protocol nmtrc009
references

1. Ries, L.A.G. and SEER Program (National Cancer Institute (U.S.)), *Cancer* incidence and survival among children and adolescents : United States SEER
program 1975-1995 /[edited by Lynn A. Gloecker Ries ... et al.]. SEER pediatric monograph. 1999, Bethesda, MD: National Cancer Institute, SEER Program. vi, 182 p.  
2. Arndt, C.A., et al., Common musculoskeletal tumors of childhood and adolescence. Mayo Clin Proc, 2012. 87(5): p. 475-87.  
3. Pappo, A.S., et al., Metastatic nonrhabdomyosarcomatous soft-tissue sarcomas in children and adolescents: the St. Jude Children's Research Hospital experience.  
Med Pediatr Oncol, 1999. 33(2): p. 76-82.  
4. Geller, D.S. and R. Gorlick, Osteosarcoma: a review of diagnosis, management, and treatment strategies. Clin Adv Hematol Oncol, 2010. 8(10): p. 705-18.  
5. Potratz, J., et al., *Ewing sarcoma: clinical state-of-the-art.* Pediatr Hematol Oncol, 2012. 29(1): p. 1-11.  
6. Davidoff, A.M., *Wilms' tumor.* Curr Opin Pediatr, 2009. 21(3): p. 357-64.  
7. Castellino, S.M., A.R. Martinez-Borges, and T.W. McLean, Pediatric genitourinary tumors. Curr Opin Oncol, 2009. 21(3): p. 278-83.  
8. *American Cancer Society: Cancer Facts and Figures. 2008 Atlanta, GA.* 2008.  
9. Bernstein, M.L., et al., A population-based study of neuroblastoma incidence, survival, and mortality in North America. J Clin Oncol, 1992. 10(2): p. 323-9.  
10. Brodeur, G.M., et al., Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol, 1993. 11(8): p. 146677.  
11. Park, J.R., A. Eggert, and H. Caron, Neuroblastoma: biology, prognosis, and treatment. Pediatr Clin North Am, 2008. 55(1): p. 97-120, x.  
12. Matthay, K.K., et al., Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13cis-retinoic acid. Children's Cancer Group. N Engl J Med, 1999. 341(16): p.  
1165-73.  
13. Hartmann, O., et al., Prognostic factors in metastatic neuroblastoma in patients over 1 year of age treated with high-dose chemotherapy and stem cell transplantation: a multivariate analysis in 218 patients treated in a single institution. Bone Marrow Transplant, 1999. 23(8): p. 789-95.  
14. Wagner, L.M. and M.K. Danks, *New therapeutic targets for the treatment of highrisk neuroblastoma.* J Cell Biochem, 2009. 107(1): p. 46-57.  
15. Mosse, Y.P., et al., *Identification of ALK as a major familial neuroblastoma* predisposition gene. Nature, 2008. 455(7215): p. 930-5.  
16. Hansford, L.M., et al., Neuroblastoma cells isolated from bone marrow metastases contain a naturally enriched tumor-initiating cell. Cancer Res, 2007.  
67(23): p. 11234-43.  
17. Nieder, C., et al., Therapeutic options for recurrent high-grade glioma in adult patients: recent advances. Crit Rev Oncol Hematol, 2006. 60(3): p. 181-93.  
18. American Cancer Society., *Cancer facts & figures - 2012*. 2012, Atlanta, GA:
American Cancer Society. 36 p.  
19. Gajjar, A. and B. Pizer, Role of high-dose chemotherapy for recurrent medulloblastoma and other CNS primitive neuroectodermal tumors. Pediatr Blood Cancer, 2010. 54(4): p. 649-51.  
20. Fleming, A.J. and S.N. Chi, *Brain tumors in children.* Curr Probl Pediatr Adolesc Health Care, 2012. 42(4): p. 80-103.  
21. Fangusaro, J., Pediatric high grade glioma: a review and update on tumor clinical characteristics and biology. Front Oncol, 2012. 2: p. 105.  
22. Jones, D.T., et al., *Dissecting the genomic complexity underlying* medulloblastoma. Nature, 2012. 488(7409): p. 100-5.  
23. Northcott, P.A., et al., Medulloblastoma comprises four distinct molecular variants. J Clin Oncol, 2011. 29(11): p. 1408-14.  
24. Northcott, P.A., et al., *Subgroup-specific structural variation across 1,000* medulloblastoma genomes. Nature, 2012. 488(7409): p. 49-56.  
25. Robinson, G., et al., Novel mutations target distinct subgroups of medulloblastoma. Nature, 2012. 488(7409): p. 43-8.  
26. Beaty, O., 3rd, et al., *A phase II trial and pharmacokinetic study of oxaliplatin in* children with refractory solid tumors: a Children's Oncology Group study.  
Pediatr Blood Cancer, 2010. 55(3): p. 440-5.  
27. Warren, K.E., et al., A phase II study of O6-benzylguanine and temozolomide in pediatric patients with recurrent or progressive high-grade gliomas and brainstem gliomas: a Pediatric Brain Tumor Consortium study. J Neurooncol, 2012. 106(3): p. 643-9.  
28. *Comprehensive genomic characterization defines human glioblastoma genes and* core pathways. Nature, 2008. 455(7216): p. 1061-8.  
29. Zhu, X., M. Gerstein, and M. Snyder, Getting connected: analysis and principles of biological networks. Genes Dev, 2007. 21(9): p. 1010-24.  
30. Jones, S., et al., Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science, 2008. 321(5897): p. 1801-6.  
31. Huang, S., *Back to the biology in systems biology: what can we learn from* biomolecular networks? Brief Funct Genomic Proteomic, 2004. 2(4): p. 279-97.  
32. Aranda-Anzaldo, A., Cancer development and progression: a non-adaptive process driven by genetic drift. Acta Biotheor, 2001. 49(2): p. 89-108.  
33. Wang, E., A. Lenferink, and M. O'Connor-McCourt, Cancer systems biology:
exploring cancer-associated genes on cellular networks. Cell Mol Life Sci, 2007.  
64(14): p. 1752-62.  
34. Michor, F., M.A. Nowak, and Y. Iwasa, *Evolution of resistance to cancer* therapy. Curr Pharm Des, 2006. 12(3): p. 261-71.  
35. Sjoblom, T., et al., *The consensus coding sequences of human breast and* colorectal cancers. Science, 2006. 314(5797): p. 268-74.  
36. Balakrishnan, A., et al., Novel somatic and germline mutations in cancer candidate genes in glioblastoma, melanoma, and pancreatic carcinoma. Cancer Res, 2007. 67(8): p. 3545-50.  
37. Heng, H.H., *Cancer genome sequencing: the challenges ahead.* Bioessays, 2007.  
29(8): p. 783-94.  
38. Overington, J.P., B. Al-Lazikani, and A.L. Hopkins, *How many drug targets are* there? Nat Rev Drug Discov, 2006. 5(12): p. 993-6.  
39. Wishart, D.S., et al., *DrugBank: a knowledgebase for drugs, drug actions and* drug targets. Nucleic Acids Res, 2008. 36(Database issue): p. D901-6.  
40. George, R.E., et al., *Activating mutations in ALK provide a therapeutic target in* neuroblastoma. Nature, 2008. 455(7215): p. 975-8.  
41. Anderson, M.J., A new method for non-parametric multivariate analysis of variance. Austral Ecology, 2001. 26: p. 32-46.  
42. McArdle, B.H. and M.J. Anderson, *Fitting Multivariate Models to Community* Data:
A Comment on Distance-Based Redundancy Analysis. Ecology, 2001. 82: p. 290-297.  
43. Love, M.I., W. Huber, and S. Anders, *Moderated estimation of fold change and* dispersion for RNA-seq data with DESeq2. Genome Biol, 2014. 15(12): p. 550.  
44. Eisenhauer, E.A., et al., *New response evaluation criteria in solid tumours:*
revised RECIST guideline (version 1.1). Eur J Cancer, 2009. 45(2): p. 228-47.
protocol nmtrc009
appendix i performance status/scores

| Performance Status Criteria                                                              |                                  |                                       |                                |                                   |                                   |
|------------------------------------------------------------------------------------------|----------------------------------|---------------------------------------|--------------------------------|-----------------------------------|-----------------------------------|
| Kamofsky and Lansky performance scores are intended to be multiples of 10                |                                  |                                       |                                |                                   |                                   |
| ECOG (Zubrod)                                                                            | Karnofsky                        | Lansky*                               |                                |                                   |                                   |
| Score                                                                                    | Score                            |                                       |                                |                                   |                                   |
| Description                                                                              | Description                      | Score                                 | Description                    |                                   |                                   |
| 100                                                                                      | Normal, no complaints, no        | 100                                   | Fully active, normal.          |                                   |                                   |
| evidence of disease                                                                      |                                  |                                       |                                |                                   |                                   |
| Fully active, able to cany on all                                                        |                                  |                                       |                                |                                   |                                   |
| 0                                                                                        | pre-disease performance          | 90                                    | Able to cany on normal         | 90                                | Minor restrictions in physically  |
| without restriction.                                                                     | activity, minor signs or         | strenuous activity.                   |                                |                                   |                                   |
| symptoms of disease.                                                                     |                                  |                                       |                                |                                   |                                   |
| 80                                                                                       | Normal activity with effort;     | 80                                    | Active, but tires more quickly |                                   |                                   |
| Restricted in physically                                                                 | some signs or symptoms of        |                                       |                                |                                   |                                   |
| strenuous activity but                                                                   | disease.                         |                                       |                                |                                   |                                   |
| ambulatory and able to carry                                                             |                                  |                                       |                                |                                   |                                   |
| !                                                                                        | out work of a light or sedentary | 70                                    | Cares for self, unable to cany | 70                                | Both greater restriction of and   |
| nature, e.g., light housework,                                                           |                                  |                                       |                                |                                   |                                   |
| office work.                                                                             | on normal activity or do active  | less time spent in play activity.     |                                |                                   |                                   |
| work.                                                                                    |                                  |                                       |                                |                                   |                                   |
| 60                                                                                       | Required occasional              | 60                                    | Up and around, but minimal     |                                   |                                   |
| assistance, but is able to care                                                          | active play, keeps busy with     |                                       |                                |                                   |                                   |
| Ambulatory and capable of all                                                            | for most of his/her needs.       | quieter activities.                   |                                |                                   |                                   |
| self-care but unable to carry out                                                        |                                  |                                       |                                |                                   |                                   |
| 2                                                                                        | any work activities. Up and      | 50                                    | Requires considerable          | 50                                | Gets dressed, but lies around     |
| about more than 50% of waking                                                            | assistance and frequent          | much of the day; no active play,      |                                |                                   |                                   |
| hours                                                                                    | medical care.                    | able to participate in all quiet play |                                |                                   |                                   |
| and activities.                                                                          |                                  |                                       |                                |                                   |                                   |
| Mostly in bed; participates in                                                           |                                  |                                       |                                |                                   |                                   |
| 40                                                                                       | Disabled, requires special care  | 4                                     |                                |                                   |                                   |
| Capable of only limited self-                                                            | and assistance.                  | quiet activities.                     |                                |                                   |                                   |
| care, confined to bed or chair                                                           |                                  |                                       |                                |                                   |                                   |
| 3                                                                                        | more than 50% of waking          | 30                                    | Severely disabled,             | 30                                | In bed; needs assistance even for |
| hours.                                                                                   | hospitalization indicated.       | quiet play.                           |                                |                                   |                                   |
| Death not imminent.                                                                      |                                  |                                       |                                |                                   |                                   |
| 20                                                                                       | Very sick, hospitalization       | 20                                    | Often sleeping; play entirely  |                                   |                                   |
| limited to very passive activities.                                                      |                                  |                                       |                                |                                   |                                   |
| indicated. Death not                                                                     |                                  |                                       |                                |                                   |                                   |
| Completely disabled. Cannot                                                              | imminent.                        |                                       |                                |                                   |                                   |
| 4                                                                                        | cany on any self-care. Totally   |                                       |                                |                                   |                                   |
| confined to bed or chair.                                                                | 10                               | Moribund, fatal processes             | 10                             | No play; does not get out of bed. |                                   |
| progressing rapidly.                                                                     |                                  |                                       |                                |                                   |                                   |
| The conversion of the Lansky to ECOG scales is intended for NCI reporting purposes only. |                                  |                                       |                                |                                   |                                   |
protocol nmtrc009
standard pathology sample

1. Obtain first core biopsy (if possible; otherwise 2nd core)
a. At least 18 gauge guiding needle or larger in caliber b. Remove excess blood from the sample with sterile saline rinse (provided in the kit) without damaging the sample 2. Place core (for standard pathology) in neutral buffered formalin container per standard practices 3. Send tissue to pathology
protocol nmtrc009
primary sample - sent to

4. Obtain second core biopsy from same track according to institutional protocol 5. *DO NOT PLACE SAMPLE INTO FORMALIN*. Flash Freeze sample within 20 minutes of extraction and place in Tube 1 provided by the NTMRC. Note time of extraction and time of flash freezing.  
6. Upon generation of a pathology report, please e-mail the NMTRC with the percent tumor in Sample 1.
protocol nmtrc009
pathologist

Place Tube 1 with the patient sample and Ashion Shipping Form in the shipping kit. Affix the label to FedEx shipping bag and place the kit in the bag for shipment. Please ship via FedEx (if you do not have a scheduled FedEx pickup, call 800-GO-FEDEX - 800-463-3339).  
Note- Ashion is unable to generate a patient molecular report without knowing the percent viable *tumor in the sample (Must be >50% to move forward) -*
Thank you For questions please call the NMTRC at (616) 267-0327 or e-mail nmtrc@helendevoschildrens.org
protocol nmtrc009
a. do not place sample

b. Please place sample directly (not wrapped) into the provided sterile saline (0.9% NaCl without dextrose), place in a labeled container and note time of extraction on container label.  
2. Send to Pathology/Histology ASAP; please hand deliver to pathology within 20 minutes. NEVER AT ANY TIME PUT SAMPLE IN TIN/ALUMINUM FOIL.
protocol nmtrc009
pathologist/pathology assistant

1. Do not ink specimen (prior to completing steps 2-4 below) 2. Section specimen with a sterile blade on a clean surface. Select tumor tissue that appears non-necrotic; avoid normal tissue. Obtain a specimen for histopathological analysis at your institution, avoiding normal or necrotic tissue.  
3. Obtain a sample with a maximum dimension of 0.5 cm3 (approximately the size of a pencil eraser). Minimum amount of tumor sample required is 50-200mg of tissue.  
4. Flash Freeze sample within 20 minutes of extraction and place in Tube 1 provided by the NTMRC. Note time of time of flash freezing.  
5. Place Tube 1 with the patient sample and Ashion shipping form in the shipping box with dry ice. Affix a shipping label to the FedEx shipping bag and place the kit in the bag for shipment. Please ship via FedEx (if you do not have a scheduled FedEx pickup, call 800-GO-FEDEX - 800-463-3339)
6. Handle any remaining tissue as per your institutional protocols and in accordance with physician order 7. Upon generation of a pathology report, please e-mail the NMTRC with the percent tumor in Sample 1.  
Note- Ashion is unable to generate a patient molecular report without knowing the percent viable tumor in the sample (Must be >50% to move forward) - Thank you For questions please call the NMTRC at (616) 267-0327 or e-mail nmtrc@helendevoschildrens.org
protocol nmtrc009
appendix iii pharmacopeia library drug

| Acetazolamide       | Dinutuximab        | Leflunomide            | Raloxifene hydrochloride   |
|---------------------|--------------------|------------------------|----------------------------|
| Adalimumab          | Docetaxel          | Lenalidomide           | Ramipril                   |
| Albendazole         | Donepezil          | Lomustine              | Rifampin                   |
| Aldesleukin         | DOXOrubicin        | Lovastatin             | Rituxumab                  |
| Alemtuzumab         | Doxycycline        | Luteolin               | Romidepsin                 |
| Alfuzosin           | Enalapril          | Mechlorethamine        | Rosuvastatin calcium       |
| Anakinra            | Epirubicin         | Mercaptopurine         | Ruxolitinib                |
| Anastrozole         | Ergocalciferol     | Mesalamine             | Simvastatin                |
| Atorvastatin        | Eribulin           | Methotrexate           | Sirolimus                  |
| Atovaquone          | Erlotinib          | Methyl CCNU            | Sorafenib                  |
| Azacitidine         | Ethambutol         | MitoMYCIN              | Streptozocin               |
| Balsalazide         | Ethosuximide       | Mitotane               | Sulfasalazine              |
| Bevacizumab         | Etodolac           | Mitoxantrone           | Sulindac                   |
| Bexarotene          | Etoposide          | MK4                    | Sunitinib                  |
| Bortezomib          | Everolimus         | Nabumetone             | Tacrolimus                 |
| Brentuximab         | Famotidine         | Naproxen               | Tamoxifen                  |
| Bumetanide          | Felodipine         | Nelarabine             | Temozolomide               |
| Bupropion           | Fenoprofen         | Nilotinib              | Temsirolimus               |
| Capecitabine        | Fexofenadine       | Nivolumab              | Teniposide                 |
| Captopril           | Flucytosine        | Octreotide             | Thalidomide                |
| Carboplatin         | Fludarabine        | Ofatumumab             | Thioguanine                |
| Carmustine          | Fludrocortisone    | Olaparib               | Thiotepa                   |
| Celecoxib           | Fluorouracil       | Omeprazole             | Trabectidin                |
| Ceritinib           | Fuoxetine          | Orlistat               | Trametinib                 |
| Cetuximab           | Fluvastatin        | Oxaliplatin            | Tofacitinib                |
| Cisplatin           | Fluvoxamine        | Oxaprozin              | Tolcapone                  |
| Cladribine          | Gabapentin         | Paclitaxel             | Tolmetin                   |
| Clofarabine         | Gefitinib          | Palbociclib            | Topiramate                 |
| Clonidine           | Gemcitabine        | Pamidronate            | Topotecan                  |
| Clopidogrel         | Griseofulvin       | Panitumumab            | Torsemide                  |
| Clotrimazole        | Guanfacine         | Panobinostat           | Trastuzumab                |
| Colchicine          | Hydroxyurea        | Pazopanib              | Trazodone                  |
| Cortisone           | Ibuprofen          | Peg-asparaginase       | Tretinoin                  |
| Crizotinib          | IDArubicin         | Pembrolizumab          | Ursolic acid               |
| Cyclophosphamide    | Ifosfamide         | Pemetrexed             | Valproic acid              |
| Cyclosporine        | Imatinib           | Pentostatin            | Valrubicin                 |
| Cytarabine          | Indomethacin       | Phenelzine             | Vandetanib                 |
| Dacarbazine         | Irinotecan         | Piperazine             | Vemurafenib                |
| Dantrolene          | Ipilimumab         | Piroxicam              | Verapamil                  |
| Dasatinib           | Isotretinoin       | Plerixafor             | VinBLAStine                |
| DAUNOrubicin        | Ixabepilone        | Pravastatin sodium     | VinCRIStine                |
| Decitabine          | Ketoprofen         | Prazosin hydrochloride | Vinorelbine                |
| Denileukin diftitox | Ketorolac          | PrednisoLONE           | Vismodegib                 |
| Dexamethasone       | Lansoprazole       | Prochlorperazine       | Vorinostat                 |
| Diazoxide           | Lapatinib          | Propylthiouracil       | Zolendronic acid           |
| Diclofenac          | Rabeprazole sodium |                        |                            |  
Naturopathic Drug Table:  
| ALA                      | Calcitriol                    | Garlic              | Modified citrus pectin       |
|--------------------------|-------------------------------|---------------------|------------------------------|
| Ascorbic acid            | Cordyceps                     | Ginger (6-gingerol) | Myrrh (Guggulsterone)        |
| Astragalus membranaceu s | Coriolus (versicolor)         | Green Tea           | Pycnogenol                   |
| Berberine                | Curcumin                      | Indole-3-Carbinol   | Quercetin                    |
| Black Cumin              | Fermented wheat germ  extract | Lycopene            | Rosemary                     |
| (thymoquinone) Bromelain | Frankincense                  | Melatonin           | Soy Isoflavones Sulforaphane |
protocol nmtrc009
appendix iv- example expression file

\#\#fileformat=VCFv4.1
\#\#DESeq
\#\#ALT=<ID=EXP,Description="Differential Expression">
\#\#INFO=<ID=IMPRECISE,Number=0,Type=Flag,Description="Imprecise ">
\#\#INFO=<ID=END,Number=1,Type=Integer,Description="End position of gene ">
\#\#INFO=<ID=GENEID,Number=1,Type=String,Description="Ensembl gene ID "> \#\#INFO=<ID=GENE,Number=1,Type=String,Description="Common gene symbol ">
\#\#INFO=<ID=BASEMEAN,Number=1,Type=Float,Description="Average of baseMeanA and baseMeanB ">
\#\#INFO=<ID=BASEMEANA,Number=1,Type=Float,Description="Average normalized counts for control sample(s) ">
\#\#INFO=<ID=BASEMEANB,Number=1,Type=Float,Description="Average normalized counts for affected sample(s) ">
\#\#INFO=<ID=FOLDCHANGE,Number=1,Type=Float,Description="Fold Change (Affected/Control)
">
\#\#INFO=<ID=LOG2FC,Number=1,Type=Float,Description="Log2 Fold Change (Affected/Control)
">
\#\#INFO=<ID=PVALUE,Number=1,Type=Float,Description="p-value ">
\#\#INFO=<ID=PADJ,Number=1,Type=Float,Description="Multiple hypothesis testing corrected pvalue ">
\#CHROM POS ID REF ALT QUAL FILTER INFO
X 99883667 . N <EXP> 0.644187137606379 .  
IMPRECISE;END=99894988;GENEID=ENSG00000000003;GENE=TSPAN6;BASEMEAN=3831.61375 995454;BASEMEANA=4951.27397871424;BASEMEANB=2711.95354119485;FOLDCHANGE=0.54 7728433702853;LOG2FC=-
0.868467319327676;PVALUE=0.644187137606379;PADJ=1;REFERENCE=SU2C002_MRN.melano cyte_control Example variant from a VCF file for BRAF V600E mutation:
7 140453136 rs113488022 A T 38.6 PASS
TYPE=somatic_SNV;DP1=194;DP2=411;AR1=0.010;AR2=0.679;LN=1;PILEUP1=AAAAAaaT
aa AaaaAAaAaaaAAaaAaaAAAAaAAaAaAAaAAaAAaaAAaAAaaAAAAAaaaAAAaAaaaAAaAAAAAAAAaA aA
aaAAtAaAAAAaaaaAAAAAaAaaaaAaaAAAAAAaAaAAAAAAAaaaAAAAAAaaaAAAAAAaaaaAAaaAaA
aa aaaAaAAAAAAAAAAAAaaaAAaAAaaAaaAa;PILEUP2=TTTtaATaaATtTtTttTtttttttttaaAaat At ataTATAttTTTatTttTtTtTATTTtttttatTtATTTTTtTTTAATTtATTtTttTATtTtTAAATtTttTT
Ta aTAtaTAtaTTTtATTTtTTAttTTTATAttTATttATATTTattTAATTTtTatATttTTtAAtttAAttTAt aa aaAttAaTttaTTttttAtTtaattATtaaatttATTtTTTttatttTTtTAATtTTtatTaAatAtatTTtAA
Aa ttTTaATTAATaAtTTataTtTtttaaattatataTaaattAaTTtTaAtattTtAATATaTTttTTATTtTaT
tA
AtTaTATtAtAAAtAAAtaAAtTttttatTtatTTtATatTtttTTtatAttTTattAaatttTTatTTATT;M
VC
1=34.0;MVBQ1=NaN;MVMQ1=60.0;MVC2=34.0;EFF=EXON(MODIFIER|||||BRAF|nonsense_
me diated_decay|CODING|ENST00000479537|2|1),NON_SYNONYMOUS_CODING(MODERATE|MI
SS
ENSE|gTg/gAg|V600E|766|BRAF|protein_coding|CODING|ENST00000288602|15|1),
ENSE|UTR_
3_PRIME(MODIFIER|||||BRAF|nonsense_mediated_decay|CODING|ENST00000497784|1 6| 1);GENEINFO=BRAF:673;LSD;NSM;OM;OTHERKG;PM;PMC;REF;RSPOS=140453136;RV;S3D;
SA O=3;SSR=0;VC=SNV;VP=050268000a01000002110100;WGT=1;dbSNPBuildID=132