Jeremy Jass Lecturre September 15, 2014 What Is the Tumor Telling Us? (Is it whispering or shouting?)

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1 Jeremy Jass Lecturre September 15, 2014 What Is the Tumor Telling Us? (Is it whispering or shouting?) Stan Hamilton, M.D. Head, Pathology and Laboratory Medicine, MDACC Deputy Chair for Laboratory Science, ECOG-ACRIN

2 What is the tumor telling us? Which story do we want to hear? In what language? How confidently are we being told?

3 Colorectal tumor characterization Which story do we want to hear? Fit-for-purpose testing Risk assessment (including family members) Surveillance Diagnosis Prognostication Neoadjuvant /surgical/post-op adjuvant therapy Prediction of therapeutic response Monitoring

4 Colorectal tumor characterization In what language? Pathology Genomics Mutation Copy number variation Rearrangements Alternative splicing Gene product expression Transcriptome Proteomics Immunohistochemistry

5 Colorectal tumor characterization How confidently are we being told? Levels of evidence Multiple integral marker clinical trials Integral marker clinical trial Multiple retrospective-prospective studies Retrospective-prospective study Multiple retrospective studies Retrospective study Case series Case report

6 Pathology as the language Diagnosis Inherited syndromes Prognosis: Stage and additional features

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8 Schmieden and Westhues. Deutsche Zeitschrift fur Chirurgie 1927:202,

9 Sanctity of the muscularis mucosae In situ carcinoma High-grade dysplasia Intramucosal carcinoma

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11 Genomics as the language

12 Encyclopedia Genomica, University of Leicester

13 CRC indication and stage Lynch syndrome Prognosis Chemotherapy Anti- EGFR Adjuvant aspirin I II III IV or recurrent MSI IHC & PCR X X X X X MSI IHC & PCR X X X X X MSI IHC & PCR X X X? KRAS/NRAS mutation X X BRAF mutation BRAF mutation BRAF mutation X (MLH1 loss) X (non-msi- H) X X X X X X X X?? X PIK3CA mutation? X PIK3CA mutation X X X X? PTEN expression? X MLH1 methylation X X X X X

14 CRC indication and stage Lynch syndrome Prognosis Chemotherapy Anti- EGFR Adjuvant aspirin I II III IV or recurrent MSI IHC & PCR X X X X X MSI IHC & PCR X X X X X MSI IHC & PCR X X X? KRAS/NRAS mutation X X BRAF mutation BRAF mutation BRAF mutation X (MLH1 loss) X (non-msi- H) X X X X X X X X?? X PIK3CA mutation? X PIK3CA mutation X X X X? PTEN expression? X MLH1 methylation X X X X X

15 CRC indication and stage Lynch syndrome Prognosis Chemotherapy Anti- EGFR Adjuvant aspirin I II III IV or recurrent MSI IHC & PCR X X X X X MSI IHC & PCR X X X X X MSI IHC & PCR X X X? KRAS/NRAS mutation X X BRAF mutation BRAF mutation BRAF mutation X (MLH1 loss) X (non-msi- H) X X X X X X X X?? X PIK3CA mutation? X PIK3CA mutation X X X X? PTEN expression? X MLH1 methylation X X X X X

16 CRC indication and stage Lynch syndrome Prognosis Chemotherapy Anti- EGFR Adjuvant aspirin I II III IV or recurrent MSI IHC & PCR X X X X X MSI IHC & PCR X X X X X MSI IHC & PCR X X X? KRAS/NRAS mutation X X BRAF mutation BRAF mutation BRAF mutation X (MLH1 loss) X (non-msi- H) X X X X X X X X?? X PIK3CA mutation? X PIK3CA mutation X X X X? PTEN expression? X MLH1 methylation X X X X X

17 CRC indication and stage Lynch syndrome Prognosis Chemotherapy Anti- EGFR Adjuvant aspirin I II III IV or recurrent MSI IHC & PCR X X X X X MSI IHC & PCR X X X X X MSI IHC & PCR X X X? KRAS/NRAS mutation X X BRAF mutation BRAF mutation BRAF mutation X (MLH1 loss) X (non-msi- H) X X X X X X X X?? X PIK3CA mutation? X PIK3CA mutation X X X X? PTEN expression? X MLH1 methylation X X X X X

18 CRC indication and stage Lynch syndrome Prognosis Chemotherapy Anti- EGFR Adjuvant aspirin I II III IV or recurrent MSI IHC & PCR X X X X X MSI IHC & PCR X X X X X MSI IHC & PCR X X X? KRAS/NRAS mutation X X BRAF mutation BRAF mutation BRAF mutation X (MLH1 loss) X (non-msi- H) X X X X X X X X?? X PIK3CA mutation? X PIK3CA mutation X X X X? PTEN expression? X MLH1 methylation X X X X X

19 Microsatellite instability: Lynch syndrome Favorable prognosis Avoidance of 5-FU adjuvant therapy? Irinotecan benefit

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21 Mismatch after slippage New Strand G-T -G-T-G-T G-T-G-T- -C-A-C-A-C-A-C-A- Template -G-T-G-T-G-T-G- -C-A-C-A C-A-C- C-A 8bp + 10bp 8bp + 6bp

22 Normal Tumor

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24

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27 Giardiello et al, Am J Gastroenterol 2014, 109:

28 Giardiello et al, Am J Gastroenterol 2014;109:

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30 Hypermutated:? Favorable prognosis

31 TCGA CRC: Ranked frequency of mutated and expressed genes Gene Non-hypermutated (84%) Hypermutated (16%) APC 1 (81%) 2 (51%) TP53 2 (60%) - KRAS* 3 (43%) - TTN 4 (31%) - PIK3CA 5 (18%) - FBXW7 6 (11%) - SMAD4 7 (10%) - NRAS* 8 (9%) - TCF7L2 9 (9%) 8 (31%) ACVR2A - 1 (63%) TGFBR2-3 (51%) BRAF* - 4 (46%) MSH3-5 (40%) MSH6-6 (40%) MYO1B - 7 (31%) CASP8-9 (29%)

32 Microsatellite instability: Lynch syndrome Favorable prognosis Avoidance of 5-FU adjuvant therapy? Irinotecan benefit

33 MSI-H and improved stage-specific survival in colorectal cancer Gryfe R, et al. New Engl J Med 2000; 342: 69-77

34 WHO Classification of Tumours of the Digestive System, 2010 Poor differentiation with MSI-H: Low-grade, not high-grade

35 Microsatellite instability: Lynch syndrome Favorable prognosis Avoidance of 5-FU adjuvant therapy? Irinotecan benefit

36 % Disease Free % Disease Free Loss of MMR protein by IHC with or without adjuvant 5-FU Stage II (N=102) Stage III (N=63) Untreated 87% Treated 72% Years HR: 2.80 ( ) p= Untreated 62% Treated 67% HR: 1.08 ( ) p= Years HR: 1.08 ( ) p=0.86 (Pooled specimens from RCT; Sargent DA et al. )

37 Microsatellite instability: Lynch syndrome Favorable prognosis Avoidance of 5-FU adjuvant therapy? Irinotecan benefit

38 Irinotecan

39 BJC 2008:99,

40 MSI-H and irinotecan, 5-FU, and leucovorin (IFL): CALGB Stage III adjuvant trial (89803) Bertagnolli MM, et al. JCO 27: 1814, 2009 Not confirmed in PETACC-3: Tejpar S, et al. J Clin Oncol 2009; 17: 15s (abstract 4001)

41 KRAS or NRAS mutation: Contraindication to anti-egfr antibody therapy

42 cetuximab High Priority Targets and DCTD/CTEP Agents bevacizumab ziv-aflibercept VEGF Surface antigens SGN 35 (CD30) HA 22 (CD22) CDX-011 other c-kit EGF-R VEGF-R receptors imatinib Notch P13 K MK-2206 AT-101 obatoclax navitoclax TL32711 fenretinide erlotinib AZD9291 Ceramide tipifarnib Akt BCL-2 XIAP Ras Raf dasatinib sorafenib dabrafenib tramitinib selumetinib TORC MLN0128 temsirolimus mtor MEK SRC Bcr dasatinib saracatinib imatinib Btk Abl PCI sorafenib sunitinib cediranib pazopanib CD105 TRC105 Angiopoietins AMG386 CDKs dinaciclib Microtubules brentuximab vedotin CHK1 SCH Aurora kinase A MLN 8237 Wee1 kinase MK-1775 IGF-1R ganitumab cixutuzumab linsitinib HER2 Lapatinib Pertuzumab trastuzumab sunitinib sorafenib Met tivantinib AMG337 cabozantinib rilotumuab ERa z-endoxifen PDGFR sunitinib imatinib pazopanib cediranib Flt3,RET bfgfr cediranib BCR ibrutinib Hsp90 AT PU-H71 Proteasome bortezomib PD1 pembrolizumab nivolumab imid thalidomide lenalidomide pomalidomide CTLA44 ipilimumab ticilimumab IDO 1-Methyl-[D]- tryptophan Notch RO Hedgehog vismodegib Stem cell signaling PARP veliparib BMN673 olaparib HDAC belinostat entinostat vorinostat Topoisomerases LMP400/776 Alkylating Dimethane sulfonate Methylation inh. FdCyd/THU TRC102 Apoptosis Survival/ Proliferation Angiogenesis Protein turnover Immunomodulation Migration/ invasion Mitosis DNA repair epigenetics

43 Cetuximab

44 Copyright American Society of Clinical Oncology Bardelli, A. et al. J Clin Oncol; 28:

45 EGFR inhibitor predictive testing Test Prediction TMUGS level of evidence EGFR expression None III (FDA required) KRAS mutation c12,13,59, 61, 117,146; NRAS c12,13,59,61 Adverse BRAF mutation exons 11 and 15 Adverse III Loss of PTEN expression Adverse III Amphiregulin expression Favorable IV Epiregulin expression Favorable IV PI3 kinase mutation Adverse IV EGFR amplification Favorable IV I - IIII I Prospective trial of marker or meta-analysis/overview of II/III studies II Prospective trial including marker III Large retrospective studies IV Small retrospective studies V Pilot studies J Natl Cancer Inst, 88: , 1996.

46 The Author Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please Gene expression signature of KRAS 12/13-mutated versus KRAS/NRAS/BRAF wild-type tumors using RNA sequencing data from the CRC TCGA project. Morris V K et al. Ann Oncol 2014;annonc.mdu252

47 Main recommendations for RAS testing of colorectal carcinoma to guide anti- EGFR therapy: Network arrangements should be established to ensure rapid and robust tissue pathways from referral centres to testing laboratories. Either primary or metastatic CRC tissue can be used for RAS testing. Either biopsy or resection specimen tissue can be used for RAS testing, though if both are equally available, use of resection tissue is preferable. The minimum neoplastic cell content tested should be at least two times the assay's LOD. RAS analysis should include at least KRAS codons 12, 13, 59, 61, 117 and 146 and NRAS codons 12, 13, 59 and 61. Turnaround time for RAS testing (of the above panel) should be 7 working days from receipt of the specimen in the testing laboratory to issuing of the final report, for >90% of specimens. J Clin Pathol 2014; 67: 715-7

48 BRAF mutation: An adverse prognostic marker in microsatellite-stable or -low CRC; adverse outcome after standard therapies

49 The Author Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please Overall survival of patients according to RAS mutational status and BRAF mutation (global P< 0.001). Morris V K et al. Ann Oncol 2014;annonc.mdu252; mcrc

50 Lochhead et al, JNCI 105:1152 (2013)

51 Figure 1 Overall Survival According to Stage at Diagnosis. ( A ) Median Overall Survival From the Time of Initial Diagnosis According to the Initial Stage of Diagnosis. ( B ) Kaplan-Meier Curves Stratified According to Stage at the Time of Initial Diagnosi... Van Morris, Michael J. Overman, Zhi-Qin Jiang, Christopher Garrett, Shweta Agarwal, Cathy Eng, Bryan Kee,... Progression-Free Survival Remains Poor Over Sequential Lines of Systemic Therapy in Patients With BRAF Colorectal Cancer Clinical Colorectal Cancer,

52 PIK3CA mutation: Effective post-operative adjuvant therapy with aspirin

53 Liao et al, NEJM 367:1601, 2012.

54 Wide gap between the state of the molecular science and clinical applications Targets for agents Biomarkers for effects Panels of assays

55 Companion diagnostic (CoDx) => Companion therapeutics (CoRxs) from assay panels

56 What is the tumor telling us?

57 Whimpering: Transcriptomics Commercially available gene expression profiles Disappointing prognostication No prediction of response to therapy

58 Whispering: Genomics BRAF mutation PIK3CA mutation Loss of PTEN protein expression No therapy with cetuximab or panitumumab anti-egfr antibodies

59 Shouting: High-quality pathology Diagnosis Inherited syndromes Lynch syndrome Polyposis syndromes Prognosis Pathologic stage Additional features MSI-H/dMMR Lymphatic and vascular invasion Tumor budding

60 Shouting: Genomics High levels of microsatellite instability (MSI-H)/deficient mismatch repair (dmmr) Lynch syndrome More favorable prognosis No 5-FU monotherapy

61 Shouting: Genomics KRAS mutation in codon 12 or 13 (?G13D) Shouting softly: KRAS mutation in codon 59, 61, 117, or 146 NRAS mutation in codon 12, 13, 59, or 61 extended RAS No therapy with cetuximab or panatumumab anti-egfr antibodies

62 Shouting: Genomics BRAF V600E mutation in the absence of MSI-H/dMMR Aggressive therapy

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64 The tumor has much more to tell us. The end of the beginning Heterogeneity: Inter-tumoral, intratumoral, progression, primaries/mets Co-alterations Drivers and passengers Signaling in pathways Therapy-induced changes Immunotherapy Circulating nucleic acids

65 Thanks for your attention.