I have no conflicts of interest

Transcription

1 Deploying lung cancer molecular pathology guidelines in real life Neal Lindeman, MD Director, Molecular Pathology Brigham & Women s Hospital USCAP Annual Meeting AMP Companion Meeting San Antonio, T Sunday, March 5, 2017 Disclosure of Relevant Financial Relationships USCAP requires that all planners (Education Committee) in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. I have no conflicts of interest 2013 Lung Cancer Testing Guidelines Why? Questions still raised regarding who, what, and how to test? Which patients to test: clinical selection? histologic selection? Which samples to test: cytology? Fixation? Small samples? Which genes to test: EGFR, ALK, KRAS? What methods to use: PCR, Sanger, FISH, IHC, RT-PCR, NGS? How to operationalize: TAT, validation, QA? Evidence had been accumulated to answer most of these Who? AMP, CAP, and IASLC each sent a co-chair, a steerer, and three experts Strict COI policy When? This took 2.5 years: Dec, 2010 Apr, 2013 But there s always hope 2013 Guidelines: Who? Who & When to test? Clinical determinants: Giaccone, Kwiatkowski Pathology determinants: Cagle, Thunnissen How to test? Sample selection, handling and processing General: Beasley, Chitale Molecular: Lindeman, Saldivar, Dacic FISH: Squire, Jenkins EGFR testing Molecular: Lindeman, Ladanyi IHC, FISH: Dacic, Ladanyi Resistance testing: Ladanyi, Kwiatkowski ALK testing FISH: Squire, Jenkins IHC: Cagle, Beasley RT-PCR: Ladanyi Other genes: Lindeman, Ladanyi, Giaccone Logistics Turnaround time: Thunnissen, Saldivar, Giaccone Testing algorithms: Thunnissen, Chitale, Saldivar Reporting: Lindeman, Kwiatkowski, Cagle, Jenkins QA, Validation: Thunnissen, Squire, Saldivar Enough about the process: What were the recommendations? 1.1: Clinical characteristics (e.g., age, gender, ethnicity, smoking history) are not sufficiently sensitive to be used to select patients for EGFR or ALK testing. 1.2: EGFR and ALK testing should be performed on all NSCLC that contain an adenocarcinoma component, regardless of histologic grade or histologic subtype. 1.3: EGFR and ALK Testing are not recommended for pure squamous cell carcinomas, pure small cell carcinomas, or pure neuroendocrine carcinomas. 1.4: Small biopsies or incomplete excisions showing only squamous or small cell histology may be tested for EGFR and ALK mutations 1.5: Poorly differentiated tumors or tumors that otherwise cannot be classified as pure squamous, pure small cell, or pure neuroendocrine carcinomas should be tested for EGFR and ALK mutations. 1.6: Non-small cell lung carcinoma (NSCLC) is no longer acceptable as a pathologic diagnosis or as an operational category for clinical management. 1.7: In the absence of interceding therapy with a targeted inhibitor, primary tumors and metastatic lesions are equally suitable for testing 1.8: Both tumors should be tested in patients with apparently separate primary cancers 1.9: Testing multiple different areas within a tumor is not recommended 2.1: Patients with stage III or stage IV disease should be tested at the time of diagnosis. 2.2: The decision whether to test patients with stage I or II disease for EGFR/ALK should be made locally by each institution, in collaboration with its oncology care team. 2.3: Tissue must be allocated for EGFR and ALK testing in all patient samples. 3.1: EGFR and ALK results should be available within two weeks (10 working days) of receiving the sample in the molecular diagnostics laboratory. 3.2: Laboratories with typical turnaround times beyond two weeks need to make available a more rapid test either in-house or by send-out in instances of clinical urgency. 3.3: Samples should be sent to the molecular pathology laboratory within 3 working days of receiving requests 4.1: Formalin-fixed, paraffin-embedded samples are adequate for PCR-based EGFR mutation tests, as are fresh, frozen, or alcohol-fixed samples. 4.2: Other tissue treatments (e.g., acidic or heavy metal fixatives, mordants, or decalcifying solutions) may hamper EGFR testing, and should be mentioned in the surgical pathology report. 4.3: Specimens should be fixed in 10% neutral buffered formalin for between 6-48 hrs. Prolonged fixation should be noted in the surgical pathology report. 4.4: Cell blocks are preferred over smear preparations for the analysis of cytology specimens. 5.1: Adequacy for EGFR testing is not determined by sample type, but rather by malignant cell content and DNA quality. 5.2: Each lab must establish during validation, and perform quality control in production, the minimum amount and concentration of cancer cells needed for precise mutation detection 5.3: In the absence of proven disomy, more than one sample should be used to determine test sensitivity during validation. 5.4: Tumor content of each sample must be assessed by a pathologist; this should be included in test validation. 5.5: Cancer cell enrichment may be performed on heterogeneous samples by a pathologist or trained technologist under a pathologist s guidance 5.6: Manual dissection of unstained slides is recommended for cancer cell enrichment 6.1: Any validated method with sufficient performance characteristics may be performed. 6.2: EGFR tests must be able to detect mutations in samples with at least 50% cancer cell content, with absolute precision. 6.3: Laboratories with tests based on unmodified Sanger sequencing only are strongly encouraged to employ a more sensitive method, or must make available sendout to a more sensitive method for patients 6.4: Specificity of positive results must be confirmed, particularly for highly sensitive methods 6.5: Methodology should permit reporting of results within the turnaround time goals established within each laboratory s institution and within the two week (10 working days) recommendation set forth in this guideline. 6.6: EGFR mutation testing should capture all individual mutations reported in at least 1% of EGFR-mutant lung adenocarcinomas. 6.7: EGFR Immunohistochemistry is not recommended for selection of EGFR TKI therapy 6.8: EGFR copy number analysis (i.e., FISH or CISH) is NOT recommended for selection of EGFR TKI therapy. 7.1: KRAS mutation testing is not recommended as a sole determinant of anti-egfr therapy 8.1: EGFR tests in patients with acquired resistance should be able to detect T790M mutation in as few as 5% of cells. 8.2: Screening for other secondary EGFR mutations is not recommended for the routine analysis of samples with acquired resistance. 8.3: MET copy number analysis is not recommended for the routine analysis of samples with acquired resistance. 9.1: ALK testing should be performed in the same patients and tumor types that are candidates for EGFR testing 9.2: Tumor architecture and cytology, as assessed by a pathologist, are more critical than tumor percentage as determinants of adequacy for ALK testing 9.3: Formalin-fixed, paraffin-embedded samples are adequate for ALK FISH testing. 9.4: FISH should be performed with split-apart, dual-labeled ALK probes. 9.5: ALK FISH requires the same slide pretreatment and hybridization procedures as other FISH tests performed on similar types of samples. 9.6: ALK FISH slides should be interpreted by two independent scorers who have specialized training in solid tumor FISH analysis, with guidance from a pathologist with training or experience in FISH. 9.7: ALK FISH should be interpreted in areas with clearly interpretable hybridization signals in the majority of cells 9.8: For split-apart ALK FISH assays, narrowly split signals can be seen in the absence of ALK rearrangement, and therefore interpretive criteria regarding the magnitude of signal splitting in positive samples must be established in validation. 9.9: Other abnormal FISH findings of uncertain significance should be reported as such. 9.10: Evidence of rearrangement in >15% of 50 analyzed cells should be interpreted as evidence of an ALK-positive tumor. 9.11: Each case should be reviewed by two independent observers and confirmed by a cytogeneticist or pathologist with training in FISH. 9.12: Quality control for ALK FISH should be performed similarly to quality control for other solid tumor FISH assays. 10.1: Immunohistochemistry is not currently recommended as an alternative to FISH for selecting patients for ALK inhibitor therapy 10.2: RT-PCR is not recommended as an alternative to FISH for selecting patients for ALK inhibitor therapy 10.3: Mutation testing for acquired resistance to ALK inhibitors is not currently recommended for clinical management 11.1: Testing for molecular biomarkers other than EGFR and ALK is not currently indicated for clinical management in lung adenocarcinoma 12.1: Sequential testing algorithms may be implemented to enhance the efficiency of molecular testing of lung adenocarcinomas, provided the overall turnaround time requirements are met. 13.1: The reports of EGFR mutation testing should contain the following elements: 13.2: The ALK Rearrangement FISH report should include the following elements: 14.1: EGFR and ALK test validation should follow the same guidelines as for other molecular diagnostics and FISH tests. 14.2: Validation samples should include all types of sample processing that will be applied to clinical samples, but validation of different tissues of origin that are processed identically is not necessary. 14.3: All relevant mutations or rearrangements that are to be reported should be included in the validation set, to whatever extent is possible. 14.4: The entire analytic process should be assessed for precision 14.5: Comparison of results obtained in another accredited laboratory performing a properly validated assay is required. 14.6: Analytical sensitivity, both the number of tumor cells required as well as the percentage of tumor cells required, should be established in more than one validation sample. 14.7: Analytical specificity studies should establish criteria for distinguishing between true positive and false positive results. 15.1: EGFR and ALK testing in lung cancers is subject to the same policies and procedures for quality assurance and quality improvement as other clinical laboratory assays. 15.2: External proficiency testing is a required element of quality assurance 1

2 CI Mutant No Mutant/ Wild typ High Low High Low I Mutant No Mutant/ Wild typ Mutant No Mutant/ Wild typ 3/27/2017 Did you get all that? 2013 Guideline: Major Points Who to test: Advanced stage lung cancers with an adenocarcinoma component Regardless of age, gender, smoking history, ethnicity Early stage testing is an institutional policy decision EGFR What to test: KRAS EGFR and ALK ALK Optimal sample according to quantity and quality of cancer DNA Cytology samples (cell block preferred) or tissue samples Fresh, frozen, or fixed (avoid acids and heavy metals) Primary or metastasis Each of multiple primaries if histologically distinct A summary of the salient points 2013 Guideline: Major Points How to test: EGFR by Molecular Diagnostics, ALK by FISH Platform selected by performance characteristics, not technology Sensitivity > 50% malignant cell content Must make available more sensitive methods (>10%) All testing completed within 10 working days Mol Dx FISH IHC Lung Guidelines: Real world challenges Selecting patients Communicating orders Selecting samples Limited samples Sensitivity for EGFR mutations Scope of genetic alterations tested Turnaround time IHC vs FISH for ALK Selecting Patients: who to test No-brainer: stage IV adeno, Rx candidate What about? Early stage adeno Squamous cell Miscellaneous/ambiguous NSCLC Small cell Smokers? Selecting Patients Clinical criteria: inadequate predictors AGE GENDER TOBACCO ETHNICITY I Asia <65 F No West >65 M Yes Afr Mutant No Mutant/ Wild typ Mutant No Mutant/ Wild typ 2

3 Selecting Patients Histology Test any tumor with lung adenocarcinoma May be mixed (adenosq, adeno/small cell) NO pure squamous, small cell,neuroendocrine Except maybe incomplete small biopsies If clinically high risk, and requested by oncologist Poorly differentiated tumors are tested Stage Generally, advanced stage patients Communicating orders Med Onc Radiologist Surgeon Cytopathologist Surgical Pathologist Molecular Pathologist Order communication Challenges: Paperwork/Communication 12/5/12 Custom IS solution needed: LIMS did not provide needed function Courtesy of Frank Kuo Communicating orders Med Onc Challenges: Paperwork/Communication Radiologist Surgeon 12/5/12 LIMS Cytopathologist Surgical Pathologist 12/15/13 Molecular Pathologist Courtesy of Frank Kuo 3

4 Selecting Samples Pathologist is needed! Oncologist cannot tell from reports Surgeons cannot tell from procedure Radiologists cannot tell from imaging Selecting Samples Quality and quantity are key determinants A cellular FNA is better than a necrotic resection Primary vs. metastasis Quality is determinant, tempered by interval therapy If metastasis after initial TKI response, then test metastasis Multiple primaries If histologies differ, then test BOTH/ALL Patients will benefit even if 1 of multiple tumors responds Testing multiple areas in a tumor is unnecessary Fixation: the weekend problem The weekend problem A satellite hospital noticed that tests often failed on samples obtained on Fridays All other days no problem Started scheduling procedures for earlier in the week Unbuffered formalin oxidizes to formic acid Acid hydrolyzes DNA Samples in acid fail PCR Limited samples 5 x 5 micron sections 80% fail rate Buffered Formalin Solution Friday samples worked 20% fail rate Headache gone A cellular cytology specimen is as good or better than a small biopsy. In a pinch, even a single, stained slide can suffice 4

5 Limited Samples Challenges: Limited samples EGFR mutation? EGFR, KRAS, HER2, BRAF, PIK3CA (and 35 more genes) mutations Oncomap, 8/11-12/12 Requires 250 ng, 30% tumor 156 4% 100 ng DNA needed Sensitive to 50% tumor 200ng DNA needed Sensitive to 30% tumor % % % withdrawn insufficient OSH into lab Oncopanel, 9/13-4/14 Requires 50 ng, 20% tumor Heterogenous samples: 30% tumor Ultrasensitive EGFR for resistance: Cell-free circulating DNA Liquid biopsy UNMODIFIED PNA-enriched Flow cytometry distinguishes mutant from normal DNA in blood samples Can detect very low amounts of tumor DNA Agrees with biopsy few false positives, but some false negatives WHEN should results be available? TKI therapy no longer empirically started without evidence of a sensitizing mutation TAT for delivery of blocks/unstained slides: In-house: 24 hours Outside: 3 working days TAT once sample is received within the lab Goal: 10 working days Slower labs: make a faster method available when needed BWH experience: ALK FISH launch: Oncologists requested it on pretty much everyone Rapidly developed major backlog 4-6 week TAT Introduced sequential testing, including IHC screening step LUNG PANEL (Sequential EGFR, KRAS, ALK, and ROS1; testing stops after first positive result) IHC results available in 2-3 days Prompt FISH if positive Eliminated 30-40% of ALK FISH volume EGFR and KRAS mutated tumors culled out 5

6 Testing algorithms 8 USS to IHC lab Lung panel requested 1 HE and 18 USS ordered Critical to understand TAT needs Testing should be completed within 10 days ALK IHC If POSITIVE: Immediately initiate FISH HE forwarded to pathologist for tumor adequacy review HE + 10 USS to lab for dissection/dna extraction Rapid EGFR analysis Exons 19, 21 EGFR ALK if neg Continue molecular testing EGFR screen first (i.e., melt curve) Reflex to ALK or EGFR confirmation KRAS EGFR if neg ALK if neg ROS IHC RET IHC MET IHC ROS FISH RET FISH RET FISH If POSITIVE: Report Results. Testing Done KRAS pyrosequencing Sanger Sequencing: EGFR KRAS BRAF ERBB2 MET NRAS Lung Cancer Algorithm 8 USS to IHC lab Lung panel requested 1 HE and 18 USS ordered HE forwarded to pathologist for tumor adequacy review ALK IHC HE + 10 USS to lab for dissection/dna extraction If POSITIVE: Immediately initiate FISH Rapid EGFR analysis Exons 19, 21 Continue molecular testing ROS IHC ROS FISH KRAS pyrosequencing RET IHC RET FISH If POSITIVE: Report Results. Testing Done MET IHC RET FISH Sanger Sequencing: EGFR KRAS BRAF ERBB2 MET NRAS Multiplex panels Scope of genetic alterations: Evidence regression Query multiple variants at once Reduced DNA requirements Acceptable TAT Reduced administrative burden DFCI/BWH Oncomap 470 mutations in 41 genes EGFR: multiple Phase III RCTs, PFS ALK: Phase II, ORR RET: case reports NTRK1: mice MEK1: cell lines 6

7 ARHGAP35 CDC73 ERBB3 GATA4 KRAS NK2 1 PTK2B SF3B1 TSC1 ARHGEF12 CDH1 ERBB4 GATA6 LIG4 NK3 1 PTPN11 SH2B3 TSC2 3/27/2017 Oncopanel Gene List (POPv1) Exons: ABL1, AKT1, AKT2, AKT3, ALK, ALO12B, APC, AR, ARAF, ARID1A, ARID1B, ARID2, ASL1, ATM, ATR, AURKA, AURKB, AL, B2M, BAP1, BCL2, BCL2L1, BCL2L12, BCL6, BCOR, BCORL1, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BUB1B, CARD11, CBL, CBLB, CCND1, CCND2, CCND3, CCNE1, CD274, CD58, CD79B, CDC73, CDH1, CDK1, CDK2, CDK4, CDK5, CDK6, CDK9, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK2, CIITA, CREBBP, CRKL, CRLF2, CRTC1, CRTC2, CTNNB1, CU1, CYLD, DDB2, DDR2, DICER1, DIS3, DMD, DNMT3A, EGFR, EP300, EPHA3, EPHA5, EPHA7, ERBB2, ERBB3, ERBB4, ERCC2, ERCC3, ERCC4, ERCC5, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, ET1, ET2, EZH2, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBW7, FGFR1, FGFR2, FGFR3, FGFR4, FH, FKBP9, FLCN, FLT1, FLT3, FLT4, GATA3, GATA4, GATA6, GLI1, GLI2, GLI3, GNA11, GNAQ, GNAS, GPC3, GSTM5, H3F3A, HNF1A, HRAS, ID3, IDH1, IDH2, IGF1R, IKZF1, IKZF3, JAK2, JAK3, KDM6A, KDM6B, KDR, KIT, KRAS, LMO1, LMO2, LMO3, MAP2K1, MAP2K4, MAP3K1, MAPK1, MCL1, MDM2, MDM4, MECOM, MEF2B, MEN1, MET, MITF, MLH1, MLL, MLL2, MPL, MSH2, MSH6, MTOR, MUTYH, MYB, MYBL1, MYC, MYCL1, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKBIA, NFKBIZ, NK2-1, NOTCH1, NOTCH2, NPM1, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PARK2, PA5, PDCD1LG2, PDGFRA, PDGFRB, PHF6, PHO2B, PIK3C2B, PIK3CA, PIK3R1, PIM1, PMS1, PMS2, PNRC1, PRAME, PRDM1, PRF1, PRKAR1A, PRKCI, PRKCZ, PRKDC, PRPF40B, PRPF8, PSMD13, PTCH1, PTEN, PTK2, PTPN11, RAD21, RAF1, RARA, RB1, RBL2, REL, RET, RFWD2, RHPN2, ROS1, RPL26, RUN1, SBDS, SDHAF2, SDHB, SDHC, SDHD, SETBP1, SETD2, SF1, SF3B1, SH2B3, SMAD2, SMAD4, SMARCA4, SMARCB1, SMC1A, SMC3, SMO, SOCS1, SO2, SO9, SRC, SRSF2, STAG1, STAG2, STAT3, STAT6, STK11, SUFU, SUZ12, SYK, TCF3, TCF7L1, TCF7L2, TERT, TET2, TNFAIP3, TP53, TSC1, TSC2, U2AF1, VHL, WRN, WT1, PA, PC, PO1, ZNF217, ZNF708, ZRSR2. Introns: ABL1, AKT3, ALK, BCL2, BCL6, BRAF, CIITA, EGFR, ETV1, EWSR1, FGFR1, FGFR3, FUS, IGH@, IGK@, IGL@, JAK2, MLL, MYC, NPM1, PA5, PDGFRA, PDGFRB, RAF1, RARA, RET, ROS1, TRA@, TRB@, TRG@. New to POPv2 SNV/CNV TR BCL2L1 LINC00894 PTPRD ERG CADM2 LMO2 QKI FGFR2 CDKN1A LMO3 RECQL4 NTRK1 CDKN1B MAP2K4 RHEB SS18 CDKN1C MAP3K1 SDHA PPARG CSF1R MAPK1 SETD2 TMPRSS2 CSF3R NEGR1 SLITRK6 DEPDC5 NPRL2 SO9 GNB2L1 NPRL3 SQSTM1 IDH2 NTRK1 STAT6 INSIG1 NTRK2 TCF7L1 KDM5C NTRK3 TCF7L2 KDM6A PBRM1 TLR4 KEAP1 PRKCZ KCNIP1 ZNF217 POP v3: 447 genes ABCB11 CBFA2T3 DNMT3A FLCN KDM6A NBN PPM1D RUN1T1 TL3 ABL1 CBFB DOCK8 FLT1 KDR NEIL1 PPP2R1A SBDS TMEM127 ACVR1 CBL EGFR FLT3 KEAP1 NEIL2 PRDM1 SDHA TMPRSS2 AKT1 CBLB EGLN1 FLT4 KIF1B NEIL3 PRF1 SDHAF2 TNFAIP3 AKT2 CCND1 ELANE FOA1 KIT NF1 PRKAR1A SDHB TOPBP1 AKT3 CCND2 EME1 FOL2 KLF2 NF2 PRKCI SDHC TP53 ALK CCND3 ENG FUS KLF4 NFE2L2 PRKDC SDHD TP53BP1 APC CCNE1 EP300 GALNT12 KLLN NFKBIA PRSS1 SERPINA1 TRAF3 AR CD274 EPCAM GATA2 KMT2A NFKBIE PTCH1 SETBP1 TRAF7 ARAF CD79B ERBB2 GATA3 KMT2D NFKBIZ PTEN SETD2 TRIM37 Scope of genetic alterations: Evidence regression EGFR: multiple Phase III RCTs, PFS ARID1A CDH4 ERCC1 GBA LMO1 NOTCH1 PTPN14 SH2D1A TSHR ARID1B CDK12 ERCC2 GEN1 LMO2 NOTCH2 PVRL4 SLC25A13 U2AF1 ARID2 CDK4 ERCC3 GLI1 MAF NOTCH3 QKI SLC34A2 UBE2T ASL1 CDK6 ERCC4 GLI2 MAFB NPM1 RAC1 SL1A UIMC1 ATM CDK8 ERCC5 GNA11 MAP2K1 NR0B1 RAD21 SL1B UROD ATR CDKN1A ERCC6 GNAQ MAP2K2 NRAS RAD50 SL4 USP28 ATR CDKN1B ERG GNAS MAP2K4 NRG1 RAD51 SMAD2 USP8 AURKA CDKN1C ESR1 GPC3 MAP3K1 NSD1 RAD51C SMAD4 VEGFA AURKB CDKN2A ETV1 GREM1 MAPK1 NT5C2 RAD51D SMARCA4 VHL AIN2 CDKN2B ETV4 H19 MA NTHL1 RAD52 SMARCB1 WAS AL CDKN2C ETV5 H3F3A MBD4 NTRK1 RAD54B SMARCE1 WHSC1 B2M CEBPA ETV6 H3F3B MCL1 NTRK2 RAF1 SMC3 WHSC1L1 BABAM1 CHEK1 EWSR1 HABP2 MCM8 NTRK3 RARA SMO WRN BAP1 CHEK2 EO1 HELQ MDM2 OGG1 RASA1 SOCS1 WT1 BARD1 CIC ET1 HFE MDM4 PALB2 RB1 SOS1 PA BCL11B CIITA ET2 HIST1H3B MECOM PARK2 RBBP8 SO2 PC BCL2 COL7A1 EZH2 HIST1H3C MED12 PA5 RBM10 SO9 PO1 BCL2L1 CREBBP FAH HMBS MEF2B PAIP1 RECQL4 SPOP RCC1 BCL2L12 CRKL FAM175A HNF1A MEN1 PBRM1 REL SRSF2 RCC2 BCL6 CRLF2 FAM46C HOB13 MET PDCD1LG2 RELA SRY RCC3 BCOR CRTC1 FAN1 HRAS MGA PDGFRA RET SS18 RCC4 BCORL1 CSF3R FANCA ID3 MITF PDGFRB RHBDF2 STAG2 RCC5 BLM CTCF FANCB ID4 MLH1 PHF6 RHEB STAT3 RCC6 BMPR1A CTLA4 FANCC IDH1 MLH3 PHO2B RHOA STAT6 YAP1 BRAF CTNNA1 FANCD2 IDH2 MPL PIK3C2B RHOH STK11 ZNF217 BRCA1 CTNNB1 FANCE IGF1R MRE11A PIK3CA RHOT1 SUFU ZNRF3 BRCA2 CU1 FANCF IGF2 MSH2 PIK3R1 RICTOR SUZ12 ZRSR2 BRCC3 CCR4 FANCG IKZF1 MSH6 PIM1 RIF1 TAL1 BRD3 CYLD FANCI IL7R MTA1 PML RINT1 TAL2 BRD4 DA FANCL ITK MTAP PMS1 RIT1 TAZ BRE DCLRE1C FANCM JAK1 MTOR PMS2 RMRP TCEB1 BRIP1 DDB1 FAS JAK2 MUS81 PNKP RNF43 TCF3 BUB1B DDB2 FAT1 JAK3 MUTYH POLB RNF8 TCF7L2 C17ORF70 DDR2 FBW7 JAZF1 MYB POLD1 ROS1 TDG C19ORF40 DICER1 FGFR1 KAT6A MYBL1 POLE RPA1 TERC C1ORF86 DIS3 FGFR2 KAT6B MYC POLH RPTOR TERT CALR DIS3L2 FGFR3 KCNQ1 MYCL1 POLQ RSPO2 TET1 CARD11 DKC1 FGFR4 KDM5A MYCN POT1 RSPO3 TET2 CASP8 DMC1 FH KDM5C MYD88 PPARG RUN1 TFE3 ALK: Phase II, ORR RET: case reports NTRK1: mice MEK1: cell lines Oncopanel interpretation Interpreting ~450 genes in a cancer sample 1999: PhD thesis 2013: 3 hours 2016: 5 minutes 7

8 Turnaround Time TAT in Anatomic Pathology Develop core biopsy protocols: Indicate when molecular testing needed on the req BRIGHT PINK STICKERS! Cut unstained slides up front to avoid refacing block 1 H&E 18 USS 1 H&E Minimize surg path workup as needed Accurate triaging Too many unnecessary orders will sink histology TAT: system improvement Sept, 2011 Jan, 2012 How should ALK be tested? 2013: ALK is a FISH test Reliable IHC antibodies not currently available RT-PCR not recommended Molecular and chromosomal variants Mutation testing in resistance: not yet 8

9 2016: Is immunohistochemistry reliable for screening for ALK translocations? 2016: Is immunohistochemistry reliable for screening for ALK translocations? YES Wynes, et al., JTO, 2014 How to test for ALK? FISH is not perfect Neither is IHC Concordance: Either method can be performed Cabillic, JTO, 2014 FDA Approval VENTANA ALK (D5F3) CDx Assay is intended for the qualitative detection of the ALK protein in FFPE NSCLC tissue stained with a BenchMark T automated staining instrument It is indicated as an aid in identifying patients eligible for treatment with ALKORI (crizotinib) We are not recommending both 3/27/2017 Lung Guidelines: Real world challenges Selecting patients nonsquamous NSCLC Communicating orders custom IS Selecting samples pathologist job Limited samples NGS Sensitivity for EGFR NGS, ddpcr Scope of genes tested liberal standards Turnaround time never good enough IHC vs FISH for ALK IHC works well Q&A 9