Genetic/genomic diagnosis for personalized cancer treatment

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1 Genetic/genomic diagnosis for personalized cancer treatment Yao-Shan Fan, MD, PhD Professor, Pathology Director, Cytogenetics & Molecular Diagnostics

2 The Achilles heel

3 A one-step remedy. Cancer cells acquire abnormalities in multiple oncogenes and tumor suppressor genes (A, B, C, and D). Inactivation of a single critical oncogene (A) can induce cancer cells to differentiate into cells with a normal phenotype or to undergo apoptosis. This dependence on (addiction to) A for maintaining the cancer phenotype provides an Achilles heel for tumors that can be exploited in cancer therapy.

4 Signaling pathways in cancer

5 Signaling pathways in cancer MAPK interacts with other pathways (PI3K, AKT, mtor, PTEN) to regulate cell growth and apoptosis. Oncogenic (driver) mutation in a growth factor receptor gene, or in RAS or RAF genes activates the MAPK pathway. Dysregulation of the pathways leads to tumor proliferation and metastasis.

6 Her2 Breast Cancer treatment Estrogen (ER+)/progesterone receptor (PR+) 60-70% -> Tamoxifen 20% have Her2 amplification (by IHC, FISH or CISH), -> Herceptin (Trastuzumab, 1998) ER/PR/Her2 (-) ->chemotherapy

7 TKI imatinib (gleevec, 2001) for CML

8 The Philadelphia chromosome 1959, Ph 1973, t(9;22) 1990, BCR-ABL1 2001, Imatinib, WHO classification David Hungerford, Fox Chase Cancer Center Peter Nowell, University of Pennsylvania School of Medicine

9 Chronic Myelogenous Leukemia (CML), BCR-ABL1 positive

10 Targeted TKI therapies Imatinib (gleevec, 2001): first line, PFS rate of 93% at 5 years, estimated OS rate of 85% at 8 years (93% when only CML related deaths are considered) Dasatinib (sprycel, 2010), nilotinib (tasigna, 2010): superior response rates and higher rates of PFS and MMR as first line than imatinib Bosutinib (bosulif, 2012) and ponatinib (Iclusig, 2102) for 2 nd and 3 rd line treatment

11 Bone marrow cytogenetics (NCCN ) Analyze 20 metaphase cells at diagnosis At 3 months, if RT-qPCR is not available At 12 months, if neither CCyR (Absence of Ph in 20 cells) nor MMR is achieved. At 18 months, if no MMR and no CCyR at 12 months 1-log increase in BCR-ABL1 level without MMR.

12 FISH FISH using blood and dual probes for BCR-ABL1 fusion at diagnosis if collection of BM is not feasible Every 3-6 months, commonly used for monitoring in practice, but not recommended by the latest NCCN

13 RT-qPCR RT-qPCR for BCR-ABL transcripts, Sensitivity: 1/ Establishing a baseline at diagnosis and then monitor log reduction MMR: 3 log reduction (100% 0.1%); CMR : 5 log reduction ( 0.001%) Every 3 months (NCCN ) Hughes et al: NEJM 2003; Blood 2006; Press et al: Blood, 2006; 2007; 2009; White et al: Blood 2010; Zhen et al; JMD, 2013;

14 Variations of RT-qPCR results C J Zhen and YL Wang, JMD, 2013 Contributing factors Sample collection Cell preparation RNA isolation RT Internal control selection Standard curve construction Data reporting 2006 CAP survey 34 labs, 2 samples, reported 34 values in different units, spanning 7 logs for each sample (1%~0.000,000,1%) 2012 CAP survey 143 labs: the highest value was 500,000 folds higher than the lowest value reported.

15 Mutations in BCR-ABL1 Associated with Imatinib Resistance >136 amino acid changes T315I resistant to imatinib, dasatinib, nilotinib, sensitive to ponatinib 50% of patients with imatinib resistance do not have BCR-ABL1 mutation

16 Eosinophilia FISH for PDGFRA, B & FGFR1 PDGFRA rearranged Myeloid neoplasm with eosinophilia PDGFRB rearranged Myeloid neoplasm with eosinophilia imatinib FGFR1 rearranged Myeloid neoplasm with eosinophilia Chronic eosinophilic leukemia PB blast >2% BM blast >5% Abn cytogenet Yes No Hypereosinoph Syndrome

17 Genetic Changes in AML Gulley et al: J Mol Diagn,2010

18 Monitor of APL Treatment 1 billion leukemic cells

19 KIT mutations in AML KIT D816V in 50% of patients with t(8;21) or inv(16)(p13q22) TKI inhibitor dasatinib, nilotinib or multikinase inhibitor sorafenib in combination with conventional chemo

20 Favorable Abnormal Karyotype t(15;17); t(8;21); inv(16) Intermediate +8 only t(9;11) MILLT3-MLL only Unfavorable Complex karyotype -5, 5q-, 7, 7q, rea11q23, inv(3), 17p-, t(9;22), t(6;9) Prognosis of AML t(8;21) + c-kit [1/3 of t(8;21)] Normal Karyotype Favorable NPM1 mut CEBPA mut 2 Intermediate FLT3 + NPM1 Unfavorable FTL3 MLL partial tandem dup

21 Genetic Changes in Childhood AML t(8;21) inv(16); t(16) t(15;17) MLL rea -7 MLL ptd FLT3 itd FLT3 mut NRAS/KRAS NPM1 CKIT PTPN11 RUX1 mut CEBPA WT1

22 Childhood B cell precusor ALL 1% 2% 5% t(9;22) BCR-ABL1 t(12;21) ETV6-RUNX1 t(1;19) TCF3-PBX1 21% 21% MLL rea hyperd hypod <44 10% 2% 5% 21% 2% Szczepanski et al: Lancet Oncol % 5% iamp21 rea IGH &CEBP IKZF1 del/mut CDKN2A del JAK2 mut IGH-CRLF2, del centromeric part of CRLF2

23 Genetic changes Childhood B-cell Precursor ALL Inciden ce Prognosis t(9;22)(q34;q11.1) 2 3% Unfavorable, EFS <30% t(12;21)(p13;q22) 20 25% Favorable t(1;19)(q23;p13.3) 5 6% Favorable, intensified Rx 5-Y EFS 84% MLL rea 5 8% poor /infants; t(4;11) poor in all age; others not conclusive Hyperdipl (51-65) 20 30% Favorable, + 4,+10,+17 Hypodipl ( 44) 5 6% Unfavorable: 5-Y EFS <40% iamp21 2 3% Unfavorable

24 FISH Panel for PCM (MM) IGH break apart CND1-IGH, t(11:14)(q32;q13) FGFR3-IGH, t(4;14) IGH-MAF, t(14;16) TP53 (17p13), CEP17 D13S319(13q14),13q34 CEP3, CEP7, CEP9, CEP15

25 Lymphomas with specific gene rearrangement Disease Chromosome Gene % FL t(14;18) IGH/BCL2, 80-90% DLBCL t(3q27;v) t(14;18); t(8;14) BCL6/>25 genes IGH/BCL2 MYC Burkitt t(8;14) variants MYC/IGH, MYC 30% 40% (GCB not ABC) 5-10% 100% Mantle t(11;14) CCND1/IGH >95% MALT t(11;18); variant BIRC3/MALT1, MAL1 26% Anaplastic large cell t(2;5), variants ALK 92% (children); 72% in adults

26 B cell lymphoma, unclassifiable, with features of intermediate between DLBCL and BL- The Gray Zone FL BCL2, BCL6, other MYC +, double -hit BL IG-MYC Atypical morphology Atypical immunolphenotype MYC-complex (non-ig/myc, BCL2/BCL6 &MYC (double hit) Intermediate lymphomas Double-hit MCL, other t(11;14) ; t(14;19) DLBCL BCL2, BCL6, other Atypical morphology Atypical immunophenotype MYC +, double- hit

27 Lung cancer Small cell lung cancer 20% Non small cell lung cancer 80% Adenocarcinoma 40% Squamous cell carcinoma 30% Large cell carcinoma 10% >1 million deaths/year worldwide. Is there an Archilles Heel?

28 EGFR EGFR as the target of NSCLN EGFR mutation in 10% of Caucasian and 30% Asian patients. >60% of EGFR mutant tumors respond to EGFR TKI, OS m FDA approval of erlotinib (2003), getifinib (2004), afatinib (2013) EGFR mutation detection for selection of EGFR-TKI

29 Sharma et al. Nature Reviews Cancer 7, , 2007

30

31 ALK fusion and ALK inhibitor ALK 2~4% patients have fusion of ALK with EML4 or other partner genes ALK fusion activates KRAS; Patients with ALK fusion respond to ALK inhibitor FDA approved crizotinib (Xalkori) and Vysis ALK Break-Apart FISH Probe Kit (Abbott), 2011

32 CAP, IASLC, AMP Guideline June, Testing EGFR +ALK on all NSCLC containing an adenocarcinoma component. 2. Specimens be fixed in 10% neutral buffered formalin for 6-48 hs and sent to lab within 3 days. 3. EGFR /ALK results be reported within 10 days. 4. KRAS not be tested as a sole determinant of EFGR TKIs. 5. Testing for other biomarkers not indicated.

33 FDA approved EGFR mutation detection kits/devices May 14, 2013 Cobas EGFR Mutation Test (Roche) Erlotinib treatment of metastatic NSCLC with exon 19 deletion or exon 21 (L858R) July 12, 2013 Therascreen EGFR RGQ PCR kit on Rotor- Gene Q MDx (Qiagen) Afatinib for NSCLC with exon 19 deletion or L858R

34 The frame of standard of care EGFR & ALK for lung cancer with adenocarcinoma component EGFR+ ALK FISH + EGFR-/ALK- EGFR TKI ALKI Chemo or other

35 Management of advanced EGFR-mutant NSCLC Cadranel et al: Crit Rev OncolHematol 2013

36 Resistance to EGFR TKI 10~30% primary resistance 50% of initial responders develop secondary resistance within 9-12 months. Mechanisms of resistance: Clonal selection of resistance mutation (T790M) Amplification of other growth factor receptors (MET, HER2, HER3) Alteration in downstream pathways (PI3K, PTEN) Transition to small cell cancer

37 The driver mutations 2013 KRAS EGFR ALK fusion ROS1 fusion BRAF V600E BRAF non-v600e PIK3CA HER2 ampl HER2 mut AKT MAP2K1 MET ampl RET fusion Unknown Pao & Girard: Lancet Oncol 2011

38 Targeted or potentially targetable therapies for lung adenocarcinoma (base on AJRCCM article in press July 2013) Target Detection (%) Therapies EGFR Erlotinib***, Gefitinib***, Afatinib*** ALK, ROS1, MET 3-5 Crizotinib*** BRAF V600E 2-4 Vemurafenib**, Dabrafenib**, Trametinib**, dasatinib** HER2 ins 2 Neratinib**, Dacomitinib** RET fusion 1-2 Cabozatinib**, Vandetanib**, Sorafenib**, Sunitinib** KRAS mut 20 Selumetinib* *** Approved for lung cancer **Approved for other cancers or * investigational agent on clinical trials

39 Paris arrows for individualized Archilles heel

40 Patients response to targeted therapies EGFR ALK ROS1 MET amp KRAS BRAF V600E BRAF non-v600e HER2 Ampl HER2 Mut RET fusion MAP2K1 AKT PIK3CA other/ unknown

41 Companion tests available for lung adenocarinoma in UM Pathology Mutation by PCR/sequencing KRAS 20% EGFR 10~15% BRAF v600e 1~2% PI3KCA * 1~2% HER2 1~2% Testing in-house Yes Yes Yes Yes No No Fusion/amplification detected by FISH ALK fusion 2~4% ROS1 fusion 1~2% RET Fusion 1% HER2 Amp 1-2% Testing in house Yes Yes No Yes No MAP2K1 (MEK1) <1% MET Amp <1% * Offered for CRC recently

42 Searching for driver mutations by sequencing 188 lung adenocarcinomas 623 cancer related genes 1013 nonsynonymous somatic mutations L Ding et al. Nature 455, (2008)

43 Significantly mutated pathways in lung adenocarcinomas. L Ding et al. Nature 455, (2008)

44 The spectrum of somatic mutations in a lung adenocarcinoma (Lee et al Nature 2010) A. Structural changes B. LOH (Affy SNP 6.0) C. CNVs (Agilent 244K) D. SNVs >50,000 somatic SNVs (530 validated: 1 KRAS; 391 others in coding regions)

45 Targetable or potentially targetable Genetic changes in squamous carcinoma of the lung FGFR1 amp FGFR2/3 FGFR fusion PIK3CA PDGFRA amp EGFR amp DDR2 BRAF HER2 amp unknown

46 Anti-EGFR treatment for CRC

47 PIK3CA mutation and aspirin use in CRC PIK3CA mutation in 15-20% of CRC; resistant to anti-egfr therapies Aspirin down regulates PI3K signaling activity Regular use of aspirin among patients with PIK3CA mutant CRC associated with a 46% reduction in overall mortality and an 82% reduction in CRC specific mortality: 2/26 (3%) with aspirin VS 13/90 (26%) without died within 5 ys P<0.001) Liao et al: N ENG J Med 2013; 367:1596

48 Genetic markers Companion tests in UM Pathology Year started KRAS % 35% NSCLC CRC Breast Gastric Melanoma BRAF ~2% 5~10% 60% EGFR % PIK3CA ~2% 15~20% HER2 mu 1~2% ALK ~7% HER ~2% 20~30% 20% ROS ~2% RET 1% Green: PCR/sequencing; Red: FISH; %: reported mutation detection rate

49 Growing companion testing in UM Pathology PCR/seq FISH

50 KRAS mutation analysis Exon 2 codon12 GGT>AGT, CGT, TGT, GTT(Gly12Val), GAT, GCT Exon 2 codon13 GGC>CGC, TGC, GAC; GCC, GTC

51 INFINITY test work flow DNA extraction Analysis & Report Multiplex PCR Scan Detection primer extension Target DNA Hybridization to oligonucleotides on chip Wash

52 PyroMark Q24 A Small Smart Affordable Pyrosequencing System with a 24 plate format A Complete solution for Mutation and Methylation Analysis CpG methylation Allele quantification Mutation analysis 10/1/2013

53 Next generation sequencing for cancer treatment

54 Challenges Identification of the driver genetic change tumor is the prerequisite for targeted cancer treatment. Each tumor is different and only a few have the same driver genetic change (Achilles heel) as the therapeutic target. Difficulties in evaluation of the efficacy of new agents and the best combinations due to small number of patients with the same target.

55 Challenges for diagnostic labs Labor intensive when multiple markers tested spontaneously (current approach) Long turn around time when multiple markers tested sequentially (current approach) High throughput tech versus low volume when multiple markers tested by next-generation sequencing in a hospital lab High cost versus low reimbursement in general for labs although great savings for the society

56 SECTION TITLE HEADER 14PT HELVETICA BOLD Thank you! 4