation of morphology, and the presence of any undesirable material, such as necrotic or inflammatory material. The H&E slide may be used as a guide to isolate specific portions of the sample for molecular analyses (i.e. viable tumor; adjacent normal tissue; etc).
appendix b information on possible
database annotation for fresh/frozen tissue

When possible, the following should be recorded on the specimen submission form with respect to fresh/frozen tissue collection:
Time before freezing: if >30 minutes, note time in 15-minute increments beyond 30 minutes.  
Freezing temperature If previous punch biopsy(ies) were performed on the patient or noted in the specimen Time point of sample - e.g., after which cycle of therapy
appendix b information on possible
labeling

Vials, including cryovials, should be labeled with the study name or number, a specimen ID number that is linked to the subject's study ID, contents of the vial, and date of collection. The subject's study ID should not be on the vial unless patient confidentiality is determined to be secure according to the clinical trial protocol. Specific procedures for labeling specimens should clearly be defined in the protocol. The central bank itself should have standardized labeling (printed or written) for archiving samples, such as unique sample IDs and or barcodes. The information included on a sample label must not include patient-identifying information, and should be compliant with the Health Insurance Portability and Accountability Act (HIPAA). The information should be sufficiently specific such that the encoded information (e.g., tracking number) can be linked to the sample in the database.
appendix b information on possible
recommended procedures for oct embedding

Specimen Preparation Prior to Fixation
- Using universal precautions, remove any excess blood from the tissue using a paper towel or KimWipe.  
- Tissue should be cut on a dissection board using a razor blade or scissors with at least one dimension at a maximum thickness of 0.5 cm.  
- Discard any unused tissue according to procedures for disposing of biological waste material.  
- Discard the blade according to procedures for disposing of contaminating sharps. - Weigh each section of the specimen prior to fixation - For labeling, Markers specifically made for cryo temperatures are recommended
(alcohol-based permanent markers will smudge).  
- *Note:* if any of the tissue will be used for RNA work later use RNAse free technique.
appendix b information on possible
1. oct embedding

a. Pre-Procedure Preparation: label cryomolds with permanent marker or printed cryolabel, and prepare (preferably print) sticker labels for foil wrappers. Labels should include sample ID number, tissue type, and date. Place cryomolds on a flat surface in a manner that will allow for easy differentiation of those intended for normal tissue and those for tumor tissue.  
b. When sample is available, fill the bottom of each cryomold with a thin layer
(less then 1 mm) of OCT by slowly and carefully filling the mold. It is important to avoid formation of bubbles and avoid uneven surfaces.  
c. Weigh each section of the specimen prior to placing it in a cryoomold. Record the weight in appropriate log and database d. If possible, pre-chill the prepared cryomold (step b). This can be done by: (1)
setting on ice for 2-5 minutes, or (2) just prior to transferring specimen (step e) partially freezing by holding over liquid nitrogen until OCT starts to become cloudy.  
e. Transfer the specimen to the OCT-filled cyromold using forceps.  
f. Cover the tissue with OCT; ensure the top surface of the OCT compound completely covers the tissue and is level.  
g. The OCT can be hardened by placing a Petri dish on the surface of liquid nitrogen and, using forceps, place the filled cryomold in the Petri dish. Avoid allowing the cryomold to come in direct contact with the liquid nitrogen. Specimen(s) are sufficiently frozen when the OCT has become completely white and hard.  
h. After the OCT has hardened, place the mold in pre-labeled aluminum foil
(heavy duty is best) with the appropriate label with the sample identification information.  
i. Place samples on dry ice until transferred to -80° freezer for storage.
appendix b information on possible
2. snap freezing

a. Pre-Procedure Preparation: Label 1.8 ml cryovials using a permanent cryomarker (EtOH- and freezer-resistant).  
b. To determine specimen weight: record weight of the empty vial, add tissue, then re-weigh. Subtract weights to determine the weight of the specimen, and record.  
c. Place specimen in a 1.8ml cryovials using forceps. Use separate forceps for each type (tumor, normal) of specimen to avoid cross contamination.  
d. Tightly secure the cap and submerged in liquid nitrogen for "snap freezing".  
Use freezer gloves and a face shield during this procedure. Take precautions to avoid accidental spillage or spattering of liquid nitrogen.  
e. Place samples on dry ice until transferred to -80° freezer for storage.
appendix b information on possible
ffpe tissue processing

Fixation and paraffin infiltration One fixative and one buffer type should be used across participating centers. All tissue samples should be fixed in 10% neutral phosphate-buffered formalin (i.e., 3.7% formaldehyde), pH 7. Resection specimens should be grossly dissected (macrosectioned) prior to fixation, to ensure adequate penetration of the fixative; ideally, the sections should be approximately 3 to 5 mm thick prior to placement in tissue cassettes for fixation. It is essential that surgical margins are appropriately marked and that these steps are carried out by a pathologist or their designate. Different blades should be used when dissecting normal tissue vs. tumor tissue. The time that elapses from resection to dissection and formalin fixation (the warm ischemia time) should be minimized, and typically should be no longer than 4 hours. Placing the specimen in fixative without dissection for overnight or longer is NOT adequate. The following are recommended acceptable ranges of duration of fixation for FFPE specimens:  
Biopsies (core, needle, and skin biopsies): 8-24 hours Excision specimens: 12-24 hours in formalin (36 hours - absolute maximum)
Tissue sections (0.25-1.0 grams): Overnight-24 hours in formalin (36 hours -
absolute maximum)
Weekend specimens: Although fixing and shipping of specimens over the weekend (i.e., Friday-Monday) is discouraged, if specimens must be left in formalin over the weekend, they should be oriented, "blocked", bread-loaf sectioned and placed in a large amount of formalin in closed containers.
If the collecting center is not associated with a pathology group and does not have access to a tissue processor, then the specimen should be either 1) shipped in formalin on the day of collection for next-day delivery to the central bank, or 2) fixed in formalin for no more than 24 hours and then transferred to 70% ethanol, then shipped to the central bank within a few days. Samples should be protected from excessive heat when appropriate by packaging them in a styrofoam container (without ice packs). See Shipping section for more detailed information about appropriate packaging of samples. The following are recommended ranges of time from formalin to paraffin on the processor (variable according to the specific manufacturer):  
5-8 hrs for biopsies 6-14 hrs for other specimens (time depending on the instrumentation and tissue size)
Completion of the processes of dehydration with alcohols, clearing with xylene, and impregnation with paraffin is important. Some findings have suggested that extended processor times may result in higher-quality analytes, although which step should be prolonged has not yet been determined (Stephen Hewitt, personal communication, 2006). Use of low-melt paraffins has been recommended. Contamination with beeswax should be avoided (Hewitt et al., 2008).
appendix b information on possible
storage of ffpe tissue

FFPE blocks should be stored at temperatures below 80ºF (below 26ºC) in an "officelike" environment - i.e., a controlled-temperature environment with room temperature typical for an office, and protected from excessive heat (>28C), humidity (>70%), and dryness (<30% humidity). FFPE tissue should not be stored in basements (danger of water) or warehouses (danger of insects). Light exposure for FFPE tissue is a key problem and should be minimized. Storage of unstained FFPE slides (whether from a single block or from a TMA block) should be discouraged due to antigen loss (DiVito et al., 2004; Fergenbaum et al., 2004). Biomarker analyses may best be carried out on freshly cut FFPE sections.
appendix b information on possible
annotation of laboratory methods for

We recommend that fixative type, buffer type, and time from resection to dissection and formalin fixation should be reported and recorded in the central bank's database.
appendix b information on possible
recommended procedures for formalin-fixation of

1. Preserving the tissue in formalin enables the embedding of specimens into paraffin blocks. Neutral buffered formalin is used to stabilize protein in fresh tissue, and prevent autolysis and putrefaction.  
a. Specimens intended for formalin fixation should be processed after the completion of other fresh tissue procedures such as snap freezing, embedding in OCT compound, and submersion in RNA stabilizing reagent.  
b. Minimize time interval i. The time interval from of removal of tissues to fixation is very important in this procedure. The faster the tissue is placed in fixative, the better. Artifact will be introduced by drying, so if tissue is left out, please keep it moist with saline. The longer the interval between excision and fixation, the more cellular organelles will be lost and the more nuclear shrinkage and artifactual clumping will occur.
c. The volume of formalin should be a minimum of 15-20 times the volume of the tissue sample - e.g., 20ml of formalin per 1cm3 of tissue.  
d. Sectioning tissue for better penetration i. Penetration of tissues depends upon the diffusibility of each individual fixative, which is a constant. One way to get around this problem is sectioning the tissues thinly (3 to 5 mm). To preserve tissue and process for paraffin embedding, cut fresh tissue into appropriate size pieces.  
ii. **NOTE:** Tissues to be fixed and processed should be cut to a size no larger than 3-5 mm in thickness. Larger sized tissues will not permeate well with formaldehyde and will result in poor fixation and poor cellular morphology.  
e. Record weight of each section i. Weigh the specimen prior to placing it in a tissue cassette. Record the weight in appropriate log and database f. Labeling i. Place specimen in tissue cassette using proper orientation. Be sure to label the cassette using the cassette labeler. Include the tissue ID number and the tissue type on the cassette.  
ii. *Note*: If the tissue is for a primary investigator, include their last name and protocol number on the side of the cassette.  
g. Fixation i. Place cassette in a specimen cup containing a 10:1 ratio of 10%
Neutral buffered formalin to tissue. Let tissue fix in the 10% formalin at room temperature from a minimum of 16 hours up to 24 hr.  
ii. *Note:* If unable to embed after 24 hours of 10% formalin fixation, transfer specimen in specimen cassette to 70% alcohol and embed within 72 hrs.
appendix b information on possible
shipping

Shipping personnel must receive training and be current in certification for biological specimen shipping. International Air Transport Authority (IATA) requires recertification every 2 years. For international studies, each country should consider identifying a tissue bank where tissue can be held before final shipping to a central bank across borders. A site should consult the central bank to determine the best times to ship samples that are frozen. This will help to avoid inadvertent thawing due to the evaporation of dry ice. Batch shipping of samples will help to reduce the time required for organizing shipments and, in the case of frozen samples, dry-ice shipping costs (see "Note on Nucleic Acid Extraction", below.) A good guideline for the interval of time between procurement and shipment is one month.
appendix b information on possible
packaging for all specimens

Packaging should comply with International Air Transport Association (IATA) criteria (please see http://www.iata.org). If ground overnight is used for FFPE samples, then shipment should conform to ground transportation standards (e.g., Department of Transportation packaging standards if in the US). The shipping box should be secured and appropriate stickers should be placed, such as "Biological Substance, Category B UN 3373", and the type of shipment, e.g., next-day. The IATA shipping category appropriate to the specimens collected should be used, both in labeling and in the training required for packaging. In addition to "Biological Substance, Category B, other IATA categories include "Exempt Human Specimens" and "Infectious Substance, Category A".