Clinical trial NCT01550367
Conditions: Metastatic Renal Cell Carcinoma
Sponsor: Leonard Appleman
Start date: 2012-03-01; Completion date: 2019-02-01
Enrollment: 30 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The main goal of the research study is to determine whether treating renal cell cancer patients with the study drug, hydroxychloroquine, along with IL-2, a standard treatment of kidney cancer that has spread to other parts of the body, can make the cancer easier to kill and eliminate. Another goal is to see how the study drug affects the body's immune cells which fight cancer cells.

Detailed description:
The rationale for combining the high dose bolus aldesleukin with hydroxychloroquine includes potential positive interactions on the immune regulatory side, non-overlapping toxicities, and potential for prolongation and increased number of responses based on murine studies conducted at the University of Pittsburgh. This study is a multi-center phase II study designed to estimate the efficacy of combination therapy of standard high dose bolus IL-2 and various doses of hydroxychloroquine therapy in metastatic RCC patients.

Arms: arm 1: One course of treatment (84 days) will consist of high dose (600,000 IU/kg) bolus IL-2 administered intravenously every 8 hours on days 1-5 and 15-19 (maximum 14 doses/5 days of administration) and hydroxychloroquine (HCQ) orally started two weeks prior to IL-2 infusions and continued while able to take oral medication for up to 3 courses.
Interventions: intervention 1: Hydroxychloroquine intervention 2: IL-2
Intervention descriptions: intervention 1: Continuous oral administration (at 600 mg/d) will be initiated prior to the first dose (day -14) given 14 days prior to initiation of the first dose of IL-2 and then daily or twice a day throughout all three treatment courses. intervention 2: 600,000 IU/kg IV bolus q 8 hrs x days 1-5 and 15-19 (maximum 28 doses - 14 per 5 day cycle) of each 84-day course

Protocol documents:
Prot_SAP_000.pdf: 
1 
 
 
Inhibiting the Systemic Autophagic Syndrome - A Phase II Study of Hydroxychloroquine 
and Aldesleukin in Renal Cell Carcinoma Patients (RCC). 
A Cytokine Working Group (CWG) Study 
Prometheus protocol #: IIT11PLK01 
Principal Investigator: 
Leonard J Appleman, MD, PhD 
UPMC Cancer Pavilion 
5150 Centre Avenue Pittsburgh, PA 15232 
Phone: 412-648-6538 
 
Co-Investigators 
Location 
Joseph Clark, MD 
Loyola University 
Brendan Curti, M.D. 
Earle A. Chiles Research Institute 
Marc S. Ernstoff, M.D. 
Dartmouth-Hitchcock Medical Center 
John Kirkwood, MD 
University of Pittsburgh 
Theodore Logan, M.D. 
Indiana University 
Michael Lotze, MD 
University of Pittsburgh (Local Co-Investigator) 
Jodi Maranchie, MD 
University of Pittsburgh (Local Co-Investigator) 
David McDermott, MD 
Beth Israel Deaconess Medical Center 
Paul Monk, MD 
The Ohio State University 
Stergios Moschos, MD 
University of North Carolina 
Andrea Facciabene, PhD 
University of Pennsylvania 
 
Pathology Core Director 
Rajiv Dhir, M.D. 
UPMC Hillman Cancer Center  
 
Immunologic Monitoring and Cellular Products Laboratories (IMCPL) 
Theresa Whiteside, PhD 
 
Statistician 
Daniel P. Normolle, PhD 
UPMC Hillman Cancer Center & 
Biostatistics Dept, University of Pittsburgh 
 


2 
 
Rheumatology 
Larry Moreland, MD 
University of Pittsburgh 
 
Program Manager for GU Clinical Research 
Clare Grzejka, RN, BSN 
University of Pittsburgh 
 
Consultant 
Kim Margolin, MD 
University of Washington, CITN, CWG 
 
Prometheus/Clinigen Contact 
Nancy Gregory 
 
Phone: 832-326-3256 
 
Commercially Available Agent(s): Hydroxychloroquine/Plaquenil® (Sanofi Aventis); 
Interleukin 2/Proleukin (Prometheus/Clinigen) 
 
IND #: exempt 
 
Protocol Version: 27  
 
Protocol Version Date: March 5, 2019 


3 
 
Table of Contents 
 
 
Summary and Schema ...................................................................................................... 4 
1.0 
OBJECTIVES ......................................................................................................... 6 
2.0 
BACKGROUND AND RATIONALE .................................................................. 7 
3.0  
ELIGIBILITY ....................................................................................................... 25 
4.0 
REGISTRATION PROCEDURES..................................................................... 28 
5.0 
PRE-STUDY TESTING/STAGING ................................................................... 30 
6.0 
TREATMENT PLAN & SCHEMA .................................................................... 31 
7.0  
CORRELATIVE STUDIES ................................................................................ 38 
8.0.  DATA MANAGEMENT ...................................................................................... 41 
9.0  
EVALUATION AND MANAGEMENT OF TOXICITY 
 
(DOSE MODIFICATION) .................................................................................. 42 
10.0   DETAILED RESPONSE ASSESSMENT .......................................................... 47 
11.0   PARTICIPANTS EVALUABLE FOR RESPONSE ......................................... 51 
12.0 REPORTING OF ADVERSE REACTIONS ..................................................... 52 
13.0 STATISTICAL ANALYSIS AND STUDY SIZE .............................................. 55 
14.0 BIBLIOGRAPHY ................................................................................................. 58 
APPENDIX A .................................................................................................................. 70 
APPENDIX B-Schedule of Events ................................................................................. 71 
APPENDIX C .................................................................................................................. 73 
APPENDIX D .................................................................................................................. 74 
APPENDIX E .................................................................................................................. 75 
APPENDIX F....................................................................................................................76 


4 
 
Summary and Schema 
 
Treatment Arm: One course of treatment (84 days) will consist of high dose (600,000 IU/kg) 
bolus IL-2 administered intravenously every 8 hours beginning on day 1 and day 15 (maximum of 
14 doses per cycle of administration); and hydroxychloroquine (HCQ) orally started two weeks 
prior to IL-2 infusions (+/- 1 day) [to allow for weekends/holidays etc] and continued while able 
to take oral medication throughout all courses. For patients with evidence of stable disease or 
response up to three courses can be administered as shown below. Patients will be administered 
600mg/d of HCQ at all sites in a total group of 21. 
 
Course 1 
Drug 
Days: 
-14 
1 
15 
29 
43 
57 
71 
84 
 
Hydroxychloroquine 
X --------------------------------------------- X 
IL-2 
X 
X 
Biomarker 
X 
X 
X 
 
 
Course 2 
Drug 
Days: 
85 
99 
113   127   141   155   168 
 
Hydroxychloroquine 
X ---------------------------------------- X 
IL-2 
X 
X 
Biomarker 
X 
 
Course 3 
Drug 
Days: 
169   183   197   211   225   239   252 
 
Hydroxychloroquine 
X ---------------------------------------- X 
IL-2 
X 
X 
Biomarker 
X 


5 
 
Agents 
- Hydroxychloroquine oral at 600 mg/d throughout the treatment. Continuous oral administration 
will be initiated prior to the first dose (day -14), given a minimum of 14 days (maximum of 21 
days) prior to initiation of the first dose of IL-2 and then daily or twice a day throughout all three 
treatment courses. 
- Aldesleukin (IL-2) 600,000 IU/kg IV bolus q 8 hrs beginning on day 1 and day 15 (maximum 
14 doses per cycle). 
 
Response Assessment 
- Patients will undergo CT scans at week 8 (+/- 1 week) and week 12 (+/- 1 week), then after both 
subsequent courses (every 10-12 weeks). After the final course (week 36), patients will undergo 
CT scan evaluation every 12 weeks (+/- 2 weeks) for up to one year or until tumor progression 
(whichever comes first). If additional scans are performed for clinical reasons, the study schedule 
may be altered to perform the requisite scans at a more acceptable time point. The change in timing 
of study scans must be approved by the local PI and documented. For patients who have not shown 
progression at the end of one year, response will be captured during follow-up for survival, which 
will be done on all patients via telephone contact and medical record review. 
 
- Hydroxychloroquine will continue daily until documented disease progression or 12 weeks 
following documented complete response. 
 
- Patients with evidence of tumor shrinkage may receive up to 3 courses of IL-2 therapy. Course 2 
will start on or within 2 weeks following day 85 and follow the same schedule as in Course 1. 
Course 3 can start within 2 weeks of completion of course 2. Patients not receiving IL-2 during 
course 2 who have some evidence of tumor shrinkage by week 24 can receive a 2nd course of IL- 
2 beginning within 2 weeks of week 25. 
 
- Patients with no evidence of progression by RECIST 1.1 criteria may receive continued 
treatment with hydroxychloroquine through one year of initiation of HCQ treatment (Day -14) or 
until disease progression. 
 
- Patients with progressive disease by RECIST 1.1 criteria will be taken off-study. 
 
-  Both responding and non-responding patients will be followed until death or final closure of 
the study (6.4.2). Attention will be paid to the time of disease progression and time of 
initiation of alternate therapies. 


6 
 
1.0 OBJECTIVES 
We hypothesize that IL-2 administration enhances systemic autophagy, limiting the effectiveness 
of immune effectors and that coadministration of the autophagy inhibitor hydroxychloroquine 
will enhance response, diminish toxicity other than possibly GI toxicity, and increase the ability 
to administer IL-2. 
1.1 Primary: 
 
1. 
Estimate the proportion of patients with metastatic RCC treated with IL-2 combined with 
hydroxychloroquine at 600 mg/d who experience a clinical complete response. 
 
1.2 Secondary: 
 
2. 
Compare the distribution of clinical responses, overall survival and progression-free 
survival of patients with metastatic RCC treated with IL-2 combined with 
hydroxychloroquine to the published response of patients treated with high dose IL-2 alone 
(Proclaim database and publication). 
 
3. 
Characterize the safety and toxicity of IL-2 combined with hydroxychloroquine: 
a. 
Number of doses of IL-2 administered during the first course of therapy 
b. 
Toxicity after the scheduled 9th dose of IL-2 
c. 
Frequency of grade III and IV or unexpected or rare toxicities [Section 9.5] 
 
4. 
Determine the relationship between laboratory parameters (Sections 7.2-7.5) evaluated at 
baseline and during treatment and toxicity, clinical response, and survival. 
 
5. 
Evaluate the utility of known prognostic criteria for RCC patients on clinical outcome. (1). 
Pretreatment features associated with a shorter survival are low Karnofsky performance 
status; (<80%), high serum lactate dehydrogenase (> 1.5 times upper limit of normal), low 
hemoglobin (< lower limit of normal), high "corrected" serum calcium (> 10 mg/dL), and 
absence of prior nephrectomy (see 13.2.4). 


7 
 
2.0 BACKGROUND AND RATIONALE 
 
2.1 Treatment of RCC- Background 
In 2010, it is estimated that there will be 35,710 new cases of kidney cancer and approximately 
12,480 kidney cancer-specific deaths in the United States (2). One-third of patients have metastatic 
disease at the time of diagnosis. Two recent randomized studies in metastatic RCC patients have 
shown survival benefit of nephrectomy and cytokine therapy over cytokine therapy alone (3, 4). 
For this reason, and for symptom control, many patients with metastatic disease at presentation 
undergo nephrectomy as a component of their standard care. The prognosis for recurrent or 
metastatic renal cell carcinoma is poor. Median survival was 10-13 months prior to the 
development of agents targeting the VEGF and mTOR pathways, and is now approximately 2 
years in good- and intermediate-prognosis patients (5) and 5-year survival is less than 5%. These 
figures underscore the need for effective systemic therapy in this disease. 
 
Medical treatment for RCC has primarily focused on biological therapies designed to mobilize 
immune effector cells that recognize and destroy cancer cells (3-9). Several randomized studies 
have suggested that interferon produces modest antitumor benefit in patients with advanced renal 
cancer, while IL-2 has received FDA approval due to its ability to produce durable responses, 
albeit in only a minority of patients. Tumor infiltrating lymphocytes are often NK cells, which, 
when present in high numbers, are associated with improved prognosis (see below). 
 
2.2 High-dose IL-2 Therapy for Patients with RCC 
Investigations of IL-2 (Aldesleukin, Chiron) in patients with RCC were initiated by our group 
[Surgery Branch] at the NCI in the mid-1980s,and continued there and within the Cytokine 
Working Group. Initial studies evaluated high-dose bolus IL-2 therapy in combination with the 
infusion of autologous lymphocytes activated ex vivo in IL-2 (lymphokine-activated killer or LAK 
cells) (10, 11). While dramatic and durable responses were reported in some patients, subsequent 
studies found the efficacy of high-dose bolus IL-2 alone to be equivalent to that of the combination 
of IL-2 and LAK cells, prompting the abandonment of this more complex cellular therapy 
component. 
 
In 1992, high-dose bolus IL-2 was approved by the FDA for the treatment of patients with 
metastatic renal cell cancer based on data presented on 255 patients entered onto 7 phase II clinical 
trials (8, 12). In these studies, 600,000-720,000 IU/kg of recombinant human IL-2 was 
administered by 15-minute infusion every 8 hours x 14 doses, thereby constituting a course of 
therapy. Patients received a course of therapy, consisting of two 5-day treatment cycles separated 
by 5-9 days of rest (maximum of 28 doses) (cycle 1A/1B). Courses were repeated every 8-12 
weeks in stable or responding patients. Although 35% of patients received 720,000 IU/kg of IL-2 
per dose and the remainder received 600,000 IU/kg per dose, the median cumulative amount of 
IL-2 administered was the same in both groups, since patients receiving 720,000 IU/kg per dose 
tolerated fewer IL-2 doses. Ninety-six percent of these patients had an ECOG performance status 
of 0 or 1, 85% had undergone a nephrectomy prior to starting IL-2 therapy, none had received 
prior immunotherapy, and the median time from diagnosis to treatment was 8.5 months. 
 
 
 


8 
 
Objective responses were seen in 37 of the 255 patients (RR 15%). There were 17 (7%) complete 
responses (CRs) and 20 (8%) partial responses (PRs). Fourteen of the responding patients (38%) 
began therapy with tumor burdens >50 cm2 on pretreatment scans and 60% of PRs had >90% 
regression of all measurable disease. The median duration of response was 54 months for all 
responders, 20 months for PRs and has not yet been reached for CRs. The median survival was 16 
months for all 255 patients. 
 
Follow-up data on these patients has now been accumulated through June 2010, with a median 
follow-up of over 10 years. Although some late relapses are still being observed, the response 
duration curve appears to have leveled off after the 30 month time point and 60% of complete 
responders remain in remission. In addition, 4 PRs who underwent surgical resection of residual 
disease while still in response remain alive and disease-free at a minimum of 65+ months. 
Therefore, many CRs remaining free from progression for more than thirty months and those PRs 
resected to NED after a response to high-dose IL-2 are unlikely to progress and may actually be 
cured. Thus, although the response rate to high-dose bolus IL-2 is modest, it can produce 
meaningful benefit in a small proportion of patients. Means to enhance this response are clearly in 
order. 
 
The High Dose Aldesleukin "Select" trial was conducted to prospectively assess the ability of 
histologic, immunohistohemical and other pathologic tumor features to predict overall response 
rate and other outcomes (McDermott et al. 2011). This study enrolled 123 patients between 
November 2006 and July 2009, at many of the same Cytokine Working Group Institutions 
participating in the present study. Patients received high dose aldesleukin at a standard dose and 
schedule, and with standard recommendations for treatment modification for toxicity 
(Schwartzentruber 2001).  The High Dose Aldesleukin ="Select" study therefore represents a 
contemporaneous population of patients with similar baseline characteristics and aldesleukin 
treatment with which to compare to the current study with regard to outcomes and toxicity." 
 
In that trial, overall response rate was 25% by independent review  (95% CI 17.5-33.7) and 28% 
by investigator review (as to be performed in this study) (95% CI 20.5-37.3%).  The distribution 
of MSKCC 2002 risk was 19/70/11% (Good/Int/Poor).  Median PFS was 4.3 months (95% CI 2.5-
4.7 mo)  (comparable to prior studies where about half the patients progressed on the first scan). 
As seen previously, there was a "tail" of durable progression-free survival (PFS rate at 3 years was 
11%).  Median overall survival was 42.8 months (95% CI 35.6-51.9 months). Median duration of 
response was 20.6 months (CI 6.9-42.7 mo) 
  
2.3 Randomized Trials with IL-2 
Other regimens involving lower doses of IL-2 with and without interferon (IFN) have been 
reported to produce similar response rates to those observed with high dose (HD) IL-2, but these 
results have not been reproducible and the quality of responses has been less impressive (9,13,14). 
Before accepting lower dose regimens as equivalent to HD IL-2, it was imperative to establish that 
the quality of tumor responses with lower dose regimens was not inferior. Three large-scale 
randomized trials have now been completed that provide clinicians with useful insights into the 
relative merits of these various regimens and thus the optimal management of patients with 
metastatic renal cell carcinoma. 
 
 
 


9 
 
Investigators in France conducted a large-scale, phase III randomized trial comparing 
intermediate-dose IL-2 administered by continuous intravenous infusion plus subcutaneous IFNα 
with either IL-2 or IFNα administered alone (9). Four hundred twenty-five patients were enrolled. 
The three treatment groups were well balanced for age and gender as well as known predictors of 
response and survival. The response rate and 1-year event-free survival were significantly greater 
for the combined IL-2 and IFNα arm than for either of the single-agent arms, although there was 
no significant difference in overall survival among the three groups. Of note, responses were seen 
in only 6.5% and 7.5% of patients receiving IL-2 or IFN alone, respectively, with only 2.9% and 
6.1% of these patients still responding at the week 25 evaluation. Although more anti-tumor 
activity was seen with the combination arm, this was largely due to the rather limited activity of 
the single-agent regimens. How an intermediate-dose combination of IL-2 and IFNα would 
compare with high-dose IL-2 alone remained to be established. 
 
The NCI Surgery Branch investigators performed a randomized trial comparing standard HD IL- 
2 and the intermediate dose (low dose – LD) intravenous bolus IL-2 regimen (LD IV IL-2) (15). 
After randomizing 117 patients, a third arm was added involving subcutaneous IL-2 administered 
in a fashion similar to that previously described by Sleijfer et al. Results were analyzed and 
reported according to groups that were concurrently randomized. Among the 306 patients 
concurrently assigned to either HD or LD IV IL-2, the response rate was significantly higher with 
high-dose therapy (21 versus 13 percent), with a trend towards more durable responses. Response 
durability and survival in complete responders was superior in patients who received the HD 
intravenous IL-2 compared to those who received the LD IV IL-2. There were no differences in 
overall survival. Although toxicities were also significantly greater in the high dose group 
(particularly hypotension), there were no deaths attributable to IL-2 in either arm and patient 
assessments of quality of life were found to be roughly equivalent. Among the patients 
concurrently assigned to either subcutaneous IL-2 or HD intravenous bolus IL-2, a higher response 
rate was seen with HD IL-2 (21 versus 10 percent) but the difference was of borderline statistical 
significance. Once again there were no differences in overall survival. 
 
In an effort to determine the value of outpatient subcutaneous IL-2 and IFNα relative to high dose 
IL-2, the Cytokine Working Group performed a phase III trial in which patients were randomized 
to receive either outpatient IL-2 (5 MIU/m2 subcutaneously every 8 hours x 3 doses on day 1 then 
daily 5 days/week for 4 weeks) and IFN (5 MIU/m2 subcutaneously thrice weekly for 4 weeks) 
every 6 weeks or standard high dose inpatient IL-2 (600,000 IU/kg/dose every 8 hours 
intravenously, days 1-5 and 15-19 [max 28 doses]) every 12 weeks (16). One hundred and ninety 
three patients were enrolled and 192 were evaluable. Patients were stratified for bone or liver 
metastases, primary in place and performance status 0/1 and the treatment arms were evenly 
balanced for these characteristics as well as other factors. 45% of patients had bone or liver 
metastases, 31% had primary in place and 60% were PS 0. Toxicities seen were typical for these 
regimens, including one treatment related death from progressive disease and ARDS on IL-2 and 
IFN arm and one death from capillary leak syndrome on high dose IL-2. 
 
The response rate for high-dose IL-2 was 23% (22/95) versus 10% (9/91) for IL-2/IFN (p=0.018). 
Eight patients achieved a complete response on high dose IL-2 versus only 3 on low dose IL- 
2/IFN. The median response durations were 24 months for high dose IL-2 and 15 months for IL- 
2/IFN (p=0.18). Median overall survivals were 17 and 13 months (p=0.21), favoring high dose IL-
2. Ten patients on high dose IL-2 were progression free at 3 years versus 3 on IL-2/IFN (p=0.08). 
Durable CRs favored HD IL-2 (7 vs. 0). Median progression free survival was 3 months for each 


10 
 
treatment arm. Of note, responses to high dose IL-2 were seen with equal frequency across the 
stratification criteria, while low dose IL-2/IFN appeared to produce fewer responses in patients with 
liver and/or bone metastases and in those who had not undergone prior nephrectomy to remove the 
primary tumor. For patients with bone or liver metastases (p=0.001) or primary in place (p=0.04) 
survival was superior with high dose IL-2 compared to IL-2/IFN, while no significant survival 
differences between the two treatments were noted for patients who had undergone prior 
nephrectomy or were without bone or liver metastases. Moreover, patients who had undergone a 
recent nephrectomy (debulking nephrectomy in the setting of stage IV disease) appeared to fare as 
well with IL-2/IFN as with HD IL-2. 
 
Taken together these studies suggest that HD bolus IL-2 is superior in terms of response rate and 
response quality to regimens involving either low dose IL-2 and IFN or intermediate or low dose 
IL-2 or subcutaneous IFN. Furthermore, the benefit of high dose IL-2 relative to the lower dose 
regimens may be most pronounced in patients with unresected primary tumors or bone and/or liver 
metastases. This suggests that high dose IL-2 is required to produce responses in typically 
refractory sites and to eliminate the last tumor cell. This data suggests that high dose IL-2 should 
remain the preferred therapy for appropriately selected patients with access to such therapy. 
However, given the toxicity and limited efficacy of high dose IL-2 therapy, additional efforts 
should be directed at better defining the patient population for whom this therapy is appropriate 
and finding means to ameliorate toxicity and increase efficacy. 
 
2.4 Predictors of Clinical Benefit from Cytokine-Based Therapy 
Many groups have attempted to determine reliable predictors of response and survival for patients 
with metastatic renal cell carcinoma who were receiving immunotherapy. Factors that have been 
variably associated with response include performance status, number of organs with metastases 
(one versus two or more), absence of bone metastases, prior nephrectomy, degree of treatment- 
related thrombocytopenia, absence of prior interferon therapy, thyroid dysfunction, rebound 
lymphocytosis, erythropoietin production, and post-treatment elevations of blood TNF-α and IL- 
1 levels (17-20). 
 
Motzer and colleagues have shown in patients receiving IFN that poor survival is associated with 
low Karnofsky performance status, high serum lactate dehydrogenase, low hemoglobin, high 
“corrected” serum calcium and time from initial renal cell carcinoma diagnosis to start of therapy of 
less than one year (20). In a cohort of 453 patients who received IFN as initial therapy, the median 
survival for the favorable (no risk factors), intermediate (one or two risk factors) and poor (three or 
more risk factors) risk groups were 30, 14 and 5 months respectively. Negrier and associates also 
identified independent predictors of rapid disease progression, defined as progression within 10 
weeks of initiation of therapy (19, 21). These included greater than one metastatic site, disease-free 
interval of less than 1 year, and presence of liver metastases or mediastinal nodes, as well as type of 
immunotherapy used. Patients with liver metastases, more than one site of disease, and disease-free 
interval of less than 1 year had a lower response rate and a median survival of only 6 months, even 
while receiving combination IL-2 and interferon α therapy. Figlin and colleagues identified prior 
nephrectomy and time from nephrectomy to relapse as important predictors of survival in patients 
receiving IL-2-based therapy (10). In their series, patients who received systemic immunotherapy 
for metastatic disease more than 6 months after nephrectomy had the best median survival and had a 
3-year survival rate of 46%. A recent multivariate analysis by the same group of investigators that 
was confined to patients who received IL-2 after nephrectomy revealed survival to be inversely 


11 
 
associated with lymph node involvement, constitutional symptoms, sarcomatoid histology, metastases 
involving sites other than bone or lung or multiple sites, and a TSH level > 2.0 mIU/L (10). They 
proposed a scoring algorithm based on these features in which survival at one year was predicted to 
vary from 1- 92%. Recent data from the Cytokine Working Group phase III trial, mentioned above, 
suggested that disease site factors such as primary in place or hepatic or bone metastases may be 
more predictive of a poor response to low dose IL-2 and IFN regimens, than to high dose IL-2. 
Furthermore, this study suggested the greatest benefit from high dose IL-2 relative to lower dose 
regimens might be seen in patients with primaries in place and/or liver and bone metastases. These 
data call into question some of the prior studies and suggests that additional predictors of response 
and survival in patients receiving cytokine-based immunotherapy are necessary. 
 
2.5 Influence of subtype on response to IL-2 
Responses to immunotherapy are most frequently seen in patients with renal cell carcinoma of 
clear cell histology. This observation was recently detailed in a retrospective analysis by Upton et 
al of pathology specimens obtained from 231 patients who had received IL-2 therapy on Cytokine 
Working Group clinical trials (22). For patients with tumor specimens available for review, the 
response rate to IL-2 was 21 percent (30 of 146) for patients with clear cell histology primary 
tumors, compared to 6 percent for patients with non-clear cell histology (1 responder in 17 
patients). Among the patients with clear cell cancer in their kidney specimen, response to IL-2 was 
also associated with the presence of > 50% alveolar features, no papillary features or granular 
features in the pathologic material. The response rate was 39 percent (14 of 36) in patients with all 
these features. In addition, patients with clear cell tumors with < 50% alveolar or granular features 
but no papillary features in the specimen had a response rate of 19 percent (15 of 77) compared to 
3 percent (1 of 33) in the other patients. 
 
From this data, a model was developed that placed patients with clear cell cancers into good, 
intermediate and poor response categories based on the analysis of their primary tumors. 
 
When this model was then applied to the 68 patients with specimens from other metastatic sites, 
all 7 tumor responses were seen in the 42 patients with clear cell cancer and intermediate or good 
prognostic features, thus confirming the validity of the model developed from the primary kidney 
tumor specimens. In addition, median survivals for all patients with clear cell tumors by risk group 
were 2.87, 1.36 and 0.87 years, respectively (p <0.001), indicating that the model may have 
prognostic as well as predictive value. 
 
 
 
 


12 
 
Table 1. Histologic Predictors of Response to IL-2 Therapy 
Category 
Pathology Type 
N 
Response 
RR % 
Good 
Clear Cell 
Alveolar > 50% 
No granular 
No papillary 
36 
14 
39 
Intermediate 
Clear Cell 
Alveolar <50% 
Granular <50% 
No papillary 
77 
15 
19 
Poor 
Others 
33 
1 
3 
 
As a result of these data, it has been conjectured that patients with non-clear cell histology and 
those with clear cell histology but adverse pathologic features (papillary and/or no alveolar 
features, and/or >50 percent granular features) receive alternative non-immunotherapy-based 
treatments; however, this supposition requires confirmation using an independent data set. In 
addition, the encouraging results in the favorable pathology types require prospective 
confirmation to determine the true response rates in a selected population. The association of 
response with pathologic features suggests that more intensive evaluation of tumor tissue might 
yield more robust predictors of response. Given that even in the most favorable prognostic group 
only 39% of patients responded to IL-2 therapy, additional investigations into tumor-associated 
predictors of responsiveness to IL-2 are still warranted. 
 
2.6 Molecular Markers of Response to IL-2 
Some investigators have begun to examine tumor tissue to identify molecular markers that might 
predict the outcome of patients with renal cell carcinoma. Recently, carbonic anhydrase IX (CAIX) 
has been identified as one potential marker (23). CAIX is thought to play a role in cell proliferation 
in response to hypoxic conditions. CAIX expression is mediated by the HIF-1 alpha transcriptional 
complex and induced in many tumors types, but is absent in most normal tissue with the exception 
of epithelial cells of the gastric mucosa. Bui et al used a monoclonal antibody designed to detect 
CAIX expression to perform an immunohistochemical analysis of paraffin embedded renal cell 
carcinoma specimens (23). They showed that > 90% of renal cell carcinomas express CAIX and 
that its expression decreases with advancing stage. In their analysis, high CAIX expression in 
primary tumors was seen in 79% of patients and was associated with improved survival and, 
possibly, response to IL-2-based therapy. In addition, all long-term responders to IL-2-based 
treatment had high CAIX expression. In this study, low CAIX expression was associated with a 
worse outcome for patients with locally advanced renal cell carcinoma and was an independent 
predictor of outcome in patients with metastatic disease. 
 
Building on this work, Atkins et al performed a nested case-control study within the larger Upton 
pathology cohort (24). Paraffin-embedded tissue sections of RCC were obtained from patients who 
had previously been included in the pathology review and were immunostained with the same CAIX 
antibody used by Bui et al. CAIX expression levels were correlated with type of IL-2 treatment, 
response to IL-2, pathologic risk categorization and survival. As in the report by Bui et al, the 
percentage of CAIX positive tumor cells was utilized to separate high (> 85%) and low (< 85%) 
expressors. Tissue specimens were obtained from 66 patients. Twenty-seven of the selected 
patients (41%) achieved a response (10CR/17PR) to high-dose IL-2 (32) or low-dose  IL-2-based 


13 
 
(34) regimens with 20 (30%) remaining alive at a median follow-up of 2.6 years. Fifty-eight 
specimens were assessed as clear cell, with 56, 33 and 4 having alveolar, granular and papillary 
features, respectively. Twenty-four (36%), 31 (47%) and 11 (17%) were classified into good, 
intermediate, or high-risk groups according to the pathology model created by Upton et al that is 
described above. Forty-one specimens (62%) had high CAIX expression. Twenty-one of 27 (78%) 
responding patients had high CAIX expression, compared to 20/39 (51%) of non-responders 
(OR=3.3, p=0.04). A similar survival benefit was observed (p=0.04). Median survival was 3 and 
1 years for high and low CAIX expressers, respectively. Survival greater than 5 years was only 
seen in the high CAIX expressing group. High CAIX staining was associated with better pathology 
prognosis and, when stratified by group, the odds ratios of CAIX expression were unequal across 
groups. The strongest association was in the intermediate pathology group where 9/9 responders 
had high CAIX expression vs. 11/22 non-responders, suggesting a possible refinement of the 
Upton pathology model. A resultant group with good pathology or intermediate pathology and 
high CAIX expression contained 26 of 27 (96%) responders compared to only 18/39 (46%) non- 
responders (OR = 30; p <0.01). Survival benefit was also seen (p< 0.01). The authors concluded 
that based on this data, patients with low CAIX staining and intermediate or poor risk pathology 
classification should be considered for non-IL-2 based treatments. 
 
Table 2. CAIX Staining and Response Rate 
Category 
N 
Response Rate % 
Low CAIX staining 
Intermediate or poor path 
22 
<5% 
Other 
44 
59% 
 
While this model requires validation, it is possible that IL-2 therapy could ultimately be reserved 
for patients with favorable or intermediate pathology classification and high CAIX staining who 
appear to be more likely to benefit. 
 
Additional studies to explain these preliminary observations and correlate results with pathologic 
features, sites of disease and previously described clinical features are necessary. In addition, gene 
expression profiling of tumor specimens to identify new proteins or patterns of gene expression 
that might be associated with IL-2 responsiveness may eventually help narrow the application of 
IL-2 therapy to those who will benefit the most. The SELECT Cytokine Working Group has 
recently concluded that combined histologic subtype and CAIX predictors were unable to define 
a predictor of response (McDerrmott et al, 2017; 150). 
 
2.7 Application of models to high dose IL-2 population 
The pathology and combined CAIX plus pathology models were retrospectively applied to the 
population of patients treated with high dose IL-2 on the Phase III Cytokine Working Group trial. 
Fifty-five patients who received high dose IL-2 and had their pathology reviewed as part of the IL-
2 pathology analysis were classified into either the good or intermediate prognostic category. In 
this group of patients, the overall response rate was 31% (17 of 55), the CR rate was 11% (6 of 
55), the 3 year progression free survival rate was 14.5% (8/55), and the median progression free 
survival was 4.7 months. In addition, thirty-two patients who received high dose IL-2 and were 
included in the CAIX staining project had either good pathology or intermediate pathology and 
high CAIX staining. These 32 patients had an overall response rate of 37.5% (12/32), a complete 
response rate of 16% (5/32), a 3-year progression free survival rate of 19% (6/32) and a median 


14 
 
PFS of 6.4 months. Considering that the CAIX staining population was enriched for responding 
patients, these latter numbers probably overestimate the true efficacy of high dose IL-2 in this 
population. On the other hand, the pathology analysis contains patients with intermediate 
pathology and low CAIX staining that may depress the overall clinical efficacy. Based on this data 
one would predict that patients selected for IL-2 therapy according to the combined CAIX and 
pathology model would exhibit a response rate of approximate 30-35%, a CR rate of 10-15%, a 3- 
year progression free survival rate of 12-18%, and a median PFS of 4.5-6 months. As both analyses 
are heavily confounded with the data used to create the models, prospective confirmation is 
required. Although supported by substantial information, the recent studies from the Cytokine 
 Working Group [David McDermott, ASCO 2010] do not substantiate an improved outcome for 
patients predicted to have better response based on these criteria although a 25% response rate was 
confirmed. We have also suggested little value of the administration of antibodies to VEGF with 
IL-2 treatment (25-28). 
2.8 Autophagy serves as a survival pathway for stressed tumor cells enhancing 
survival following cytotoxic chemotherapy. 
Autophagy is an important highly evolutionarily conserved catabolic process by which cells shuttle 
effete or damaged organelles and proteins to the autophagosome for sequestration and destruction. 
In addition to its homeostatic role, the autophagic process also allows cells to survive periods of 
nutrient deprivation and stress. Autophagy is thought to maintain cellular metabolism through 
increased turnover of cell components during times of nutrient deprivation. A growing body of 
literature suggests that autophagy is in fact a form of programmed cell survival and is induced to 
prevent apoptosis or necrosis (29-31). As is the case in normal cells, autophagy is induced in tumor 
cells during times of metabolic stress. In fact, several studies have demonstrated that markers of 
increased autophagic flux localize to metabolically stressed areas of tumor (32). In particular, a 
recent study by Fujii et al. examined 71 resected adenocarcinomas and demonstrated a high 
correlation between worse prognosis and increased levels of autophagy in the specimens (33). That 
autophagy can protect tumor cells during times of stress has led to increased interest in blocking 
this process as a means of anti-tumor therapy. Inhibition of autophagy as a single agent is unlikely 
to have much clinical impact since only a small fraction of the tumor cells are under metabolic 
stress at any given time. Therefore, most strategies have focused on the combination of autophagy 
inhibition in combination with agents that induce cellular stress (34, 35). Most modern neoplastic 
therapies fall into this category inducing cellular stress either by genotoxic injury, metabolic insult 
or blockade of growth factor activity. As shown in Figure 1, administration of chloroquine, an 
inhibitor of autophagy at 50mg/kg/d limits the weight gain in mice observed with high dose IL-2 
administration [6x105 IU bid x 5D] concurrent with administration. 


15 
 
Figure 1. Chloroquine limits IL-2 Induced Vascular Leak and Weight Gain. Weight gain 
observed with IL-2 administration was ameliorated with chloroquine coadministration. 
Shown in the ordinate/y axis is the change in weight for each animal over the days of treatment. IL-
2+CQ versus IL-2, Days 3 and 6; p=0.04 
 
 
2.9 Chloroquine and its derivatives inhibit autophagy and enhance immunotherapy 
and chemotherapy effects. 
Chloroquine (CQ) is a synthetic 4-aminoquinoline that has been used for 60 years in humans 
initially for malaria prophylaxis and treatment, and with emergence of resistance, in patients with 
systemic lupus erythematosus, rheumatoid arthritis, and human immunodeficiency virus (HIV). It 
is an inexpensive orally available drug with a large therapeutic index. Chloroquine derivatives 
such as HCQ are still used extensively in patients with rheumatoid arthritis and lupus 
erythematosus. Chloroquine blocks acidification of the lysosome, thus inhibiting the last step in 
autophagy. With this last step blocked, a cell reliant on autophagy will increase the generation of 
autophagosomes and will eventually undergo either apoptotic or non-apoptotic cell death. 
Evidence in mouse models and human cancer cell lines suggest CQ may have significant anti- 
tumor activity by inhibiting autophagy induced by cancer therapy (35). Hydroxychloroquine is 
generally considered the safest disease-modifying agent currently used to treat rheumatic diseases. 
Up to 10% of patients will experience nausea, headache, or bizarre dreams, but these are 
infrequently dose-limiting. Retinal toxicity is a rare complication, estimated to occur in <1% of 
patients treated for years at doses not exceeding 6.5 mg/kg/day lean body weight, or approximately 
400 mg/day (36, 37). Given the prolonged half-life of this drug (t1/2=40=45 d), treatment with 
daily doses of up to 1200 mg daily for a maximum of one month would achieve plasma 
concentrations approximating steady state with 400 mg daily dosing, and the risk of retinal toxicity 
for this relatively short-term exposure is thus low. While data is sparse regarding precise risk at 
higher daily dosing, Shearer and Dubois reported only a single patient with retinopathy in a survey 
of 94 patients treated with HCQ 800 mg daily for up to 54 months (38). Levy et al. identified 78 
patients receiving a daily dosage >7.8 mg/kg, and of the 3 with possible or definite retinal toxicity, 
HCQ was taken for at least 3.5 years (36). Because retinal toxicity may progress after drug 
cessation (39), HCQ will be discontinued in any patients reporting any visual complaints. A formal 


16 
 
ophthalmic evaluation plus a 3 month follow-up examination will be obtained only with ocular 
symptoms. 
 
2.10 
Murine Models of IL-2 and Chloroquine [Figure 2] 
 
We 
have 
tested 
IL-2 
in 
 
 
 
 
 
 
 
 
 
 
combination with chloroquine in mouse models of metastatic colorectal cancer to the liver using 
luciferase transfected tumor and imaging strategies. When applying high dose IL-2 [6x105IU bid 
x 5D] as opposed to low dose regimens [6x104IU bid x 5D] in the setting of these models, we 
were able to demonstrate substantial antitumor effects in combination with chloroquine at 
50mg/kg/d. Lower doses and dosing frequency were less effective. 
 
2.11 
Hydroxychloroquine is more feasible than chloroquine. 
Hydroxychloroquine is commonly prescribed for rheumatoid arthritis and lupus at a dose of 400 
mg po qd. A pharmacokinetic/pharmacodynamic study of escalating doses of HCQ at 400 mg/800 
mg/1200 mg po qd in patients with rheumatoid arthritis followed by maintenance doses of 400 mg 
po qd found that doses of up to 1200 mg po qd were well tolerated (40). Dose-limiting toxicities 
(nausea, vomiting and abdominal pain) were observed at 800 and 1200 mg po qd. This toxicity 
correlated with blood HCQ levels, but not to blood levels of the other active metabolites DHCQ, 
DCQ, or BDCQ. Improvement of symptoms in rheumatoid arthritis correlates with blood DHCQ 
levels, suggesting a dose-response relationship. Chloroquine derivatives are metabolized through 
the p450 enzyme system, and CQ may inhibit the metabolism of CYP2D6- metabolized drugs. A 
predictable cumulative toxicity associated with CQ is retinopathy, and this is another reason why 
dose escalation in chronic treatment with CQ could be limited. While a link between HCQ and 
retinopathy has also been made, it occurs infrequently and only after prolonged exposure. In a 
study using multifocal electroretinography to detect early pre-clinical retinal changes in long-term 
HCQ users, 10 out of the 11 patients that developed early pre-clinical changes had been taking 
HCQ at doses of >400 mg po qd for greater than 5 years (41). No overt retinopathy was noted in 
the 19 patients followed. This suggests that at a cumulative dose of 730 g. the risk of retinal 


17 
 
changes increase, but techniques such as multifocal electroretinography can detect early changes 
and prevent overt visual loss. 
 
 
2.12 
Clinical trials examining hydroxychloroquine and chemotherapy for the 
treatment of human cancers. 
The above cited literature, demonstrating the utility of blocking autophagy alone or in combination 
with cytotoxic chemotherapy in vitro, and preclinical models, has led to the initiation of human 
clinical trials. Currently, there are more than twelve clinical trials examining the combination of 
hydroxylchloroquine and conventional chemotherapeutic agents. These are summarized briefly on 
the next page. In dose escalation trials performed at the University of Pennsylvania, doses of 
1200mg daily have been achieved with no significant toxicity attributed to the HCQ in various 
combination including dose intense temazolamide, Velcade, or temsiloimus. In a protocol using 
temazolamide and radiation therapy in combination with HCQ for patients with Glioblastoma, 800 
mg was associated with myelosuppression and the MTD defined as 600 mg/d [Personal 
Communication, Ravi Amaravadi, MD]. 
 
Table 3. Ongoing Clinical Trials of Hydroxychloroquine in Oncology. 
 
Condition 
Intervention 
Phase 
Sponsors 
Collaborators 
ClinicalTrials.gov 
Identifier 
Title 
Prostate cancer 
Docetaxel hydroxy- 
chloroquine 
II 
CINJ, NCI 
NCT00786682 
A Phase II Study of Docetaxel and 
Modulation of Autophagy with 
Hydroxychloroquine 
for 
Metastatic Hormone Refractory 
Prostate Cancer 
Prostate cancer 
Hydroxy- 
chloroquine 
II 
CINJ, NCI 
NCT00726596 
Autophagic Cell Death in Patients 
with 
Hormone-Dependent 
Prostate-Specific 
Antigen 
Progression after Local Therapy 
for Prostate Cancer 
Multiple myeloma, 
plasma 
cell 
neoplasm 
Bortezomib hydroxy- 
chloroquine 
I/II 
University of 
Pennsylvania, 
NCI 
NCT00568880 
A 
Phase 
I/II 
Trial 
of 
Hydroxychloroquine Added to 
Bortezomib 
for 
Relapsed/Refractory Myeloma 
Brain, 
central 
nervous 
system 
tumors 
Hydroxy- 
chloroquine 
temozolomide 
I/II 
University of 
Pennsylvania, 
NCI 
NCT00486603 
A 
Phase 
I/II 
Trial 
of 
Hydroxychloroquine 
in 
Conjunction 
with 
Radiation 
Therapy and Concurrent and 
Adjuvant Temozolomide in 
Patients With Newly Diagnosed 
Glioblastoma Multiforme 
Breast cancer 
Hydroxy- 
chloroquine 
ixabepilone 
I/II 
CINJ, NCI 
NCT00765765 
Phase I/II Study of Ixabepilone in 
Combination with the Autophagy 
Inhibitor Hydroxychloroquine for 
the Treatment of Patients with 
Metastatic Breast Cancer 
Lung cancer 
Bevacizumab 
carboplatin hydroxy- 
chloroquine 
paclitaxel 
I/II 
CINJ, NCI 
NCT00728845 
Modulation of Autophagy with 
Hydroxychloroquine 
 in 
Combination with Carboplatin, 
Paclitaxel and Bevacizumab in 
Patients 
with 
Advanced/Recurrent Non-Small 
Cell Lung Cancer -A Phase I/II 
Study 


18 
 
Adult solid tumors 
Hydroxy- 
chloroquine 
temozolomide 
I 
University of 
Pennsylvania, 
NCI 
NCT00714181 
A 
Phase 
I 
Study 
of 
Hydroxychloroquine 
in 
Combination with Temozolomide 
in Patients with Advanced Solid 
Tumors 
Non-small cell lung 
cancer 
Gefitinib, hydroxy- 
chloroquine 
I/II 
National 
University 
Hospital, 
Singapore, 
Massachusetts 
General 
Hospital 
NCT00809237 
A Phase II with a Lead in Phase I 
Study 
to 
Examine 
the 
Tolerability, Safety Profile and 
Efficacy of Hydroxychloroquine 
and Gefitinib in Advanced Non- 
Small Cell Lung Cancer 
Advanced cancer 
Hydroxy- 
chloroquine, 
sunitinib malate 
I 
CINJ, NCI 
NCT00813423 
Anti-Angiogenic 
Therapy 
in 
Patients 
with 
Advanced 
Malignancies: A Phase I Trial of 
Sunitinib 
and 
Hydroxychloroquine 
B-cell 
chronic 
lymphocytic 
leukemia 
Hydroxy- 
chloroquine 
II 
North 
Shore 
Long 
Island 
Jewish Health 
System 
NCT00771056 
Autophagic 
Modulation 
with 
Phase II Study to Evaluate the 
Tolerability 
and 
Efficacy 
of 
Treatment 
of 
Previously 
Untreated 
B-Cell 
Chronic 
Lymphocytic Leukemia (B-CLL) 
Patients 
with 
Hydroxychloroquine 
Pancreas Cancer 
Hydroxychloroquine, 
Gemcitabine 
I 
University of 
Pittsburgh 
NCT01128296 
Phase I/II study of Preoperative 
Gemcitabine in Combination with 
Oral 
Hydroxychloroquine 
in 
Subjects with Resectable Stage 
IIb Or III Pancreatic 
Adenocarcinoma 
Renal Cancer 
Hydroxychloroquine 
IA 
University of 
Pittsburgh 
NCT01144169 
Neoadjuvant 
Study 
 of 
Preoperative 
Hydroxychloroquine in Patients 
with 
Resectable  Renal 
Cell 
Carcinoma 
NOTE: Study citations were obtained from the National Institutes of Health (NIH)/NCI clinicaltrials.gov website 
Abbreviations: CINJ, The Cancer Institute of New Jersey; NCI, The National Cancer Institute. 
 
 
2.13 
miRNAs as Biomarkers of RCC. 
 
The modification of cell death pathways important in cancer progression were postulated by the 
DAMP Laboratory five years ago (42) but the causes of the shift, and its regulation, were obscure. 
One possible explanation may be the regulation by micro-RNAs (miRNAs). These are 18-22 bps 
long single stranded non-coding RNA that regulate gene expression in both plant and animals by 
interacting with and degrading or silencing as many as 100 separate messenger RNAs (mRNAs) 
(43-45). We have shown that tumor expression of miRNAs can limit immune recognition (51), 
and have focused on the receptor for advanced glycation end products (RAGE) (52) as a potential 
target for miRs (see below). 
Role of miRNAs in Target Sensitivity to Lysis. miRNAs play an important role in cell 
differentiation, tumor progression, organogenesis and embrybogenesis. Many miRNA machinery 
genes including Dicer, AGO1, AGO3, AGO4 are down-regulated in tumors and play a role in 
inflammatory cells (45-50). Various miRNAs have been identified to be involved in regulation of 
the cell cycle including lin-4 and let-7 controlling cell differentiation and proliferation, miR-14 as 
an apoptosis suppressor, miR-1, miR-273, lys-6, miR-181, miR-375, miR-143, and miR-196 for 
organogenesis. Further application of miRNA as gene therapies to deliver tumor suppression as 


19 
 
miRNA, anti-miRNA oligonucleotides (AMOs), or cholesterol conjugated AMOs, so-called 
antagomirs, are also in progress. Since miRNA plays an important role in cell differentiation and 
proliferation affecting many cell types including hematopoietic cells, it will be useful to understand 
the impact of miRNAs in the immune response in human biology (52-64). 
Inhibition of miRNA processing by Dicer disruption up-regulates intercellular cell 
adhesion molecule (ICAM)-1 and enhances the susceptibility of colorectal tumor cells to antigen- 
specific lysis by cytotoxic T-lymphocytes (CTLs) whereas expression of other immunoregulatory 
proteins examined is not affected. Blockade of ICAM-1 inhibits the specific lysis of CTLs against 
Dicer-disrupted cells, indicating a pivotal role of ICAM-1 in the interaction between tumor cells 
and CTL. Both miR-222 and -339 are down-regulated in Dicer-disrupted cells and directly interact 
with the 3' untranslated region (UTR) of ICAM-1 mRNA (50). Modulation of Dicer or these 
miRNAs inversely correlated with ICAM-1 protein expression and susceptibility of U87 glioma 
cells 
to 
CTL-mediated 
cytolysis 
while 
ICAM-1 
mRNA 
levels 
remain 
stable. 
Immunohistochemical and in situ hybridization analyses of 30 primary glioblastoma tissues 
demonstrated that expression of Dicer, miR-222, or miR-339 was inversely associated with ICAM- 
1 expression. We will similarly assess expression of ICAM-1 in circulating macrophages (52-59) 
activated by DAMPs in the context of this study. 
Studies of miRNA in Renal Cancer. There have been several investigations of miRNA 
in renal cancer. miRNAs regulate renal development, activity and disease. Many kidney specific 
miRNAs and miRNAs specific to renal tumor vs normal tissue have been described (65, 66). Up- 
regulation of miR-28, miR-185, miR-27, and let-7f-2 has been identified in RCC relative to normal 
kidney (68).   miR 141 and 200c are downregulated in RCC (69). Loss of miRNAs permits 
translation of proteins that promote tumorigenesis (67). An additional 38 miRNAs have been 
identified as increased in tumor, and 48 decreased assessed by microarray and PCR based 
techniques (70). miR-155 and miR-21 and miR-210 are overexpressed in clear cell carcinoma 
(71). miR-192 and miR-377 are associated with specific processes including matrix deposition 
whereas miR-200 and miR-205 regulate the epithelial-to-mesenchymal transition (65). The four 
histologic RCC types were effectively classified through assessment of miR 424, 203 and s-has- 
miR 33 (72). Identification of miRNA targets is still a challenge. Although there are methods to 
predict  targets,  few  have been  validated  in mammals (66). New techniques available in our 
proteomic core may help advance this effort (73). miRNA can be successfully extracted from 
paraffin or frozen tissue (74). We recently completed our own studies of normal kidney, clear cell, 
oncocytoma, chromophobe, and papillary renal cell carcinoma (Bastacky SI, et al in preparation). 
miR34c and miR214. We have identified two candidate miRNAs, miR34c and miR214 
which are upregulated by HMGB1+, or HMGB1+/- tumor lysates respectively. HMGB1 is 
released from stressed or dying tumor cells in response to ischemia reperfusion injury and most 
therapeutic cytotoxic agent (55-59). HMGB1 (60-64) translocated to the cytosol in the setting of 
autophagy can bind Beclin-1 with dissociation of Beclin-1/Bcl-2. Mitochondrial HMGB1 
regulates cellular bioenergetics and mitophagy by promoting phosphorylation and activation of 
ERK1/2 (pERK1/2). Reduced but not oxidized HMGB1 suppresses SOD and mTOR expression, 
and increases mitochondrial superoxide production, which in turn induces autophagy. 
We have also identified putative miRNAs involved in HMGB1-induced signaling and/or 
differentiation including hsa-mir-155, hsa-miR-34c and hsa-mir-214 in three normal individuals 
(Table 4). These are of particular interest to us due to their computationally calculated potential 
targets. Hsa-mir-155 is predicted to target Spi-1 (or PU.1) and TLR4, as well as MAP4K5 (a 
Mitogen-activated protein kinase), whereas hsa-34c is predicted to target Nemo/IKKγ of NFκB. 


20 
 
Both Spi-1 and IRF-8 are myeloid-specific transcription factors involved in monocyte activation 
and differentiation. The MAP Kinase may be involved in early HMGB1 signaling, which in turn 
may be regulated by miR-34c or miR214. With the peripheral blood obtained in this protocol, our 
next step will be to validate expression levels of these miRNAs by quantitative RT-PCR. 
 
Table 4. Putative miRNAs involved in human myeloid cells stimulated with PAMPS/DAMPS 
 
RCC 
 
 
 
 
 
 
 
 
 
 
PBMCS IL-2 miRs. 
We would also like to predict IL-2 responsiveness by miR profiling. Recent studies have shown 
(75, 76) that miRs (miR-146a, miR-182) are differentially expressed following treatment with IL- 
2 or during T-cell activation in mouse models. Those miRs involved in regulating genes involved 
in the IL-2 production pathway (e.g. NFAT, AP-1 and NF-κB) and controlling FOXO1 expression 
and STAT5 signaling pathway are of particular interest. In our preliminary data, 9 miRNAs are 
differentially expressed during IL-2 treatment in a dose dependent manner in vivo. (5 miRs down- 
regulated and 4 miRs are up-regulated). We propose to perform miR profiling of clinical blood 
samples from RCC patients before receiving IL-2. After a clinical response has been documented, 
the miR profiles from responders and non-responders will be used to generate a training set to 
predict IL-2 responsiveness. 
 
2.14 
Natural Killer (NK) Cells in Renal Cancer. 
The role of NK cells in RCC has been considered possible since our earliest clinical trials of IL-2 
demonstrated remarkable expansion of NK cells in the peripheral blood and tissues during therapy 
in mice and humans. We now know that RCC can attract various effector cells of both the innate 
and adaptive immune system, including natural killer (NK) cells, neutrophils (77), γδ T cells, NKT 
cells, dendritic cells (DC), and regulatory T cells (Tregs). These, histology, and serum factors 
predict clinical response (78-83). These individual cell types do not act in isolation but function 
within complex networks (84). Unlike melanoma, where a clear role for T-cells has been identified, 
the case for T-cells in successful immunotherapy of renal cell cancer is far less clear (85). Although 
RCC responds to immune modalities, T-cell therapies have shown a very limited success in 
contrast with melanoma. RCC immunotherapy with NK cell infusions and agents that enhance cell 
lysis is an intriguing alternative strategy (86). Several defects in cell-mediated immune function 
are apparent in RCC patients at the time of diagnosis. These defects transiently worsen after 
nephrectomy but return to baseline by post-resection on day 8 (87). High dose Interleukin 2 (HDIL- 
2) is FDA-approved for therapy of patients with metastatic melanoma and renal cell carcinoma, 


21 
 
based on its ability to induce durable responses in 5-10% of patients but associated with substantial 
side effects (88). Efforts to develop more potent IL-2 analogues that preferentially target activated 
T-cells but not NK cells have been made. BAY 50-4798, such an IL-2-specific agonist, was tested 
in patients with RCC (89) with only 1/20 responding, suggesting with such limited response, that 
NK cells are possibly important in this disease. 
There are many infiltrating cells within RCC (90). When examining resected RCC, two 
subgroups are identified with high (>20% of the lymphocytes, n = 14), or low (<20%, n = 20), NK 
cell numbers. Although these cells are noncytolytic the majority of NK cells from tumors with 
high NK cells become cytotoxic following IL-2 exposure (113) whereas those from the low NK 
tumors do not. Significantly increased numbers of intratumoral CD56+ NK cells (p=0.008) and 
CD8+ T cells (p=0.019) compared with baseline are found after treatment with IL-2 and histamine 
(91). Low-dose IL-2, IFNα and GM-CSF, administered perioperatively (92) increased intratumoral 
numbers of CD3+ T cells, S100+ DC, CD83+ DC and IL-2R+ cells (4-fold, 2-fold, 10-fold and 20- 
fold, respectively, compared to controls). It further increased TNFα+ CD8+ T cells and DC-SIGN 
(CD209)+, CD83+, CD80+, IL-12+ and DC-CK1 (CCL18)+ DC. NK cells comprise a significantly 
higher proportion of in RCC TILs relative to peripheral blood colorectal or breast cancers, where 
these cells are found in small numbers. Notably, significantly fewer NK cells are observed in 
peripheral blood from patients with metastatic RCC (93). All the RCC lesions (n = 140) tested in 
one study by tissue microarray are cytosolic MHC class I-Related Chain A/B [MICA/B+] (94), 
important for recognition by NKG2D expressing NK cells. Substantial numbers of NK cells in 
RCC can be detected with an antibody to NKp46, exclusively expressed by all NK cells in mouse 
and man. Cytotoxicity is observed after overnight activation with low-dose IL-2, proportional to 
the number of NK cells. Infiltrating NK cells express a functional inhibitory receptor, 
CD94/NKG2A and are cytolytic following cytokine stimulation (95). 
Human TLR triggered monocytes up-regulate MICA and in turn downregulate NKG2D on 
autologous NK cells (96) and enhance production of IFN-γ. Thus myeloid cells (monocytes) and 
NK cells communicate directly during an innate immune response in humans. Two distinct 
activating NK cell synapses can be identified, a lytic and nonlytic one. Triggering NK cell 
proliferation and cytokine secretion is associated with NK cell 2B4, while macrophages are killed 
in NK cell NKG2D/DAP10 clusters (97). Interestingly, IFN-γ in turn down-regulates NKG2D 
ligand expression and subsequent cytolysis of MHC class I-deficient tumor cells by NK cells (98). 
This may be dependent on inducing a protective autophagy in these cells as we have shown in 
murine models. Expression of other MHC molecules on fresh RCC (99-101) such as HLA-G may 
be also be important for evasion from immune detection by NK cells. It is lost during cultivation 
of RCC cells and thus the tumor microenvironment and/or endothelium appear to be involved in 
its regulation, likely by IFNγ. HLA-G expression in the 27% of RCC lesions prevents lysis of RCC 
cells by cytokine immune effector cells. Enhancement of NK cytolytic activity by fever-range 
thermal stress is also dependent on NKG2D function (102). Assessment of pSTAT5 in patient 
PBMC in response to therapeutic IL-2 administration reveals persistent activation of STAT5 
within circulating NK and T cells for prolonged periods (103). Although chloroquine is said to 
diminish cytolytic activity of NK cells, this has not been tested in patients with cancer or certainly 
those with renal cancer (104). We will examine the function of NK cells in the proposed blood 
samples obtained in this study. 


22 
 
2.15 Dendritic Cells (DCs) and Renal Cancer. 
 
Multiple strategies have been employed to use DCs transfected with antigen or fused with tumor 
to generate an effective antitumor response. To date, they have been largely disappointing. 
Interestingly IL-2 treatment during DC-based immunotherapy of patients with metastatic renal 
cancer (105) leads to a transient and ‘massive’ increase of circulating natural regulatory T-cells, 
perhaps explaining their limited effectiveness; we hope to evaluate their number in the setting of 
HCQ administration. Intranodal autologous tumor lysate-DC vaccines with IL- 2 and IFNα therapy 
in RCC patients led to objective clinical responses in 9/18 patients with three CRs. Pre-IP-10 serum 
levels might have helped predict outcome (106). We [WJS] found decreased numbers of myeloid 
and plasmacytoid DC in the peripheral blood of RCC patients and were found at large numbers in 
the tumor, where they displayed an immature phenotype (DC-LAMP) (107). In another study 27 
patients with RCC were vaccinated with DCs loaded with either a cocktail of survivin and 
telomerase peptides or tumor lysate depending on their HLA-A2 haplotype, with low-dose IL-2. 
None of the patients had an objective response (108). Although not subjected to clinical trial, 
apoptotic RCC appeared to be better inducers of cross-presenting activity than necrotic cells (109). 
Another tumor lysate (TL)-pulsed DC study in patients with RCC, only 1/9 patients achieved a 
partial response (110). Electrofused allogeneic DC/autologous tumor-derived cells in patients with 
2/20 patients demonstrated only a single PR by RECIST criteria (111). As in other epithelial 
tumors, resected RCC in 25 patients, using immunohistochemistry, higher CD83+ DC in the tumor 
predicted a better survival (p = 0.0339) (112). Patients with RCC vaccinated with Carbonic 
anhydrase-IXG250/MN (CA9) -peptide-pulsed mature DCs, showed no evidence for induction of 
CA9-peptide-specific immunity or any clinical responses (113). Allogeneic DCs with or without 
cyclophosphamide have also been tested in patients with RCC. Only 2/22 patients had mixed 
responses, both in the cyclophosphamide group (114). Thus we conclude that little has been gained 
in this disease by conventional DC therapies. Little is known about chloroquine effects on 
circulating or tumor DCs but it appears to enhance human CD8+ T cell responses against soluble 
antigens in vivo (115). We are aware of the important role of autophagy as a source of antigen for 
cross-presentation but consider that in the setting of established cancer that autophagy in the tumor 
is the more critical process needing to be blocked to enable effector NK and T cells. 
 
2.16 T-cells and Renal Cancer. 
 
Human renal cell cancer (RCC) is clearly responsive to immunotherapy. Clinical responses may 
be mediated by "non-specific" (e.g. NK cells) or "specific" MHC-class-I-restricted tumor-specific 
CD8+ T lymphocytes. Typically RCC progresses, however, despite significant infiltration of 
various lymphoid cells. We (116) examined freshly isolated RCC tumor-infiltrating lymphocytes 
(TIL) derived from seven RCC patients for cytokine expression by the polymerase chain reaction 
(PCR). Established RCC tumor cell lines derived from these RCC patients were negative for 
interleukin-2 (IL-2), IL-4, IL-10, and interferon gamma and found to be positive for tumor necrosis 
factor alpha (TNF α), IL-6, IL-1 β, granulocyte/macrophage-colony-stimulating factor (GM-CSF), 
and transforming growth factor beta 1 (TGFβ1) message as detected by PCR. An identical pattern 
of cytokine mRNA expression was identified in other long-term RCC lines and in normal human 
kidney cells upon culture, but not in two Wilms tumor cell lines tested. Short-term-, and long- 
term-established RCC lines, but not Wilms tumor lines, secreted substantial levels of GM-CSF, 
TNFα, IL-1 beta, and IL-6 as detected by enzyme-linked immunosorbent assay. Both RCC lines 


23 
 
and Wilms tumor lines secreted TGF β1. In comparison, normal kidney cells secreted IL-6 and 
GM-CSF, but not IL-1 β, or TFG β 1 under identical in vitro cell culture conditions. We applied 
PCR-based methods to characterize the cytokine mRNA expression pattern in immune cells 
infiltrating into renal cell cancer without the need for expansion of such effector cells in vitro. 
Examining freshly collected RCC TIL by PCR from patients with primary cell cancer, we could 
demonstrate that such cells, but not lympho-mononuclear cells harvested from normal human 
kidney tissue, typically exhibit IL-4 and IL-10 mRNA expression. Given that alterations in tumor 
autophagy would be predicted to affect the death rate of these cells and provision of Ag for 
crosspriming of specific T cells, we propose to evaluate the HLA-A2+ patient cohort for changes 
in polarized CD8+ T cell response to a range of RCC and tumor stromal-associated antigens 
(EphA2, EGFR, HEr2, MAGE-3/6, PDGFR, RGS5, VEGFR1/2, G250 (CA-IX), DLK1, TEM1, 
and HBB) (117-121) pre- vs. post-treatment with hydroxychloroquine in the peripheral blood. In 
particular, IFN-γ response would be prioritized, but we could also consider granzyme or TNF-α 
(Tc1) vs. IL-4/-5 or IL-10. It would be possible to do this by intracellular cytokine stain in concert 
with surface staining for CXCR3 and/or VLA-4, given the importance of these markers for the 
ability of Th1 cells to home to and infiltrate tumors (113, 122). 
Relapsed/refractory NBL patients receiving the hu14.18-IL-2 IC (humanized anti-GD2 
monoclonal antibody linked to human IL-2) in a Children’s Oncology Group (COG) Phase II trial 
were genotyped for KIR and HLA to determine if KIR receptor-ligand mismatch were associated 
with anti-tumor response. Thirty-eight of 39 patients enrolled had DNA available for analysis; 24 
were found to have autologous KIR/KIR -ligand mismatch; 14 were matched. Seven of 24 
mismatched patients experienced either complete response or improvement of their disease 
following IC therapy. There was no response or comparable improvement of disease in patients 
who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement 
to IC (p = 0.03). Thus response or improvement of relapsed/refractory neuroblastoma patients after 
IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for 
NK cells in this clinical response (123,124). The NK content of TILS can be predicted by transcript 
levels of NKp46, perforin, CX3CL1, and CXCR1 with normalization using qRT-PCR and are 
prognostic indicators of survival (125). 
 
2.17 Rationale for the current trial 
 
The rationale for combining the high dose bolus aldesleukin with hydroxychloroquine includes 
potential positive interactions on the immune regulatory side, non-overlapping toxicities, and 
potential for prolongation and increased number of responses based on murine studies conducted 
at the University of Pittsburgh. This study is a multi-center phase II study designed to estimate the 
efficacy of combination therapy of standard high dose bolus IL-2 and various doses of 
hydroxychloroquine therapy in metastatic RCC patients. The combination of high dose bolus IL- 
2 and hydroxychloroquine has not been used in prior clinical trials. Hydroxychloroquine has been 
shown to be safe in other acute stressful situations such as operative procedures (127-130). 
Combinations of IL-2 with chloroquine have recently been shown in Pittsburgh to be statistically 
more effective than IL-2 alone, tested in three murine models (131). Based on this murine data, we 
could expect to double the complete response rate in patients. Use of the number of doses of IL-2 
as a composite of maximum tolerated dose and dose limiting toxicity helps us (132, 133) deal with 
assessing the tolerability of the addition of a second drug to IL-2 designed to enhance efficacy 


24 
 
or decrease toxicity. Evaluating toxicity after 8 doses allows us to compare comparable amount of 
drug across patients when all are expected to have received all eight of the doses. 
 
The primary outcomes for this study will be overall survival, progression-free survival and 
response rate. A secondary analysis will compare outcomes between the trial patients and a 
historical control set matched according to the prognostic criteria described by Motzer et al (20) 
[lactate dehydrogenase (> 1.5 upper limit of normal), hemoglobin (< lower limit of normal), 
corrected calcium (>10mg/dL), Karnofsky performance status (< 80%) and time from initial RCC 
diagnosis to start of interferon-α therapy of less than one year were significant prognostic factors. 
Patient categories are no risk factors, 1 or 2 risk factors, or 3 or more] (20). 
 
2.18 Early experience with the regimen. 
 
The first five patients were treated with 600mg of Hydroxychloroquine with one patient deemed a 
complete response. Out of the 13 patients treated with combination HCQ/IL-2 therapy using 1200 
mg of Hydroxychloroquine, two patients experienced pronounced hypotension and tachycardia. 
Furthermore, one patient died from pulmonary emboli, not attributed to therapy. The cardiac events 
are consistent with IL-2 toxicities, but were observed somewhat earlier in the course of treatment. 
For that reason we are accruing another 21 patients to assess response in addition to the first five 
patients at the lower 600mg/d dose. 
 
 
 


25 
 
3.0 ELIGIBILITY 
 
3.1 
Inclusion Criteria 
 
3.1.i. 
Histologically confirmed metastatic renal cell carcinoma with predominantly clear 
cell histology. 
 
3.1.ii. Have measurable disease by RECIST 1.1 criteria. For example, this would include 
tumor in the lung, liver, and retroperitoneum. Bone disease is difficult to follow 
and quantify and as a sole site would not be acceptable. 
 
3.1.iii. Patients must be at least 4 weeks from radiation or surgery and recovered from all 
ill effects. 
 
3.1.iv. Age ≥18 years. 
 
3.1.v. Karnofsky Performance Status ≥80%. 
 
3.1.vi. Adequate end organ function: 
 
a. Hematologic: ANC ≥ 1000 cells/L, platelets ≥ 100,000/L, hemoglobin ≥ 
9g/dl (pre transfusion values used for prognostic factor, can be transfused or 
use recombinant erythropoietin growth factors but must not have active 
bleeding). 
 
b. Liver: AST ≤ 2 x ULN (upper limit of normal), serum total bilirubin ≤ 2 x ULN 
(except for patients with Gilbert’s Syndrome). 
 
c. Renal: serum creatinine ≤ 1.5 mg/dL or estimated creatinine clearance ≥ 
60ml/min using Crockcroft-Gault estimation using the formula 
 
This 
formula expects weight to be measured in kilograms and creatinine to be 
measured in mg/dL, as is standard in the USA. 
 
d. Pulmonary: FEV1 ≥ 2.0 liters or ≥ 75% of predicted for height and age. 
(PFTs are required for patients over 50 or with significant pulmonary or 
smoking history defined as >20 pack years or history of COPD/emphysema). 
 
e. Cardiac: No evidence of congestive heart failure, symptoms of coronary artery 
disease, myocardial infarction less than one year prior to entry, serious cardiac 
arrhythmias, or unstable angina. Patients who are over 40 or have had previous 
cardiac disease will be required to have a negative or low probability cardiac 
stress test for cardiac ischemia. 
 
3.1.vii. Women should not be lactating and, if of childbearing age, have a negative 
pregnancy test within two weeks of entry to the study. 
 


26 
 
3.1.viii. Appropriate contraception in both genders (i.e., use of contraceptive strategies that 
prevent pregnancy; this most effectively would involve abstinence during the 
period of treatment but could include contraceptives, contragestives, and barrier 
methods.) 
 
3.1.ix. The patient must be competent and have signed informed consent. 
 
3.1.x. CNS: No history of cerebrovascular accident, transient ischemic attacks, central 
nervous system or brain metastases. 
 
3.2 
Exclusion Criteria 
 
3.2.i. 
Patients who have previously received IL-2 are not eligible. Patients on HCQ in 
neoadjuvant protocols or in the past for clinical indications ARE eligible, as are 
patients who have previously received CTLA-4 and/or PD-1/PD-L1 antibodies. 
 
3.2.ii. Concomitant second malignancy except for non-melanoma skin cancer, and non– 
invasive cancer such as cervical CIS, superficial bladder cancer without local 
recurrence or breast CIS. 
 
3.2.iii. In patients with a prior history of invasive malignancy, less than five years in 
complete remission. 
 
3.2.iv. Positive serology for HIV, hepatitis B or hepatitis C 
 
3.2.v. Significant co-morbid illness such as uncontrolled diabetes or active infection that 
would preclude treatment on this regimen. 
 
3.2.vi. Use of corticosteroids or other immunosuppresion (if patient had been taking 
steroids, at least 2 weeks must have passed since the last dose). 
 
3.2.vii. History of inflammatory bowel disease or other serious autoimmune disease. (Not 
including 
thyroiditis 
and 
rheumatoid 
arthritis). 
Patients 
already 
on 
hydroxychloroquine for such disorders are not eligible. 
 
3.2.viii. Patients with organ allografts. 
 
3.2.ix. Uncontrolled hypertension (BP >150/100 mmHg). 
 
3.2.x. Proteinuria dipstick > 3+ or ≥ 2gm/24 hours. 
 
3.2.xi. Urine protein:creatinine ratio ≥ 1.0 at screening. 
 
3.2.xii. Major surgery, open biopsy, significant traumatic injury within 28 days of starting 
treatment or anticipation of need for major surgical procedure during the course of 
the study. 
 
 
 


27 
 
3.2.xiii. Minor surgical procedures, fine needle aspirations or core biopsies within 7 days 
prior to starting treatment. Central venous catheter placements are permitted. 
 
3.2.xiv. History of abdominal fistula, gastrointestinal perforation, or intraabdominal 
abscess within 6 months prior to starting treatment. 
 
3.2.xv. Serious, non-healing wound, ulcer, or bone fracture. 
 
3.2.xvi. History of tumor-related or other serious hemorrhage, bleeding diathesis, or 
underlying coagulopathy. 
 
3.2.xvii. History of deep venous thrombosis, clinically significant peripheral vascular 
disease, or other thrombotic event. 
 
3.2.xviii. Inability to comply with study and/or follow-up procedures. 
 
3.2.xix. Individuals with known history of glucose 6 phosphate deficiency are excluded 
from the trial (possible issue with HCQ tolerance). No literature can be found to 
support hemolytic anemia having ever occurred in these patients and 
rheumatologists who frequently utilize this drug have not seen this as a side effect. 
Given that this has been a concern that has been raised, we believe it is prudent to 
exclude these patients with known G6PD deficiency from participation. These patients can 
receive IL-2 alone off trial. 
 
3.2.xx. Patients with previously documented macular degeneration or diabetic retinopathy 
are excluded from the trial. 
 
3.2.xxi. Baseline EKG with QTc > 470 msec (including subjects on medication). Subjects 
with ventricular pacemaker for whom QT interval is not measurable will be eligible 
on a case-by-case basis. 
 
3.3 
Minority and Women Inclusion 
The patients for this study will be drawn from the oncology patient population seen at the 
Cytokine Working Group Institutions. No one will be excluded because of gender or 
race. 
 
 
 


28 
 
4.0 REGISTRATION PROCEDURES 
 
To register a patient, the investigator or their designee will contact the Clinical Research 
Coordinators of Clinical Research Services (CRS) at the University of Pittsburgh Cancer 
Institute: 
 
Deborah Hice, RN 
Phone: 412-623-8962 
Email: hiceda@upmc.edu 
 
Kathy Mansfield, RN 
Phone: 412-623-7048 
Email: manskd@upmc.edu 
 
If the coordinators are unavailable, contact Clare Grzejka, RN, BSN, at 412-623-4891 or 
grzejkac@upmc.edu. Patients must be registered prior to the first dose of 
hydroxychloroquine (day -14). The following information will be requested: 
 
4.1 
Investigator Identification 
 
4.1.1 
Institution name and/or affiliate 
 
4.1.2 
Investigator's name 
 
4.2 
Patient's Identification 
 
4.2.1 
Patient's initials and study ID   
 
4.3 
Eligibility Verification 
 
 
Patients must meet all of the eligibility requirements listed in Section 3.0. 
Documents verifying eligibility, including a completed eligibility checklist, must 
be sent via fax or email to the coordinating center. The coordinating center 
requires 24 hours to review eligibility of potential subjects from external sites. 
 
4.4 
Cancellation Guidelines 
 
 
No patient will be cancelled. Data will be collected on all patients, even if a patient 
does not receive protocol therapy. Reasons for a patient not initiating therapy should 
be submitted in writing as soon as possible. All patients receiving at least one week 
of IL-2 treatment and two weeks of oral HCQ will be included in the final evaluation. 
 
4.5 
Central Pathology Core 
 
 
All patients will be required to have sent four representative unstained slides or 
paraffin blocks containing tumor. Consent for requesting pathology material will 
be required for enrollment in the study. This material will be batched and sent to 
the UPCI Pathology Tissue Bank Core for central pathology review. 


29 
 
 
 
All samples should be mailed by courier to: 
 
 
 
UPCI Renal Cancer Tissue Bank 
c/o Rajiv Dhir, MD 
 
Department of Pathology 
Shadyside Hospital 
 
5230 Center Ave. 
 
Pittsburgh PA 15232 
 
 
 


30 
 
5.0 PRE-STUDY TESTING/STAGING 
 
All patients considered for entry in the trial should have measurable or evaluable metastatic 
disease as described in the RECIST 1.1 criteria (see Section 11.0). Staging will include: 
 
5.1 History and physical examination within two weeks of the first day of treatment (day 
-14). 
 
5.2 Within 4 weeks prior to treatment (day -14), the following procedures are required: 
 
5.2.i 
CT scans of the chest/abdomen/pelvis 
 
5.2.ii 
Brain MRI or enhanced brain CT 
 
5.2.iii 
Bone scan (if clinically indicated) 
 
5.2.iv 
Cardiac stress test: All patients over 40 years of age or with significant 
cardiac history 
 
5.2.v 
Pulmonary function tests within 4 weeks of starting therapy for all 
patients over 50 years old or with significant pulmonary or smoking 
history 
 
5.2.vi 
Cardiac ECHO or MUGA scan to assess LVEF 
 
5.2.vii 
12-lead EKG 
 
5.3 Within 2 weeks prior to treatment (day -14) the following procedures are required: 
 
5.3.i 
Urine protein:creatinine ratio (see appendix A) 
 
5.3.ii 
Urinalysis 
 
5.3.iii 
Laboratory tests: HIV, hepatitis B and C serology, pregnancy test when 
indicated, CBC with differential, comprehensive metabolic panel 
(creatinine, BUN, albumin, total protein, glucose, electrolytes, liver 
function tests [total bilirubin, alkaline phosphatase, aspartate 
aminotransferase, alanine aminotransferase]), direct bilirubin, LDH, Ca, 
PO4, TSH, PT, PTT. A serum sample for determination of serum 
VEGF/ANG1/ANG2 levels will be drawn on day -14, prior to the dose 
of hydroxychloroquine. 
 
5.4 Within 4 weeks after enrollment, tissue blocks or four unstained slides of renal tissue 
sent to central pathology facility. 
 
 
 


31 
 
6.0 TREATMENT PLAN & SCHEMA 
 
6.1 Classification: 
 
Patients will be classified by the following criteria prior to entry and the historical cohort will be 
group-matched according to these criteria. (Each criterion is dichotomized as absent - 0 or  
present -1): 
• lactate dehydrogenase > 1.5 upper limit of normal 
= 
1 
• hemoglobin < lower limit of normal 
= 
1 
• corrected calcium (>10mg/dL) (total calcium – 0.707*[albumin-3.4]) 
= 1 
• Karnofsky performance status < 80% 
= 
1 
• time from initial RCC diagnosis to start of protocol therapy < 1 year = 
1 
Patients  
# of Risk Factors 
Median time to Death 
Good Prognosis 
= 
no risk factors 
30 months 
Intermediate prognosis 
= 
1 or 2 risk factors 
14 months 
Poor prognosis 
= 
3 or more 
5 months 
 
6.2 Protocol Design 
 
Patients will receive high dose IL-2 + hydroxychloroquine 
 
Treatment: One course of treatment (84 days) will consist of high dose bolus IL-2 administered 
intravenously every 8 hours on days 1-5 (or 6) and 15-19 (or 20) (maximum 14 doses/5 days of 
administration) and hydroxychloroquine daily starting two weeks before IL-2 therapy and 
continued through up to three courses. Dependent upon when Dose 1 is administered on Day 1/Day 
15, Dose 14 could require hospitalization on Day 6/Day 20. 
 
6.3 Agents 
6.3.1 Hydroxychloroquine 
6.3.1.1 Administration. 
Patients will receive HCQ every day starting at 14d before the first dose of IL-2. HCQ can be taken 
at any time of the day prior to or after meals. Tablets of HCQ are available in 200 mg strengths. 
HCQ will be administered in divided doses (BID) for doses above 200 mg po qd to minimize 
nausea. The lower dose (200) should be taken in the AM and the higher dose (400) at night due to 
the potential for GI distress. Patients should be told to swallow the whole capsule in rapid 
succession without chewing. Missed and vomited doses of HCQ can be skipped during therapy if 
necessary. The two-week lead in and long half-life of HCQ allow for this. Two dose reductions 
(Levels -1 and -2 in Table 5) may be allowed in a patient if treatment-limiting toxicity (Section 
9.2) is observed. A pill diary will be provided and is required. 
 
Table 5. Hydroxychloroquine Dose Schema 
Dose Level 
Dose hydroxychloroquine (mg/day) 
1 
600 mg 
-1 
400 mg 
-2 
200 mg 
 
 
 


32 
 
6.3.1.2 Description 
• Generic name: 
Hydroxychloroquine sulfate 
• Commercial name: 
Plaquenil 
• Chemical name: 
7-Chloro-4-[4-[ethyl-(2-hydroxyethyl)amino]-1- 
methylbutylamino] quinolon 
• Supplied by: Commercially available; Sanofi-Aventis 
 
6.3.1.3 Known Potential Toxicities of Hydroxychloroquine 
• Central nervous system: Irritability, nervousness, emotional changes, nightmares, 
psychosis, headache, dizziness, vertigo, seizure, ataxia, lassitude. 
• Dermatologic: Bleaching of hair, alopecia, pigmentation changes (skin and mucosal; 
black-blue color), rash (urticarial, morbilliform, lichenoid, maculopapular, purpuric, 
erythema annulare centrifugum, Stevens-Johnson syndrome, acute generalized 
exanthematous pustulosis, and exfoliative dermatitis). 
• Gastrointestinal: Anorexia, nausea, vomiting, diarrhea, abdominal cramping, pancreatitis. 
• Hematologic: Aplastic anemia, agranulocytosis, leukopenia, thrombocytopenia, 
hemolysis (in patients with glucose-6-phosphate deficiency). 
• Hepatic: Abnormal liver function/hepatic failure (isolated cases). 
• Neuromuscular & skeletal: Myopathy leading to progressive weakness and atrophy of 
proximal muscle groups (may be associated with mild sensory changes, loss of deep 
tendon reflexes, and abnormal nerve conduction). 
• Ocular: Disturbance in accommodation, keratopathy, corneal changes/deposits (visual 
disturbances, blurred vision, photophobia - reversible on discontinuation), macular 
edema, atrophy, abnormal pigmentation, retinopathy (early changes reversible - may 
progress despite discontinuation if advanced), optic disc pallor/atrophy, attenuation of 
retinal arterioles, pigmentary retinopathy, scotoma, decreased visual acuity, nystagmus. 
6.3.2 Interleukin 2 
 
6.3.2.1 Administration 
IL-2- 600,000 IU/kg IV bolus q 8 hrs beginning on day 1 and on day 15, for a maximum of 14 
doses per cycle. 
 
6.3.2.2 Description 
Aldesleukin is recombinant formulation of interleukin-2 (IL-2). Aldesleukin is a nonglycosylated 
biosynthetic interleukin-2 (also known as T-cell growth factor), which differs only slightly in 
amino acid sequence from the natural compound with a serine for cysteine substitution at position 
125 to allow correct folding. Aldesleukin is commercially available from Prometheus. 
 
6.3.2.3 Mechanism of Action 
Aldesleukin's effects are essentially identical to those of endogenous interleukin-2. Aldesleukin 
interacts with the high-affinity IL-2 receptor expressed on cells of the immune system and 
stimulates a cytokine cascade involving various interferons, interleukins, and tumor necrosis 
factors. Aldesleukin along with other cytokines induce proliferation and differentiation of B and 


33 
 
T-cells, monocytes, macrophages, and cytotoxic lymphocytes which include natural killer (NK) 
cells, cytotoxic T-cells, tumor-infiltrating lymphocytes (TIL), and lymphokine-activated killer 
(LAK) cells. Aldesleukin's antitumor activity is believed to result from activation of cytotoxic 
lymphocytes, however, the exact mechanism is unknown. Whether aldesleukin acts directly or 
through second messengers is also unclear, however, aldesleukin does elevate production of 
interleukin-1, tumor necrosis factors α and β, interferon γ, and interleukin-6. 
 
6.3.2.4 Pharmacokinetics 
Aldesleukin is administered parenterally. Following a short IV infusion, the drug is rapidly 
distributed to the extravascular and extracelluar space as well as to the liver, spleen, kidneys, and 
lungs. Approximately 30% of an administered dose is distributed within the plasma. The 
pharmacokinetics of aldesleukin may be affected by sodium dodecyl sulfate, the solubilizing agent 
in the commercial formulation. In addition, subcutaneous administration with albumin produces 
slightly higher and more prolonged serum concentrations of aldesleukin. Following distribution, 
aldesleukin is cleared from the systemic circulation by the kidneys through both glomerular 
filtration and peritubular extraction. The drug is then metabolized to amino acids by renal cells 
lining the proximal convoluted tubules. Very little drug is excreted unchanged in the urine. 
Following a 5 minute IV infusion, the serum distribution and elimination half-life in cancer 
patients was 13 and 85 minutes, respectively. 
 
6.3.2.5 Toxicities 
Abdominal pain, alopecia, anemia, angina, anorexia, anuria, arthralgia, ascites, atrial fibrillation, 
azotemia, back pain, chills, cholestasis, conjunctivitis, constipation, diarrhea, drowsiness, 
dysgeusia, dyspepsia, dyspnea, dysuria, edema, elevated hepatic enzymes, eosinophilia, erythema, 
exfoliative dermatitis, fatigue, fever, GI bleeding, GI perforation, glomerulonephritis, 
hallucinations, 
headache, 
hematuria, 
hepatomegaly, 
hyperbilirubinemia, 
hyperkalemia, 
hypoalbuminemia, 
hypocalcemia 
, 
hypokalemia, 
hypomagnesemia, 
hyponatremia, 
hypophosphatemia, hypotension, hypovolemia, infection, injection site reaction, interstitial 
nephritis, jaundice, leukocytosis, leukopenia, lymphopenia, malaise, metabolic acidosis, metabolic 
alkalosis, myalgia, myocardial infarction, nausea/vomiting, oliguria, pancreatitis, paranoia, 
petechiae, pleural effusion, premature atrial contractions (PACs), premature ventricular 
contractions (PVCs), proteinuria, pruritus, pulmonary edema, purpura, sinus bradycardia, sinus 
tachycardia, splenomegaly, stomatitis, supraventricular tachycardia (SVT), syncope, tachypnea, 
thrombocytopenia, thrombosis, urticaria, visual impairment, weakness, weight gain, weight loss, 
wheezing, xerosis. 
 
6.4 Response Assessment/ Duration of Therapy 
 
Patients will undergo CT scans at week 8 and week 12 (+/- 1 week) following initiation of IL-2 
therapy and then after each course (every 10-12 weeks). After the final course (week 36), patients 
will undergo CT scan evaluation every 12 weeks (+/- 2 weeks) for up to one year or until tumor 
progression (whichever comes first). If additional scans are performed for clinical reasons, the 
study schedule may be altered to perform the requisite scans at a more acceptable time point. The 
change in timing of study scans must be approved by the local PI and documented. For patients 
who have not shown progression at the end of one year, response will be captured during follow- 
up for survival (explained below). 
 
 
 


34 
 
Patients with evidence of tumor shrinkage with all lesions measurable and evaluable may receive 
up to 3 courses of IL-2 therapy. Course 2 will start on or within 2 weeks following day 85 and 
follow the same schedule as in Course 1. Course 3 can start within 2 weeks of completion of Course 
2. Patients not receiving IL-2 during course 2 with tumor shrinkage by week 24 can receive a 2nd 
course of IL-2 beginning within 2 weeks of week 25. 
 
Patients with no evidence of progression by RECIST 1.1 criteria may receive continued treatment 
with hydroxychloroquine through one year of initiation of HCQ treatment (Day -14) or until 
disease progression. 
 
Tumor response will be assessed by the NCI’s Response Evaluation Criteria in Solid Tumors 
(RECIST) 1.1 guidelines. Patients will undergo tumor evaluation with physical exam and CT scans 
at week 8 (+/- 1 week) and week 12 (+/- 1 week), then after each course (every 10-12 weeks). After 
the final course (week 36), patients will undergo CT scan evaluation every 12 weeks (+/- 2 weeks) 
for up to one year or until tumor progression (whichever occurs first). If additional scans are 
performed for clinical reasons, the study schedule may be altered to perform the requisite scans at a 
more acceptable time point. The change in timing of study scans must be approved by the local PI 
and documented. For patients who have not shown progression at the end of one year, response will 
be captured during follow-up for survival, which will be done on all patients via telephone contact 
and medical record review. Patients with progressive disease by RECIST 1.1 criteria will be taken 
off study. 
Table 6. Tumor Assessment 
 
Prior to therapy 
At week 8 (course 1), 
and at the end of courses 2 
and 3. 
Every 3 mos. until 
1 yrs 
At disease 
progression 
CT C/A/P, BS*, 
MRI** 
CT C/A/P, BS*, MRI** 
CT C/A/P, BS*, 
MRI** 
CT C/A/P, BS*, 
MRI** 
PE 
PE 
PE 
PE 
 
CT C/A/P = Computerized Tomography of the chest/abdomen/pelvis, BS = Bone scan, MRI = 
Brain MRI or enhanced Brain CT, PE = Physical examination. 
Tumor evaluation will include CT of chest/abdomen/pelvis. 
*Bone scan should be performed if bone metastases documented prior to study enrollment or if 
clinically indicated. 
**Brain MRI or enhanced Brain CT is required prior to therapy for evaluation of brain metastases. 
The Brain MRI or enhanced Brain CT will be repeated if clinically indicated. 
Response criteria are provided in section 10.0. 
6.4.1 Toxicity Evaluation 
Patients enrolled in this study will be evaluated clinically and with standard laboratory tests before 
and at regular intervals during their participation in this study. Safety evaluations will consist of 
medical interviews, recording of adverse events, physical examinations, blood pressure, and 
laboratory measurements. Patients will be evaluated for adverse events (all grades), serious adverse 
events (SAE), and adverse events requiring study drug interruption or discontinuation at each study 


35 
 
visit for the duration of their participation in the study. Specific monitoring procedures are as 
follows: 
• 
During IL-2 treatment, AEs will be assessed daily. 
• 
In patients with bleeding, hemostasis evaluation should be performed as clinically indicated 
• 
All patients will be followed by either history, physical examination, laboratory tests or 
telephone interviews until resolution of AEs. The frequency of these contacts will depend 
on the type and severity of the SAE and will be determined by the investigator. 
• 
Patients will have at least one telephone contact between cycles of IL-2 administration to 
assess toxicity. A CBC and complete metabolic survey will also be obtained. 
All patients on trial will have a compilation of treatment (number of doses), toxicity scoring, 
laboratory results, and response assessment monthly (see Section 8) by the Clinical Trials Office 
and this information made available at the time of the monthly teleconferences, the Cytokine 
Working Group teleconferences, and to the local oversight DSMB. In addition, the trial will be 
monitored monthly by the UPCI Multi-Center Trial DSMC, which operates under the direct 
supervision of the UPCI DSMC. Data on accrual and adverse events at all sites will be aggregated 
by UPCI Clinical Research Services so that the Multi-Center DSMC has the necessary data to 
perform its function in a timely fashion. 
 
Patients discontinued from the treatment phase of the study for any reason will be evaluated ~30 
days (28-42 days) after the decision to discontinue treatment. 
 
6.4.2 Clinical Assessment and Duration of Therapy 
 
All patients will have blood tests and a thorough physical examination prior to each cycle of IL-2. 
Further therapy will be withheld until laboratory values and performance status return to within 
the eligibility criteria. IL-2 may be re-initiated once serum creatinine reaches ≤ 1.8 mg/dL (at the 
discretion of the treating physician). 
 
Both responding and non-responding patients will be followed until death or final closure of the 
study. After the final course of treatment, follow-up procedures will be performed every 3 months 
(+/- 2 weeks) for up to one year or until disease progression (whichever occurs first). Follow-up 
procedures will include CT scan, routine laboratory tests, and physical examination. Subjects will 
then be followed for survival by telephone contact and medical record review every 3 months for 
one year following the time of disease progression or after one year off of treatment, then every 6 
months for the next two years, then annually until death. A window of +/- 2 weeks is permitted. 
This will include review of any routine procedures performed as part of the patient’s standard care 
(e.g., routine scans, physical examinations, laboratory exams, etc.). Attention will be paid to the 
time of disease progression and time of initiation of alternate therapies. 
In the absence of treatment delays due to adverse events, treatment may continue for up to 3 
courses per protocol or until one of the following criteria applies: 
 
• Disease progression (see section 10.0), 
• Intercurrent illness that prevents further administration of treatment, 
• Treatment-related toxicities (see section 9.5), 


36 
 
• Subject decides to withdraw from the study, or is non-adherent, 
• Subject becomes pregnant or begins breast-feeding, 
• General or specific changes in the subject's condition rendering the subject unacceptable 
for further treatment in the judgment of the investigator. 
 
Patients discontinued from the treatment phase of the study for any reason will be evaluated ~30 
days (28-42 days) after the decision to discontinue treatment. 
 
6.5 Ancillary Treatment 
 
6.5.1 Venous Access Catheters 
 
Institutional guidelines should be followed for venous access. However, it is 
recommended that patients with adequate peripheral venous access be treated through 
2 large bore peripheral IVs or PICCs. Patients without adequate peripheral venous 
access should have a double lumen central venous catheter or PICC placed prior to 
initiating each course of therapy. Central venous catheters will be placed preferentially 
in the subclavian vein. Infusaports should be used for neither IL-2 administration 
nor continuous IV hydration as this may create an unacceptable infection risk. 
Catheters should be removed at the end of each hospital course. Patients requiring 
central venous catheters or PICC will receive antibiotic prophylaxis (e.g. cephaloxin 
250 mg PO QID for those without penicillin allergies or ciprofloxacin, 250 mg PO BID, 
days 1-10 and 15-24 of each course). If catheter related bacteremia develops, the 
catheter should be removed and parenteral antibiotic treatment (vancomycin 1 gm IV 
q 12-24 hours depending on renal function) may be required. 
 
6.5.2 Suggested Concurrent Therapy 
Corticosteroids will not be permitted. 
Antihypertensive therapy should be discontinued at least 48 hours prior to initiating 
each cycle of IL-2 and for the duration of the cycle. Investigators may choose to taper 
beta blockers over a few days prior to each cycle. 
 
While IL-2 is administered (days 1-5 (or 6) and 15-19 (or 20)), patients may receive the 
following to abrogate toxicity related to IL-2 (start before IL-2 begins, continue for 12 
hours after last IL-2 dose): 
 
Acetaminophen 650mg PO every 4 hours. 
 
Meperidine (25-50mg) IV or IM should be given in the case of severe 
rigors. 
 
Indomethacin 25mg PO every 6 hours, or Naprosyn 750mg PO every 12 
hours, or Motrin 600mg PO every 6 hours can be given for fever, 
chills and myalgias unresponsive to acetaminophen. 
 
Ranitidine (150 mg) or Axid (150 mg) or Pepcid (20 mg) PO q 12 hours 


37 
 
for prophylaxis of gastrointestinal bleeding. 
 
Lomotil or Imodium supplemented with paregoric, 10-14 ml PO q 4-6 
hours PRN will be used for diarrhea. 
 
Benadryl (25-50 mg PO),Atarax (25 mg PO), or Gabapentin (300-600mg 
PO TID) may be used for generalized erythematous skin rash and/or 
pruritus. 
 
Furosemide (20-40 mg) IV/PO qd may be administered following 
completion of IL-2 infusion and until edema resolves and weight 
returns to baseline per local physician. 
 
Keflex 250mg PO QID for prophylaxis of catheter-related infections. 


38 
 
7.0 LABORATORY STUDIES 
 
The schedule for blood sampling is in Appendix C. Bloods will be sent directly by overnight 
courier to the UPCI for separation and cryopreservation. 
 
UPCI – IMCPL 
5117 Centre Avenue 
1.26 Hillman Cancer Center 
Pittsburgh, PA 15213 
Attn: Processor 
 
Phone: 412-624-0078 
 
7.1 Baseline tumor samples All patients will be required to have four representative unstained 
slides or paraffin blocks containing tumor available to be sent within four weeks of enrollment in 
the study. This material will be batched and sent for analysis to: 
 
UPCI Renal Cancer IL-2/HQ Tissue Bank 
c/o Rajiv Dhir, MD; Department of Pathology; UPCI 
5117 Centre Ave. 
Pittsburgh, PA 15213 
 
7.2 Serum and PBMC 
a. All serum and plasma samples will be aliquoted and stored at 
-70 to -80ºC at the UPCI. Specimens will be sent overnight to the IMCPL/UPCI 
(see Appendix D): 
 
b. Blood samples will be used for: 
1) Western Blots and EM for measures of autophagy (LC3, p62, Caveolin 1, 
HMGB1, sRAGE)-Research Laboratory (DAMP Laboratory) 
2) Serum angiogenic cytokine measurements including VEGF, ANG1, and ANG2; 
Fibronectin; Osteopontin (IMCPL) 
3) Immune Function: TCR zeta chain, serum arginase/arginine levels (IMCPL) 
4) Phenotype: KIRS, T cells, Treg, MDSC, NK cells, Measures of CD11b+IL4Rα+ myeloid 
derived suppressor cells, CD56bright and CD56dull NKp46+ NK cells, and Lineage-HLA- 
DR+ myeloid DCs, and CD123+Lineage- plasmacytoid DCs will be assessed. Measures 
of cell surface RAGE and cytosolic RAGE on permeabilized cells will also be carried 
out. (IMCPL) 
5) miRNA-Research Laboratory (DAMP Laboratory) 
6) Bank DNA and/or RNA (i.e. RNA PAX tubes) for germline SNP, gene expression 
analysis etc.) See 7.5. 
 
Autophagy assays will be run on all specimens 
 
7.3 Correlate the level of autophagy in resected renal adenocarcinomas (where available) 
with measures of clinical outcome and response. We hypothesize that levels of autophagy in 
biopsies from patients on treatment will directly correlate with response to therapy. Fixed 
specimens in 10% formalin at room temperature, and the size and gross appearance of the tumors 
will be recorded. Select serial sections of the tumor specimens will be assessed for 


39 
 
clinicopathologic correlates, HMGB1, RAGE, LC3, Caveolin 1, and p62 staining as described 
below. The immunohistochemical staining results will then be analyzed statistically to examine 
the relationship between clinicopathological factors in the tumor, disease-free survival and overall 
survival. 
 
Clinicopathological Correlates: 1) tumor size (≥3 vs <3 cm); 2) predominant differentiation of the 
tumor (well,moderately, or poorly differentiated); 3) lowest degree of tumor differentiation 4) 
angiolymphatic invasion 5) neural invasion; 6) margin status; 7) International.Union Against 
Cancer (UICC) pathological T and N stage. 
 
Immunohistochemical staining for autophagy. We will use a modification of the technique 
described by Fujii et al to evaluate LC3 staining in tumor tissues. Briefly formalin-fixed, paraffin 
embedded tissue sections containing the maximal cancer tissue area will be processed for 
immunohistochemical staining. Because nerve cells stain for both RAGE and LC3 they will be 
used as internal positive controls for each section Tumor cells whose staining intensity is equal to 
or stronger than that of nerve cells will be judged to be strongly positive, whereas those whose 
staining weaker than that of the nerve cells will be scored as weakly positive. Tumor cells that do 
not stain positively for LC3 or RAGE immunohistochemically despite a positive internal control 
will be scored as negative. We will select sections from the center and the invasive border for 
independent review and scoring. The dominant intensity level of positive cells will be evaluated 
as follows: The level of intensity of staining in each area will be determined by the percentage of 
cells that stain negative, weakly positive, and strongly positive. When more than 50% of the 
positive cancer cells are strongly positive for LC3/RAGE in each area, the area will be scored as 
strongly positive, and when more than 50% of the LC3/RAGE positive cancer cells are weak 
positive the area will be designated weakly positive. When 30% of the cancer cells are weakly 
positive and 40% are strongly positive, the predominant intensity will be recorded as strongly 
positive. Specimens will then be classified into three groups according to the dominant overall 
intensity of the cancer tissue: negative, weakly positive, or strongly positive. A similar procedure 
will be carried out to assess HMGB1 staining of the cytosol. Two independent pathologists, 
blinded to the clinical outcomes will evaluate all of the immunohistochemical slides and their 
results compared and reviewed for discrepancies by a third party. 
 
7.4 Correlate the levels of autophagic markers with measures of clinical outcome in 
peripheral blood mononuclear cells. We hypothesize that there will be biomarkers in the 
peripheral blood that will be specifically associated with induction of autophagy in renal cancer 
patients. We further hypothesize that these markers will correlate with treatment status, stage, and 
response to therapy, disease progression and overall survival. To test this hypothesis we will 
correlate the level of autophagy in the peripheral blood mononuclear cells with these measures of 
clinical outcome. Blood samples will be collected according to the schedule outlined in Table 6. 
Peripheral blood mononuclear cells are then separated from whole blood via ficoll hypaque 
separation and frozen in DMSO. Cells will be lysed for western blot analysis for RAGE, HMGB1, 
LC3, LC3-2, pre-LC3, caveolin 1, and p62. Levels of autophagic proteins, and RAGE/HMGB1 
will then be compared between time points and correlated with tumor clinicopathologic correlates, 
response to therapy, time to progression, and overall survival. 
 
7.5. Evaluation of KIR/KIR-ligand Genotype and Response to Therapy. KIR genotyping will 
be performed on patient DNA samples by PCR sequence-specific primer technique (SSP Unitray 
assay, Invitrogen Corporation, Carlsbad, CA) in the UPCI Genomics Facility. KIR-ligand typing 


40 
 
will be performed at low-resolution on the same samples for HLA-B & -C loci by reverse PCR- 
SSO methodology (LifeMatch assay, Gen-Probe, Inc, Stamford, CT) and for high-resolution HLA-
C alleles by direct sequencing (AlleleSEQR assay, Abbott Labs, Des Plaines, IL). The four 
inhibitory KIR genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1) evaluated in this study and 
their corresponding KIR-ligands (HLA-C1, HLA-C2, or HLA-Bw4). KIR/KIR-ligand mismatch 
will be defined as absence of one or more HLA alleles known to be ligands for the inhibitory KIR 
genes present, using previously published criteria. 
 
 


41 
 
8.0 DATA MANAGEMENT AND DATA SAFETY MONITORING PLAN 
 
The number of IL-2 doses administered, severe adverse events, including their relatedness to 
HCQ (definitely, probably, possibly or unlikely), clinical response (Section 10.3), date of 
progression and date of death will be forwarded immediately to the Data Coordinating Center at 
UPCI for monthly presentation to the CWG and the UPCI Multi-Center DSMC. The UPCI 
IMCPL will report to the DCC on samples received on a monthly basis. Biomarker and 
laboratory data that will be evaluated at the end of the trial need only be reported to the DCC 
when the trial is closed to accrual, but more timely reporting is advisable so that missing and 
questionable data can be resolved prior to final data analysis. The DCC will use the UPCI 
Clinical Trials Management Application to store and manage all demographic, clinical, 
laboratory and biomarker data required to achieve the Specific Aims. 
 
 
 


42 
 
9.0 EVALUATION AND MANAGEMENT OF TOXICITY (DOSE MODIFICATION) 
 
9.1 General 
Investigator/Sub-investigators, regulatory, CRS management, clinical research coordinators, 
clinical research associates, data managers, and clinic staff meet regularly in disease center Data 
Safety Monitoring Boards (DSMB) to review and discuss study data to include, but not limited 
to, the following: 
 
• serious adverse events 
• subject safety issues 
• recruitment issues 
• accrual 
• protocol deviations 
• unanticipated problems 
• breaches of confidentiality 
 
All toxicities encountered during the study will be evaluated on an ongoing basis according to the 
NCI Common Toxicity Criteria version 4.0 and recorded prior to each course of the investigational 
therapy. Any modifications necessary to ensure subject safety and decisions to continue, or close 
the trial to accrual are also discussed during these meetings. All study data reviewed and discussed 
during these meetings will be kept confidential. These reviews are summarized by each respective 
disease center and submitted to the University of Pittsburgh Cancer Institute’s Data and Safety 
Monitoring Committee (UPCI DSMC) for their monthly review of all disease centers’ studies. The 
UPCI DSMC will provide oversight for this study. At the time of renewal a summary of all serious 
adverse events and modifications will also be reported to the UPCI DSMC. 
 
Any modifications necessary to ensure patient safety will be submitted to the IRB prior to 
implementation. The IRB will be notified of any change in the risk/benefit ratio which would affect 
whether the study should continue. All protocol deviations or breaches of confidentiality will be 
reported to the IRB according to IRB guidelines. If any literature becomes available which suggests 
that conducting this trial is no longer ethical the study will be terminated and the IRB will be notified 
of the new findings in the form of an Unanticipated Problem submission. All study treatment 
associated adverse events that are serious, at least possibly related and unexpected will be reported 
to the IRB. DSMC reports and approval will be submitted to the IRB at the time of renewal. 
 
All records related to this research study will be stored in a locked environment. Only the 
researchers affiliated with the research study and their staff will have access to the research records. 
 
Safety data for each patient at the end of each cycle of IL-2 will be provided by all sites to the PI 
at UPCI. 
 
Patients with life-threatening or persistent, severe toxic reactions to IL-2 will receive no further 
treatment with this agent. Specific toxicities, which require special consideration, are discussed 
below. Previously unknown or severe toxicities will be reported to the PI as Adverse Drug 
Reactions. They will also be reported to the FDA on FDA Form 3500A Medwatch report. A 
courtesy copy of these reports will also be forwarded to Prometheus Drug Safety Department. See 
section 12.0. 


43 
 
 
9.2 Dose Modification for Toxicity: HD IL-2 and Hydroxychloroquine 
Modification of the treatment protocol will occur by withholding doses of IL-2 rather than 
continuing therapy at a reduced dose. The only dose modification for patients of 
Hydroxychloroquine will be for those individuals deemed by the treating investigator as ‘likely’ 
due to hydroxychloroquine or the combination and not to IL-2 alone based on their experience and 
dose reduction as shown in Table 5. Missed doses of IL-2 will not be made up. Dose of IL-2 will 
be withheld for: 
 
9.2.1 
Hypotension refractory to fluids and pressors or requiring unacceptably high 
pressor doses (those associated with individual decrease in extremity perfusion). We will 
use vasopressors judicially where necessary to support patients with limiting kidney/other 
organ perfusion. Requirement for vasopressors will be a relative indication for 
concluding a cycle (see 10.4.2.3 below). 
 
9.2.2 
Anuria for>24 hours and unresponsive to fluid replacement 
 
9.2.3 
Respiratory distress requiring oxygen>4 liters to maintain O2 saturation>95%. 
 
9.2.4 
Confusion (mental status changes can progress to paranoia despite discontinuation 
of IL-2; it is imperative that the IL-2 be stopped at any sign of persistent confusion or 
disorientation). 
 
9.2.5 
Sustained ventricular tachycardia or any sign or symptom of myocardial 
ischemia or myocarditis. Patients experiencing sustained ventricular tachycardia or 
myocardial ischemia should not receive further treatment with IL-2 
 
9.2.6 
Metabolic acidosis with HCO3<18, despite attempts to correct with IV HCO3. 
 
9.2.7 
Atrial fibrillation or myocarditis. 
 
9.2.8 
Documented systemic infection. 
 
9.2.9 
Management of Nausea and Vomiting. The frequency of nausea and vomiting 
with interleukin 2 administration falls within a broad category of moderate emetic risk 
(NCCN Practice Guidelines, 2010). It may be enhanced by HCQ. Patients with nausea and 
vomiting will be given 8 mg bid of ondansetron (a 5-HT3 receptor antagonist). For nausea 
and vomiting that is not controlled with regimen of ondansetron a general principle (NCCN 
Practice Guidelines, 2010) is to add one agent from a different drug class to the current 
regimen 5-HT3 and NK1 receptor antagonists are very safe drugs that are standards of care 
in this area. Although not well studied, by carefully monitoring NV and using appropriate 
treatments patients might have better nutrition and maintenance of body weight. Records 
of antiemetics given routinely or as part of symptom management will be recorded. 
 
or Any other serious toxicity that is not controlled at time of next dose. 
 
9.3 Specific IL-2 Toxicity Management 


44 
 
 
Several treatment-related toxicities have been uniquely associated with the administration of high- 
dose IL-2. Antihypertensive medication should be stopped prior to IL-2 therapy. 
 
Recommendations for management of the more significant toxicities typically seen with high-dose 
bolus IL-2 are as follows (the following are only optional guidelines for toxicity management): 
 
9.3.1 
Fluid Replacement. Excessive fluid replacement will increase the patient’s 
likelihood of developing pulmonary edema. It is suggested that when intravenous access 
has been established; begin administration of normal saline at 75cc/hour IV. Once patients 
have gained greater than 15% of baseline weight, elective discontinuation of IL-2 is 
recommended. Diuretics should not be given unless severe symptomatic fluid retention 
develops and blood pressure is adequate. 
 
9.3.2 
Hypotension. Administration of high dose IL-2 leads to decreased peripheral 
vascular resistance and consequent hypotension. In order to manage this toxicity, the 
physician may utilize the following guidelines: 
1. 
Monitor patients in a setting capable of providing level high 
intensity care. 
 
2. 
Prior to starting IL-2 therapy determine a minimum tolerated blood 
pressure (MTBP). For patients under age 40 and with no prior history of 
ischemic or valvular heart disease, the MTBP can be a systolic blood 
pressure (SBP) of 80 mmHg, while the MTBP for all other patients should 
be a SBP of 85-90 mmHg based on perceived risk of cardiac toxicity. 
 
3. 
When a patients systolic BP falls below the MTBP, suggested therapy may 
involve (in the following order): 
 
a. Begin fluid boluses (250cc normal saline IV over 15 min. may 
repeat x 2) until SBP is > MTBP. 
 
b. Should fluid boluses fail make the SBP>MTBP, vasopressor support 
such as phenylephrine could also be instituted (.1-2 ug/kg/min) to maintain 
SBP. 
 
c. If blood pressure cannot be maintained on phenylephrine alone, or if 
unacceptable tachycardia develops, treatment with dopamine (1-6 
ug/kg/min) should be added to sustain blood pressure. 
 
9.3.3 
Management of Arrhythmias/Myocarditis. If significant arrhythmias occur at 
any stage in the patient's treatment (whether on pressor agents or not), the possibility of 
myocardial ischemia/infarction must be excluded by both EKG and cardiac enzyme 
assessment. Patients who develop atrial fibrillation should have IL-2 doses held. Therapy 
may resume when the patient converts to normal sinus rhythm and is hemodynamically 
stable. If a significant supraventricular arrhythmia occurs while a patient is on dopamine 
therapy; neosynepherine should also be substituted for dopamine as initial blood pressure 


45 
 
support. Patients experiencing sustained ventricular tachycardia or documented myocardial 
ischemic episodes during therapy should not receive further treatment with IL-2. Patients 
with myocarditis may resume treatment in subsequent courses if CPK returns to normal. A 
cardiac ECHO documenting normal cardiac function should be performed prior to 
restarting therapy. 
 
9.3.4 
Management of Neurotoxicity. Doses are held rather than reduced for 
neurotoxicity. If Grade 4 neuro-cortical toxicity is encountered and is not reversible within 
48 hours, no further treatment should be given and the patient should be removed from the 
study. If Grade 4 toxicity is reversible to Grade 1 within 48 hours, future treatment may be 
considered (in subsequent courses) if the patients shows any evidence of tumor regression 
and as discussed with the patient. 
 
9.3.5 
Metabolic Acidosis. In the course of IL-2 therapy, when a patient’s HCO3 falls to 
below 20, NaHCO3 should be added to the maintenance IV infusion. Should the HCO3 
level fall below 18, IL-2 therapy should be held, and bolus infusions of NaHCO3 should be 
instituted. IL-2 therapy may resume if repeat HCO3 > 18. Sodium acetate may be 
substituted for NaHCO3 per institutional policy. 
 
9.3.6 Low Urine Ouput. Fluid boluses and ‘renal’ dose dopamine may be administered. 
Weight gains/edema may serve as a basis for holding doses. 
 
9.3.7 Other Toxicities. Increases in the serum creatinine to 2.0-3.5 mg/dl and total 
bilirubin to 3.0-10.0 mg/dl are common and reversible upon cessation of treatment. Doses 
of IL-2 have not generally been withheld for renal and hepatic dysfunction alone. Although 
toxicity may become severe, recovery usually occurs following cessation of IL-2 and 
vigorous supportive care is warranted. 
 
9.4 
Management of Grade 4 Toxicity 
Patients with Grade 4 (Life Threatening) toxicity (with the exception of those Grade 4 
toxicities listed in Section 10.5) may be treated with dexamethasone 4 mg qid until side 
effects improve to an acceptable level as indicated. Two dose reductions of 
hydroxychloroquine (Levels -1 and -2 in Table 5) may be allowed in a patient if treatment-
limiting toxicity considered ‘likely’ attributable to the combination (and not to IL-2 alone) 
by the treating investigator is observed. A pill diary will be provided and is required. 
 
9.5 
Toxicity Criteria for Discontinuing Treatment 
 
Patients will not be considered for further therapy if the following toxicities are 
encountered: 
 
9.5.1 
Pulmonary toxicity requiring endotracheal intubation 
 
9.5.2 
Renal dysfunction requiring dialysis 
 
9.5.3 
Grade 4 cardiac dysrhythmia or Grade 2 or 3 dysrhythmia not easily 
controlled with medical management. 
9.5.4 
Myocardial ischemia (Grade 3 or 4) or infarction or symptomatic myocarditis (note: 


46 
 
asymptomatic CPK or CPK-MB band elevations without EKG changes are not a 
contraindication to further treatment). 
 
9.5.5 
Coma 
 
9.5.6 
Life-threatening sepsis 
 
9.5.7 
Pericardial tamponade 
 
9.5.8 
Bowel ischemia or perforation 
 
9.5.9 
Grade 4 hypertension or reversible posterior leukoencephalopathy syndrome (RPLS) 
 
9.5.10 Grade > 2 pulmonary or CNS hemorrhage 
 
9.5.11 Grade 4 hypertension or hemorrhage, symptomatic grade 4 venous 
thromboembolic event, nephrotic syndrome 
 
9.5.12 
Any grade arterial thromboembolic event 
 
9.5.13 
Grade 4 congestive heart failure 
 
9.5.14 
Wound dehiscence requiring medical or surgical intervention 
 
9.5.15 
Inability of subject to comply with study requirements 
 
9.5.16 Any other severe or life-threatening toxicity which, in the opinion of the 
investigator, would preclude further treatment with these agents, or has not resolved 
substantially to an acceptable level as determined by the local PI. 
 
Patients who have an ongoing Grade 4 or serious adverse event at the time of discontinuation from 
study treatment will continue to be followed until resolution of the event or until the event is 
considered irreversible (see Section 7.1.3). 
 
 
 


47 
 
10.0 
DETAILED RESPONSE ASSESSMENT (also see 6.4) 
 
Response will be defined by NCI’s Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. 
 
10.1 
Disease Evaluation 
Measurable disease - the presence of at least one measurable lesion. If the measurable disease is 
restricted to a solitary lesion, its neoplastic nature should be confirmed by cytology/histology. 
 
Measurable lesions - lesions that can be accurately measured in at least one dimension with longest 
diameter (longest diameter to be recorded) as ≥20 mm using conventional techniques or ≥10 mm 
with CT scan or ≥10 mm with calipers by clinical exam. 
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 lesions - all other lesions, including small lesions (longest diameter <10 mm or 
pathological lymph nodes with ≥ 10 to < 15 mm short axis), i.e., bone lesions, leptomeningeal 
disease, ascites, pleural/pericardial effusion, inflammatory breast disease, lymphangitis 
cutis/pulmonis, cystic lesions, and also abdominal masses that are not confirmed and followed by 
imaging techniques. Non-measurable also includes lesions that are < 20 mm by chest x-ray 
 
Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules 
and palpable lymph nodes). 
 
All measurements will be taken and recorded in metric notation, using a ruler or calipers. All 
baseline evaluations should be performed as closely as possible to the beginning of 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. 
 
10.2 
Methods of Measurement 
Conventional CT and MRI should be performed with cuts of 5 mm or less in slice thickness 
contiguously. If CT scans have slice thickness greater than 5 mm, the minimum size for a 
measurable lesion should be twice the slice thickness. Spiral CT should be performed using a 5-7 
mm contiguous reconstruction algorithm. This applies to tumors of the chest, abdomen and pelvis. 
 
Baseline documentation of "Target" and "Non-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 


48 
 
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 of the 
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 or absence of unequivocal 
progression of each should be noted throughout follow-up. 
 
10.3 
Response Criteria 
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. For patients with > 90% Partial 
Response defined as at least a 90% decrease in the sum of the LD of target lesions, taking 
as reference the baseline sum LD sustained for greater than one year will be scored as a CR 
(maximal patient benefit), given the difficulty of evaluating lesions in some instances. 
* Partial Response (PR): 
At least a 30% decrease in the sum of the LD of target lesions, taking as reference the 
baseline sum LD 
* Minimal Response (MR): 
At least a 20% decrease in the sum of the LD of target lesions, taking as reference the base 
line sum LD 
* Progressive Disease (PD): 
At least a 20% increase in the sum of the LD of target lesions, taking as reference the 
smallest sum LD recorded since the treatment started. In addition to the relative increase 
of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. 
* Stable Disease (SD): 
Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for 
PD, taking as reference the smallest sum LD since the treatment started. Note: a change of 
20% or more that does not increase the sum of the diameters by 5 mm or more is coded  as SD. 
 
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 non-pathological in size (<10 mm short axis) 
* Stable Disease (SD): Persistence of one or more non-target lesion(s) or/and 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. Although a clear progression of "non target" lesions 
only is exceptional, in such circumstances, the opinion of the treating physician should prevail 
and the progression status should be confirmed later on by the review panel (or study chair). 
 


49 
 
Evaluation of best overall response: 
The best overall response is the best response recorded from the date of first protocol treatment 
until disease progression/recurrence (taking as reference for PD the smallest measurements 
recorded since the treatment started). In general, the patient's best response assignment will depend 
on the achievement of both measurement and confirmation criteria. 
 
Table 7. Response Criteria 
 
Target lesions 
Non-Target lesions 
Overall response 
CR 
CR 
CR 
CR 
Incomplete response/SD 
PR 
PR 
Non-PD 
PR 
MR 
Non-PD 
MR 
SD 
Non-PD 
SD 
PD 
Any 
PD 
Any 
PD 
PD 
Any 
Any 
PD 
 
Patients with a global deterioration of health status requiring discontinuation of treatment without 
objective evidence of disease progression at that time should be classified as having "symptomatic 
deterioration". Every effort should be made to document the objective progression even after 
discontinuation of treatment. In some circumstances it may be difficult to distinguish residual 
disease from normal tissue. When the evaluation of complete response depends on this 
determination, it is recommended that the residual lesion be investigated (fine needle 
aspirate/biopsy) to confirm the complete response status. 
 
10.4 
Confirmation 
The main goal of confirmation of objective response is to avoid overestimating the response rate 
observed. To be assigned a status of PR or CR, changes in tumor measurements must be confirmed 
by repeat assessments that should be performed no less than 4 weeks after the criteria for response 
are first met. In the case of SD, follow-up measurements must have met the SD criteria at least 
once after study entry at a minimum interval (in general, not less than 6-8 weeks) that is defined 
in the study protocol. 
 
10.5 
Duration of overall response 
The duration of overall response is measured from the time measurement criteria are first met (i.e., 
not from time of confirmation of response) for CR or PR (whichever status is recorded first) until 
the first date that recurrence or PD is objectively documented, taking as reference for PD the 
smallest measurements recorded since the treatment started. 
 
10.6 
Duration of stable disease 
SD is measured from the date of first protocol treatment until the criteria for disease progression 
are met, taking as reference the smallest measurements recorded since the treatment started. 
 
 
 


50 
 
10.7 
Time to Progression 
The time to progression (TTP) is defined as the time from the date of first protocol treatment until 
the date disease progression criteria are met (in responding patients progression criteria uses the 
reference of the smallest measurements recorded since the treatment started) or is censored at date 
of last disease assessment for those who have not progressed. 
 
10.8 
Progression-free Survival 
Progression-free survival (PFS) is defined as the time from date of first protocol treatment until 
the date disease progression criteria are met (in responding patients progression criteria uses the 
reference of the smallest measurements recorded since the treatment started) or date of death from 
any cause, or is censored at date of last disease assessment for those who have not progressed. 
 
10.9 
Survival: 
Survival is calculated from the date of first protocol treatment to the date of death, or censored at 
date of last contact. 
 
 
 


51 
 
11.0 
PARTICIPANTS EVALUABLE FOR RESPONSE 
 
Reporting of results: 
 
Clinical Results: All patients included in the study will be assessed for response to treatment, even 
if there are major protocol treatment deviations or if they are ineligible. Each patient will be 
assigned one of the following categories: 
 
1) 
complete response 
2) 
>90% partial response 
3) 
partial response 
4) 
stable disease 
5) 
progressive disease 
6) 
early death from malignant disease (within 3 months of initiating therapy) 
7) 
early death from toxicity 
8) 
early death because of other cause 
8) 
unknown (not assessable, insufficient data). 
 
All patients who met the eligibility criteria and receive at least one week of IL-2 treatment will be 
included in the main analysis of the response rate. Patients in response categories 5-8 will be 
considered as failing to respond to treatment (disease progression). Thus, an incorrect treatment 
schedule or drug administration does not result in exclusion from the analysis of the response rate. 
 
 
 


52 
 
12.0 
REPORTING OF ADVERSE REACTIONS 
ADVERSE EVENT DEFINITIONS 
Adverse event means any untoward medical occurrence associated with the use of the drug in 
humans, whether or not considered drug related. 
 
Adverse reaction means any adverse event caused by a drug. 
 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
(definitely or probably) that the drug caused the adverse event. Suspected adverse reaction implies 
a lesser degree of certainty about causality than “adverse reaction” 
 
Serious Adverse Event: Any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. Specifically, results in death, is life-threatening, 
requires inpatient hospitalization or causes prolongation of existing hospitalization, results in 
persistent or significant disability/incapacity, is a congenital anomaly/birth defect, or is an 
important medical event (defined as a medical event(s) that may not be immediately life- 
threatening or result in death or hospitalization but, based upon appropriate medical and scientific 
judgment, may jeopardize the subject or may require intervention [e.g., medical, surgical] to 
prevent one of the other serious outcomes listed in the definition above.) Also, included: any 
subject death within 30 days of the last dose of study drug, regardless of the causality. 
 
Scheduled protocol inpatient hospitalizations do not meet the criteria above for recording and 
reporting as an SAE. Only events which would prolong the scheduled inpatient stay to be resolved 
or unplanned hospitalizations due to events occurring between scheduled inpatient stays should be 
recorded as SAEs. 
 
Life-threatening, suspected adverse reaction. A suspected adverse reaction is considered “life- 
threatening” if, in the view of either the Investigator (i.e., the study site principal investigator), its 
occurrence places the patient or research subject at immediate risk of death. It does not include a 
suspected adverse reaction that had it occurred in a more severe form, might have caused death. 
 
Unexpected, suspected adverse reaction. A suspected adverse reaction is considered “unexpected” 
if it is not listed in the general investigational plan or clinical protocol; or is not listed at the 
specificity or severity that has been previously observed and/or specified. 
All observed or volunteered adverse events (serious or non-serious) and abnormal test findings, 
regardless of study group or suspected causal relationship to the study drug(s) will be recorded in 
the subjects’ case histories. For all adverse events, sufficient information will be pursued and/or 
obtained so as to permit 1) an adequate determination of the outcome of the event (i.e., whether 
the event should be classified as a serious adverse event) and; 2) an assessment of the casual 
relationship between the adverse event and the study drug(s). 
 
Adverse events or abnormal test findings felt to be associated with the study drug(s) will be 
followed until the event (or its sequelae) or the abnormal test finding resolves or stabilizes at a 
level acceptable to the Principal Investigator. 
 
In the event of an adverse event the first concern will be for the safety of the subject. 


53 
 
REPORTING OF SUSPECTED ADVERSE REACTIONS 
 
In the event of any unexpected or serious suspected adverse reaction, the PI, the institutional review 
board (per institutional reporting requirements), and Prometheus Drug Safety Department will be 
notified using the FDA Form 3500 MedWatch report. We will report all secondary malignancies 
occurring or following treatment by this protocol. 
 
All events meeting the definition of a serious adverse event should be recorded on a MedWatch 
3500A Form: 
(http://www.fda.gov/downloads/AboutFDA/ReportsManualsForms/Forms/UCM048334.pdf) 
and submitted to the Clinical Research Coordinators and Prometheus Laboratories’ Drug Safety 
Department: 
Deborah Hice, RN 
Phone: 412-623-8962 
Email: hiceda@upmc.edu 
Fax: 412-623-8974 
 
 
Kathy Mansfield, RN 
Phone: 412-623-7048 
Email: manskd@upmc.edu 
Fax: 412-623-8974 
 
Prometheus Laboratories’ Drug Safety Department 
Email:drugsafety@prometheuslabs.com 
Fax: 858-754-3046. 
 
In addition to completing appropriate patient demographic and suspect medication information, 
the report should include the following information within the Event Description (section 5) of the 
MedWatch 3500 form: 
 
Reporting of toxicity will include: 
 
1) Treatment regimen (dosing frequency, combination therapy) 
2) Protocol description (and number, if assigned) 
3) Description of event, severity, treatment, and outcome, if known 
4) Supportive laboratory results and diagnostics 
5) CTCAE Version 4.0 Term and Grade of event 
6) Investigator’s assessment of the relationship (causality) of the adverse event to each 
investigational product and suspect medication 
 
• the likelihood of the relationship between the toxicity and treatment (i.e., unrelated, 
unlikely, possibly, probably, or definitely related to treatment) 
o list alternate causality if event is unrelated or unlikely related to treatment 
(e.g. attributed to underlying disease, history of COPD, etc.) 
• whether the side effect noted falls within the expected toxicity profile of the agent 
employed 


54 
 
Follow-up information: 
 
Additional information may be added to a previously submitted report by any of the following 
methods: 
 
• Adding to the original MedWatch 3500 report and submitting it as follow-up 
• Adding supplemental summary information and submitting it as follow-up with the original 
MedWatch 3500 form 
• Summarizing new information and faxing it with a cover letter including subject identifiers 
(i.e. D.O.B. initial, subject number), protocol description and number, if assigned, suspect 
drug, brief adverse event description, and notation that additional or follow-up information 
is being submitted (The subject identifiers are important so that the new information is 
added to the correct initial report) 
 
Occasionally Prometheus may contact the reporter for additional information, clarification, or 
current status of the subject for whom and adverse event was reported. 
 
Assessing Causality: 
 
Investigators are required to assess how likely it is that hydroxychloroquine caused or contributed 
to an adverse event (definitely, probably, possibly, or unlikely). The following factors should be 
considered when assessing relatedness. 
● 
The temporal relationship of the clinical event to hydroxychloroquine 
administration 
● 
The likelihood that IL-2 provides a sufficient explanation for the observed event. 
 
● 
Other drugs, therapeutic interventions or underlying conditions that may provide a 
sufficient explanation for the observed event. 
 
 


55 
 
13.0 
STATISTICAL ANALYSIS AND STUDY SIZE 
 
13.1 Objectives and Design 
The primary objective of this Phase II trial is to estimate the proportions of patients with metastatic 
RCC treated with high-dose IL-2 combined with hydroxychloroquine experiencing complete 
response, or any response. The secondary objectives are to compare response, time to progression 
and survival to historical data to determine the toxicity of the treatment regimen, to evaluate PD, 
and to examine correlative science endpoints. Disease endpoints are defined in Section 10. 
 
The accrual goal is 39 participants. All study participants will receive the same high-dose IL-2 
treatment, which is considered standard of care. The initial dose of HCQ will be 600 mg/day for 
all patients. If no limitations on additional dosing (treatment-limiting toxicities [TLTs], as defined 
in Section 9.2), dosing will continue at 600mg/d, otherwise, HCQ will de-escalate to 400 mg/d. 
Toxicity and response will be monitored after every three patients (Section 13.4), and will be 
stopped if the probability that treatment with IL-2+HCQ is excessively toxic or ineffective. The 
study may be opened more broadly to additional Cytokine Working Group sites whenever 
sufficient evidence has accrued that treatment with IL-2+HCQ is no more toxic or less effective 
than treatment with IL-2 alone. 
 
13.2 Analysis Plan 
 
The data will be analyzed for response, overall survival and other endpoints, as described in this 
section; data will only be analyzed as specified in Section 14.3.1 to ensure the toxicity of IL- 
2+HCQ is not unexpectedly high. All analyses will be performed using the most recent versions 
of SAS and/or R available. 
 
13.2.1. Estimate the proportion of patients with metastatic RCC treated with IL-2 combined with 
hydroxychloroquine at 600mg/d who experience a clinical complete response. Response is 
defined in Section 10.3. The probability of each category of response will be estimated. 
Secondary analyses will employ cumulative logit regression for increasing levels of 
response, with adjustment for demographic and clinical covariates. All point estimates will 
be accompanied by appropriate 95% confidence intervals. 
 
13.2.2. Compare the response, overall survival and time to progression of patients with metastatic 
RCC treated with IL-2 combined with hydroxychloroquine to the published response of 
patients treated with high dose IL-2 alone. Logistic and proportional hazards (Cox) 
regression will be used to compare clinical response, and OS and PFS, respectively, to 
examine the effect of HCQ on the efficacy of IL-2. Formal tests of non-inferiority are 
beyond the scope of this Phase II study, and these secondary analyses are not statistically 
powered. 
 
13.2.3. Characterize the toxicity of IL-2 combined with hydroxychloroquine in patients with 
metastatic RCC: number of doses of IL-2 administered during the first course of therapy; 
toxicity after the scheduled 9th dose of IL-2; frequency of grade III and IV or unexpected or 
rare toxicities. Number of doses, maximal toxicity grade and incidence of treatment-
limiting toxicities (TLTs, as defined in Section 10.5) will be tabulated.  


56 
 
13.2.4 Evaluate the utility of known prognostic criteria for RCC patients on clinical outcome. 
Prognosis is evaluated by whether the patient has 0, 1, or 2, or 3 or more of the following 
risk factors: 
• lactate dehydrogenase (> 1.5 upper limit of normal) 
• hemoglobin (< lower limit of normal) 
• corrected calcium (>10mg/dL) 
• Karnofsky performance status (< 80%) 
• time from initial RCC diagnosis to start of interferon-α therapy of less than one 
year 
This prognostic classification will be added to the baseline models described in Sections 
13.2.1 to 13.2.3 to determine if any of the factors would significantly change the prognostic 
evaluation, and to the dynamic and post-treatment factors to potentially increase the 
statistical power. 
 
13.2.5 Correlate baseline laboratory parameters (miRNAs pre- and post-IL-2; KIR genotyping; 
NK enumeration in the peripheral blood; DC function, TCR- chain expression, arginase 
or arginine levels, proposed pathological/molecular prognosis classification described in 
Section 2.6) with toxicity, response, and survival. Stepwise proportional hazards or logistic 
regression will be used to add variables to the models resulting from the analyses in Section 
13.2.4. 
 
13.2.6 Assess if changes in biomarkers measured in PBMC (Section 7.4) are associated with 
clinical response, overall survival or progression-free survival. Stepwise proportional 
hazards or logistic regression will be used to determine if changes of any of the markers in 
Section 7.4 are related to patient outcomes, after adjusting for the prognostic criteria in 
13.2.4. 
 
13.3    Monitoring for Toxicity and Response 
 
13.3.1 21 additional patients will be enrolled at IL-2+HCQ 600 mg/day on this revised protocol. 
If one or more TLTs are observed, treatment will be de-escalated to IL-2+HCQ 400 
mg/day. 
 
13.3.2 The number of overall partial and complete responses (Table 8) will be compared to the 
stopping rule described in Table 10 by the UPCI Multi-Center DSMC after every three 
patients become evaluable for response. For each number of evaluable patients in the 
left-hand columns, if the number of complete responses is less than or equal to the 
number in the right-hand column, the trial will be halted for lack of efficacy. The trial is 
derived from a Bayesian beta-binomial model with the prior determined by the number of 
complete responses observed in the high-dose arm of the randomized trial described in 
Section 2.3 (8/95). The values correspond to P (partial and complete response rate < 
0.10) 
> 0.8. 


57 
 
# Patients 
#Responses 
18 
0 
21 
1 
24 
1 
27 
1 
30 
2 
33 
2 
36 
2 
 
Table 8.Monitoring rule for complete response. For the given number of patients in Column 1, if 
the number of partial or complete responses is less than or equal to the number in Column 2, the 
trial is halted for lack of efficacy. 
 
13.4 Justification of Design 
The primary objective of this study is to estimate the clinical response rate of patients with 
metastatic RCC treated with IL-2 combined with hydroxychloroquine at 600mg/d. Assuming 
no TLTs occur and the trial is not closed early for toxicity or inadequate response (Section 
13.3.2), there will be 39 patients available for this objective. A 95% exact binomial confidence 
interval on 5 responses out of 39 patients (expected if the addition of HCQ to IL-2 doubles the 
historic proportions of CR ~ 8%) would have width equal to 0.27 (0.07,0.34). For the secondary 
endpoints of OS and PFS, if patients are recruited over 15 months, observed for 15 additional 
months, and the true median survival is 16 months, then the expected width of the 95% 
confidence interval for median survival is 12.8 months. 


58 
 
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Dec;65(12):1523-1532. Epub 2016 Sep 30. PubMed PMID: 27695964; PubMed Central 
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145. Wallin JJ, Bendell JC, Funke R, Sznol M, Korski K, Jones S, Hernandez G, Mier 
J, He X, Hodi FS, Denker M, Leveque V, Cañamero M, Babitski G, Koeppen H, Ziai J, 
Sharma N, Gaire F, Chen DS, Waterkamp D, Hegde PS, McDermott DF. Atezolizumab in  
combination with bevacizumab enhances antigen-specific T-cell migration in 
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146. Choueiri TK, Escudier B, Powles T, Tannir NM, Mainwaring PN, Rini BI, Hammers 
HJ, Donskov F, Roth BJ, Peltola K, Lee JL, Heng DYC, Schmidinger M, Agarwal N, 
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PMCID: PMC4806782. 
 


70 
 
APPENDIX A 
 
Procedure for Obtaining a Urine Protein:Creatinine Ratio (Or alternatively calculate from 
the serum creatinine based on gender and age) 
 
1) Obtain at least 4 ml of a random urine sample (does not have to be a 24 hour urine). 
2) Determine protein concentration (mg/dL). 
3) Determine creatinine concentration (mg/dL). 
4) Divide #2 by #3 above: urine protein / creatinine ratio = protein concentration (mg /dL) / 
creatinine concentration (mg /dL). 
 
The UPC directly correlates with the amount of protein excreted in the urine per 24 hrs (i.e. a 
UPC of 1 should be equivalent to 1g protein in a 24hr urine collection). 
 
Protein and creatinine concentrations should be available on standard reports of urinalyses, 
not dipsticks. If protein and creatinine concentrations are not routinely reported at an 
institution, their measurements and reports may need to be requested 


71 
 
 
 
APPENDIX B - Schedule of Events 
 
Study Visit 
Screening 
Study 
start 
Each Course (1-3) 
End of 
Study 
Visit q 
Follow 
Up r 
Days -41 to 
-14 
Days -27 to 
-14 
Day 
-14 
Day 1 
Days 
7-14 
Day 
15 
Day 
29 
Day 
43 
Day 
57 
Day 
71 
Day 
84 
History/Physical exam 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse events 
X 
X 
X ---------------------------------------------------------------- X 
X 
X 
Radiologic exams 
Xa 
Xb 
X 
Xb 
Pulmonary Function Testing 
Xc 
Cardiac Stress Test 
Xd 
12-lead EKG 
X 
ECHO or MUGA for LVEF 
Xe 
Xe 
Screening labs 
Xf 
Urine protein:creatinine ratio 
X 
Urinalysis g 
X 
X 
X 
Patient Registration 
X 
Treatment labs 
Xh 
Xi 
Xj 
Xi 
Xh 
Xh 
Xh 
Xh 
Xh, k 
IL-2 l 
Xl 
Xl 
Hydroxychloroquine m 
X-----------------------------------------------------------------------------------------------------------Xk 
Research blood draws n 
X 
X 
Xn 
Pathology submission 
Xo 
Telephone contact for toxicity assessment 
X 
Subject Drug Administration Diary for HCQ 
Tablets 
X 
X 
X 
X 
X 
Xk 
a. 
Within 4 weeks prior to day –14 patients will have CT scan of chest/abdomen/pelvis and brain MRI. If clinically indicated a bone scan should be done. 
b. 
After week 8 (+/- 1 week) and week 12 (+/- 1 week) of each course patients will have CT of chest/abdomen/pelvis. If clinically indicated repeat bone scan and brain MRI. After 
the final course (week 36), patients will have CT scan evaluation every 12 weeks for up to one year or until tumor progression (whichever occurs first). For patients who have 
not shown progression at the end of one year, response will be captured during follow-up survival via telephone contact and medical record review. If additional scans are 
performed for clinical reasons, the study schedule may be altered to perform the requisite scans at a more acceptable time point. The change in timing of study scans must be 
approved by the local PI and documented. 
c. 
All patients over 50 years of age or those with significant pulmonary or smoking history. 


72 
 
 
d. 
All patients over 40 years of age or with significant cardiac history. 
e. 
ECHO or MUGA will be performed at baseline. ECHO will be performed prior to start of each new course only if clinically indicated (section 10.3.3). 
f. 
Screening labs to include CBC w/differential, CMP (creatinine, BUN, albumin, total protein, glucose, electrolytes [Na, K, Cl, HCO3], liver function tests [total bilirubin, ALP, 
AST, ALT]), direct bilirubin, calcium, phosphorus, LDH, TSH, PT, PTT, HIV, Hep B and C, Pregnancy test for women of childbearing potential. 
g. 
Proteinuria level must be checked prior to each cycle of IL-2. 
h. 
Outpatient labs to include CBC w/differential, CMP (creatinine, BUN, albumin, total protein, glucose, liver function tests [total bilirubin, ALP, AST, ALT], electrolytes [Na, K, 
Cl, HCO3]), LDH, CPK, amylase, and lipase. 
i. 
Inpatient labs to include CBC w/differential, CMP (creatinine, BUN, albumin, total protein, glucose, liver function tests [total bilirubin, ALP, AST, ALT], electrolytes [Na, K, 
Cl, HCO3]), direct bilirubin, calcium, magnesium, phosphorus, LDH, CPK, amylase, and lipase. 
j. 
Outpatient labs to include CBC w/differential, CMP (creatinine, BUN, albumin, total protein, glucose, liver function tests [total bilirubin, ALP, AST, ALT], electrolytes [Na, K, 
Cl, HCO3]), amylase, and lipase 
k. 
Patients with no evidence of progression by RECIST 1.1 criteria may receive continued treatment with hydroxychloroquine through one year of initiation of HCQ treatment 
(Day -14) until disease progression. 
l. 
IL-2 every 8 hours on days 1-5 (or 6) and 15-19 (or 20) (maximum 14 doses per 5 day cycle). Dependent upon when Dose 1 is administered on Day 1/Day 15, Dose 14 could 
require hospitalization on Day 6/Day 20. 
m. HCQ daily dosing starting on Day -14 (minimum of 14 days, plus a window of + 7 days if needed) 
n. 
See Table 6 for sampling schedule. Cycle 2 draws will only be done in Course 1. 
o. 
Blocks or slides will be sent within 4 weeks after enrollment. 
p. 
Subjects will be evaluated with physical examination ~30 days (28-42 days) after being removed from study for any reason. 
q. After the final course of treatment, follow-up procedures will be performed every 3 months (+/- 2 weeks) for up to one year or until disease progression (whichever occurs first). 
Follow-up procedures will include CT scan, routine laboratory tests, and physical examination. Subjects will then be followed for survival by telephone contact and medical 
record review every 3 months for one year following the time of disease progression or after one year off of treatment, then every 6 months for the next two years, then annually 
until death. A window of +/- 2 weeks is permitted. 
 
 
If a subject's treatment and/or testing days need to be rescheduled due to the subject’s inability to comply with the study calendar (i.e., 
hospitalizations, business and vacation travel plans, illness, transportation issues, holidays, family emergencies, etc.), a window of +/- 
one week is available for rescheduling of treatment and procedures per the discretion of the treating physician investigator, and as 
discussed with the principal investigator. 


73 
 
Appendix C 
 
 
 
Blood Draw Instructions for Research Labs for Protocol IL-2HQ 
 
A Phase II Study of Hydroxychloroquine and Aldesleukin in Patients with Advanced RCC 
 
Comments 
Treatment Day 
Timepoint 
Tube(s) to draw 
Storage 
condition 
Hydroxychloroquine is 
oral 
Day -14 
Pre-dose of 
Hydroxychloroquine 
2 lge (10ml) Red top 
1 on ice or in 
frig., 1 ambient 
10 tubes at 
ambient temp. 
on their side 
1 tube on ice or 
in frig 
10 lge (10ml) sod. Hep. Green 
top 
1 PAXgene RNA blood tube 
(2.5ml) 
Course 1 
Cycle 1 
Day 1 and 2 
Please draw blood before pre- 
treatment ancillary medication 
is given 
Pre-dose of IL-2 
(post-dose PO HCQ) 
2 lge (10ml) Red top 
10 lge (10ml) sod. Hep. Green 
top 
1 PAXgene RNA blood tube 
(2.5ml) 
1 on ice or in 
frig., 1 ambient 
10 tubes at 
ambient temp. 
on their side 
1 tube on ice or 
in frig 
Just prior to Dose 4 of IL-2 
( + / - 1 dose) 
1 lge (10ml) Red top 
On ice or in 
frig. 
Course 1 
Cycle 1 
Day 15 and 16 
Pre-dose of 
Hydroxychloroquine 
2 lge (10ml) Green top 
1 lge (10ml) Red top 
Ambient temp. 
on their side 
On ice or in 
frig. 
just prior to Dose 4 of IL-2 
( + / - 1 dose) 
1 lge (10ml) Red top 
On ice or in 
frig. 
Course 1 
Between 
Days 60-80 
During clinic visit 
2 lge (10ml) Red top 
10 lge (10ml) sod. Hep. Green 
top 
1 on ice or in 
frig., 1 ambient 
10 tubes at 
ambient temp. 
on their side 
1 tube on ice or 
in frig 
1 PAXgene RNA blood tube 
(2.5ml) 
Course 2 
Cycle 1 
Day 1 
Pre-dose of IL-2 
(post-dose PO HCQ) 
2 lge (10ml) Green top 
1 lge (10ml) Red top 
Ambient temp. 
on their side 
On ice or in 
frig. 
Course 3 
Cycle 1 
Day 1 
Pre-dose of IL-2 
(post-dose PO HCQ) 
2 lge (10ml) Green top 
1 lge (10ml) Red top 
Ambient temp. 
on their side 
On ice or in 
frig. 


74 
 
APPENDIX D 
Processing Specimens for RCC Study (hydroxychloroquine / Aldesleukin for RCC) 
 
1 Large Red Top Tube(s) for Serum (Kept on ice): Please carry out in laminar flow hood to keep serum sterile. 
 
• 
After blood is drawn, invert tube several times to mix well. Send to UPCI IMCPL overnight. 
• 
IMCPL Centrifuge for 10 minutes at 900g to 1000g to separate out serum. 
• 
Aliquot serum into prelabeled sterile freeze vials at approximately 0.5 to 1.0 ml per vial (4 vials 
maximum). 
• 
Fill in vial labels with patient ID# and date. (Please be careful to use appropriate lettered vials) 
• 
Fill in Inventory/Shipping Log for W0454 
• 
Store vials at -70 to -80C until ready to ship. 
 
1 Large Green Top Tube for Plasma (Kept on ice): UPCI/IMCPL will carry out in laminar flow hood to keep 
plasma sterile. 
• 
After blood is drawn, invert tube several times to mix well. 
• 
Centrifuge for 10 minutes at 900g to 1000g to separate out plasma. 
• 
Aliquot plasma into prelabeled sterile freeze vials at approximately 0.5 ro 1.0 ml per vial (4 vials 
maximum). 
• 
Fill in vial labels with patient ID# and date. (Please be careful to use appropriate lettered vials) 
• 
Fill in Inventory/Shipping Log for IL-2HQ 
• 
Store vials at -70 to -80C until ready to ship. 
 
1 10cc Red Top Tube for Serum (kept at ambient temp.) AND 
10 Large Sodium Heparin Green Top Tubes for PBMCs. (Kept at ambient temp.): 
 
Batch specimens from one patient and ship on dry ice along with a copy of the Inventory / Shipping Log, for next 
day morning delivery by FedEx on a Monday-Thursday to: 
 
Please call or e-mail to alert us of shipment arrival, including tracking number. 
 
UPCI – IMCPL 
5117 Centre Avenue 
1.27 Hillman Cancer Center 
Pittsburgh, PA 15213 
Attn: Specimen processor 
Phone 412-623-1418 


75 
 
APPENDIX E 
Inventory / Shipping Log for IL-2HQ Research Blood Specimens 
 
IL-2HQ: A Phase II Study of hydroxychloroquine and Aldesleukin in Patients with Metastatic RCC 
Patient 
Study ID#   
Institution 
  
 
Date sent 
to UPCI 
Day -14 
"A" 
Pre-dose hydroxychloroquine 
Date 
Time 
am/pm 
10 green tops 
2 red tops 
1 PAXgene RNA blood 
Day 1 - 2 
Cycle 1 
"B" 
Pre-dose IL-2 (Post-dose PO HCQ) 
Date 
Time 
am/pm 
Course 1 
10 green tops 
2 red tops 
1 PAXgene RNA blood 
"C" 
Prior to IL-2 Dose 4 (+/- 1 Dose) 
Date 
Time 
am/pm 
1 red top 
Day 15-16 
Cycle 2 
"D" 
Pre-dose hydroxychloroquine 
Date 
Time 
am/pm 
Course 1 
2 green tops 
1 red top 
"E" 
Prior to IL-2 Dose 4 (+/- 1 Dose) 
Date 
Time 
am/pm 
1 red top 
Between 
Days 60-80 “F” 
During clinic visit 
Date 
Time 
am/pm
Course 1 
10 green tops 
2 red tops 
1 PAXgene RNA blood 
Day 1 
Cycle 1 
“G” 
Pre-dose IL-2 (Post-dose PO HCQ) 
Date 
Time 
am/pm
Course 2 
2 green tops 
1 red top 
Day 1 
Cycle 1 
“H” 
Pre-dose IL-2 (Post-dose PO HCQ) 
Date 
Time 
am/pm
Course 3 
2 green tops 
1 red top 
Green top tube (8-10 mL/tube), Red top tube (8-10 mL/tube), PAXgene tube (2.5 mL/tube) 


76 
 
APPENDIX F 
 
Subject Drug Administration Diary for Hydroxychloroquine (HCQ) Tablets 
 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE 
TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
 
 
INSTRUCTIONS TO THE PATIENT: 
 
1. You will take a dose of hydroxychloroquine (HCQ)  
mg daily starting on day –14. 
You will take:  
200 mg tablets in the morning and  
 
200 mg tablets in the 
evening every day while you are participating in this study. 
2. You will start taking HCQ on day –14. You will take HCQ daily as directed. 
3. For every day you take HCQ, record the date, the number of capsules you took, and the 
time of day you took each capsule. 
4. You should take your HCQ tablets at about the same time each day. 
5. Swallow the tablets whole. Do not chew the tablets. 
6. Missed and vomited doses should not be made up. 
7. If you have any comments or notice any side effects, please record them in the 
“Comments” column. 
8. Please bring this form with you when you return for your doctor’s appointments. 
9. If you make a mistake on the form, please cross out your mistake with a single line through 
it. Then use the next day’s space to correct the entry. Then cross out each of the 
subsequent numbers and write in the correct number. 
10. 
DO NOT SHARE THIS MEDICATION WITH ANYONE. 


77 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: N/A (Day –14 to Day –1) 
Course Start Date: 
 
Study 
Day 
Date 
AM DOSE 
  
200 mg tablet(s) 
PM DOSE 
  
200 mg tablet(s) 
Comments 
Missed 
Doses 
 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
–14 
–13 
–12 
–11 
–10 
–9 
–8 
–7 
–6 
–5 
–4 
–3 
–2 
–1 
 
 
 
 
 
 
 


78 
 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: 
Course Start Date: 
Study 
Day 
Date 
AM DOSE 
  
200 mg 
tablet(s) 
PM DOSE 
  
200 mg 
tablet(s) 
Comments 
Missed 
Doses 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 


79 
 
23 
24 
25 
26 
27 
28 
 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: 
Course Start Date: 
 
Study 
Day 
Date 
AM DOSE 
  
200 mg tablet(s) 
PM DOSE 
  
200 mg tablet(s) 
Comments 
Missed 
Doses 
 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
29 
30 
31 
32 
33 
34 
35 
36 
37 
38 
39 
40 
41 
42 
43 
44 
45 
46 
47 
48 
49 


80 
 
50 
51 
52 
53 
54 
55 
56 
 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: 
Course Start Date: 
 
Study 
Day 
Date 
AM DOSE 
  
200 mg tablet(s) 
PM DOSE 
  
200 mg tablet(s) 
Comments 
Missed 
Doses 
 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
57 
58 
59 
60 
61 
62 
63 
64 
65 
66 
67 
68 
69 
70 
71 
72 
73 
74 
75 
76 


81 
 
77 
78 
79 
80 
81 
82 
83 
84 


82 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: N/A (Day 267 to Day 351) 
Course Start Date: N/A 
 
Study 
Day 
Date 
AM DOSE 
  
200 mg tablet(s) 
PM DOSE 
  
200 mg tablet(s) 
Comments 
Missed 
Doses 
 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
267 
268 
269 
270 
271 
272 
273 
274 
275 
276 
277 
278 
279 
280 
281 
282 
283 
284 
285 
286 
287 
288 
289 
290 
291 
292 
293 


83 
 
294 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: N/A (Day 267 to Day 351) 
Course Start Date: N/A 
 
Study 
Day 
Date 
AM DOSE 
  
200 mg tablet(s) 
PM DOSE 
  
200 mg tablet(s) 
Comments 
Missed 
Doses 
 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
295 
296 
297 
298 
299 
300 
301 
302 
303 
304 
305 
306 
307 
308 
309 
310 
311 
312 
313 
314 
315 
316 
317 
318 
319 
320 
321 


84 
 
322 
 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: N/A (Day 267 to Day 351) 
Course Start Date: N/A 
 
Study 
Day 
Date 
AM DOSE 
  
200 mg tablet(s) 
PM DOSE 
  
200 mg tablet(s) 
Comments 
Missed 
Doses 
 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
323 
324 
325 
326 
327 
328 
329 
330 
331 
332 
333 
334 
335 
336 
337 
338 
339 
340 
341 
342 
343 
344 
345 
346 
347 


85 
 
348 
349 
350 
351 
 
 
 
SUBJECT DRUG ADMINISTRATION DIARY FOR HYDROXYCHLOROQUINE TABLETS 
Protocol ID: UPCI 11-080 
Study Drug: Hydroxychloroquine (HCQ) 
Subject Identification #: 
Study Drug Start Date: 
Course #: N/A 
Course Start Date: N/A 
 
Study 
Day 
Date 
AM DOSE 
  
200 mg tablet(s) 
PM DOSE 
  
200 mg tablet(s) 
Comments 
Missed 
Doses 
 
# pills 
taken 
Time taken 
# pills 
taken 
Time taken 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


86