Molecular Pathology –You happens next?...epithelioid sarcoma ini loss (smarcb1) extrarenal rhabdoid ini loss extraskeletal myxoid chondrosarcoma ewsr1‐nr4a3 rbp56‐nr4a3 tcf12‐nr4a3

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Molecular Pathology – You ordered molecular tests, what happens next?Alain C Borczuk, MD

Professor of Pathology and Vice Chairman, Anatomic Pathology

Weill Cornell Medicine

Goals

• Definitions• Test examples

• Analytes

• Reasons for testing

• Sample types and tissue triage• Maximizing supply

• Determining molecular adequacy

• Examples

• Retesting

Definitions

• Point mutations • Single base change

• Can be activating – Oncogene• “trunk” or driver mutation

• Can be deleterious – tumor suppressor gene

• Deletion or insertions• Inframe – activate a protein ‐ Oncogene

• Frameshift – inactivates – tumor suppressor gene

• Translocation• Larger regions of DNA linking 2 unrelated genes

• Results in fusion RNA and fusion protein

Mutations

• Somatic mutations are changes in DNA that occur in tissues that do not produce germ cells and are not inherited. This is the case in sporadic cancers.

• Germline Mutation is any change from DNA sequence from “normal” or common variants (usually <1% of population) that are inherited. These can be harmful, neutral or beneficial. If beneficial or neutral they may become more common over time.

Mutation versus polymorphismPolymorphisms are common sequence variants in the population. These arose from germline mutations that presumably were neutral or beneficial and expanded through generations.

Common confusion

• Mutations versus polymorphisms• Is it the tumor or just population variation

• Non‐deleterious mutations• What do these mean?

• ALK – translocation versus point mutation

• KRAS mutations and cigarette smoking

• BRAF non—V600E mutations

• MET amplification versus splice site mutation versus polymorphisms• Target analyte matters!

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Large scale• Amplification – increases gene dose

• Deletion – gene loss – small or large, whole chromosome arms or regions in one arm.

• Translocation – exchange of parts of DNA from one chromosome to another or within one chromosome (inversion).

50% tumor

Allele frequency

Heterozygous mutationEGFR mutation in 1 alleleEGFR normal in 1 allele

TOTAL EGFR DNA tumor and normal50% of cells with 1 mutant allele ‐ tumor50% of cells with 1 normal allele ‐ tumor

50% of cells with 2 normal allele – normal

So 25% mutant EGFR allele frequency75% normal EGFR allele freqeuncy

This assumes normal DNA content (no amplifications or deletions)

POINT MUTATIONS

Point mutation detection

• DNA test • Sequencing including next generation sequencing

• Other assays using PCR and fluorescence

• Immunohistochemistry• Mutated protein, mutation specific anibody

Report examples – NGS panel

•

Block No: A3 Specimen Type: Paraffin Embedded Tissue Tumor Type: Adenocarcinoma Primary Site: Lung Tissue Tested: Lung Neoplastic Cell Content: 40% Institution: NYPH

Result: The following variants were detected in the patient's specimen:

Tier 1 There were no variants found in this tier.

Tier 2 Gene Variant: KRAS c.35G>C, p.G12A Type of Variant: SNV COSMIC ID: COSM522 Variant Allele Frequency: 23% Read Depth: 1070

Common confusion




• KRAS mutations, transversions and cigarette smoking (lung only)

• BRAF non—V600E mutations


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TranslocationsIHC, FISH and RNA

Figure 1 Formation and consequences of TMPRSS2–ERG gene fusions

Clark, J. P. and Cooper, C. S. (2009) ETS gene fusions in prostate cancerNat. Rev. Urol. doi:10.1038/nrurol.2009.127

Martelli et al, AJP, 2009

RNA test

• RNA sequencing with analysis for fusions

• Anchored Multiplex PCR (AMPTM)

Summary

• Different molecular alterations drive tumors

• Tests have to match alteration• Ability to detect ‐ sensitive and specific

• Work in diverse sample types

Why did you order the test?

• Diagnosis?• Staging?

• Guidelines for predictive testing (NCCN or CAP/IASLC/AMP)?

• Clinician asked for it?• Clinical trials eligibility?

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Diagnosis

• Use IHC as a surrogate for mutational event

• BAP1 loss in mesothelioma – deletion or mutation

• P53 – nuclear accumulation

• Beta‐catenin – stabilization and nuclear accumulation

• BRAF V600E – mutation specific antibody

• SDHB – genetic association

• ALK – expression of protein as part of translocation

• Histone methylation events – glioma, peripheral nerve sheath tumor

• Molecular test for diagnosis

• EGFR mutation in lung adenocarcinoma

• Translocation test – FISH Breakapart – EWSR1, SYT

• P16 loss –FISH – mesothelial proliferations

Tumor Translocations

Alveolar Rhabdomyosarcoma PAX3‐FOXO1PAX7‐FOXO1PAX3‐AFX1

Alveolar soft part sarcoma ASPSCR1/TFE3

ANEURSYMAL BONE CYST CDH11‐USP6COL1A1‐USP6

ANGIOMATOID FIBROUS HISTIOCYTOMA EWSR1‐ATF1 (CREB1)FUS‐ATF1

CLEAR CELL SARCOMA EWSR1‐ATF1, EWSR1‐CREB1

DESMOID TUMOR CTNNB1 MUTATION OR APC MUTATION

DFSP COLA1‐PDGFB

DSRCT EWSR1‐WT1


ENDOMETRIAL STROMAL SARCOMALOW GRADE

JAZF1‐SUZ12

ENDOMETRIAL STROMAL SARCOMAHIGH GRADE

YWHAE‐FAM22A/B

EPITHELIOD HEMANGIOENDOTHELIOMA WWTR1‐CAMTA1YAP1‐TFE3

EPITHELIOID SARCOMA INI LOSS (SMARCB1)

EXTRARENAL RHABDOID INI LOSS

EXTRASKELETAL MYXOID CHONDROSARCOMA

EWSR1‐ NR4A3RBP56‐NR4A3TCF12‐NR4A3TFG‐NR4A3

EMBRYONAL RHABDOMYO MYOD1 MUTATIONCOMPLEX

EWINGS PNET EWSR1‐FLI1 (ERG/FEV/ETV1/ETV4)FUS‐ERG (FEV)

INFLAMMATORY MYOFIBROBLASTIC ALK TRANSLOCATIONS

INFANTILE FIBROSARCOMA ETV6‐NTRK3


LIPOSARCOMA – WELL DIFF/DEDIFFERENTIATED

12q14 – amplification (MDM2 amp)

LIPOSARCOMA‐MYXOID FUS‐DDIT3EWSR1‐DDIT3

LIPOSARCOMA – PLEOMORPHIC COMPLEX

LOW GRADE FIBROMYXOID SARCOMA FUS‐CREB3L2FUS‐CREB3L1

MPNST UNKNOWN, NF1, CDKN2A, EED SUZ12

MESENCHYMAL CHONDROSARC HEY1‐NCOA2

Nodular fasciitis MYH9‐USP6

PVNS COL6A3‐CSF1

SCLEROSING RHABDOMYOSARC VGLL2‐NCOA2, OTHER NCOA2

SOLITARY FIBROUS TUMOR NAB2‐STAT6

SYNOVIAL Sarcoma SS18‐SSX1, SS18‐SSX2, SS18‐SSX4

STAGING

• Different driver mutations – separate lung primary/ separate primary• Scenario in pancreatic mass and single lung nodule

• Same driver mutation – suggests intrapulmonary metastasis

• New tumor versus recurrence

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BRAF V600EBRAF V600E KRAS Q61H

Predictive testing – per Guidelines or FDA approvals• IHC for mismatch repair proteins

• IHC for PDL1• Mutation tests – point mutations, small insertions/deletions

• DNA sequencing

• Translocation tests• FISH, RNA tests, some DNA tests

• Splice variants – e.g. MET exon 14 loss

• Copy number changes (amplifications)• FISH, sequencing

• Expression tests – e.g. Oncotype breast, mammaprint• Quantitative PCR, microarray

NCCN Guidelines/FDA approvals ‐ Lung

First Line TherapyAfatinibErlotinibGefitinibOsimertinib

Subsequent therapyT790M positiveOsimertinib

T790M negativeAfatinib +cetuximab

EGFR positive ALK positiveFirst lineAlectinibCeritinibCrizotinib

SubsequentAlectinib (if new)BrigatinibCeritinib (if new)

ROS1 positiveCeritinibCrizotinib

BRAF positiveDabrafenib+trametinibDabrafenibVemurafenib

NTRK TranslocationEntrectinib

PDL1 Expression PositiveEGFR/ALK/ROS negFirst line, >50%Pembrolizumab

PDL1 Expression PositiveSecond line or later>1% ‐ PembrolizumabOther drugs – PDL1 test optional

EGFR, BRAF, KRASALK, ROS1, NTRKTranslocationPD‐L1

MET exon 14 skippingRET TRANSLOCAMET AMPLIFIC.

ERBB2 (HER2), PIK3CA, MAP2K1/MEK1NRAS

MOLECULAR TARGETS ‐ LUNG TIERED TARGETS TESTS

Hot spot panelsPCR based NGS“50 gene panel”

IHC

FISH

Check test parametersBy laboratory

Some NGS providers offer larger panels with varied combinations

• EGFR

• KRAS

• BRAF

• HER2

• PIK3CA

• EML4‐ALK

• MAP2K1/MEK1

• ROS

• RET, NTRK

• MET amplification

• MET exon 14 skipping

• NRAS

• PD‐L1

EGFR, BRAF, KRASROS1, NTRKTranslocationALK IHCPD‐L1



MOLECULAR TARGETS ‐ LUNG TIERED TARGETS TESTS


IHC

FISH



• EGFR

• KRAS

• BRAF

• HER2

• PIK3CA

• EML4‐ALK

• MAP2K1/MEK1

• ROS

• RET, NTRK



• NRAS

• PD‐L1

EGFR, BRAF, KRASROS1, NTRKTranslocationALK IHCPD‐L1



MOLECULAR TARGETS ‐ LUNG TIERED TARGETS TESTS SLIDES


IHC

FISH



• EGFR

• KRAS

• BRAF

• HER2

• PIK3CA

• EML4‐ALK

• MAP2K1/MEK1

• ROS

• RET, NTRK



• NRAS

• PD‐L1

10

4‐8

2

?

TOTAL18‐20

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• EGFR

• KRAS

• BRAF

• HER2

• PIK3CA

• EML4‐ALK

• MAP2K1/MEK1

• ROS

• RET, NTRK



• NRAS

• PD‐L1

EGFR, BRAF, KRASALK TRANSROS1, NTRK TranslocationPD‐L1

MET exon 14 skippingRET TRANSLOCATIONMET AMPLIFICATION


MOLECULAR TARGETS – LUNG TIERED TARGETS TESTS SLIDES

Larger NGS testsDNA and RNA

IHC

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TOTAL17

STAGE 4 LUNG cancer – Non small cell, non-squamous

B i o p s y I H C r e s u l t s

F I S H r e s u l t s N G S t e s t s

Day1

Lung cancerNon-squamous non-small celldiagnosis

Day2-3

PDL1 IHC ALK IHC

Immunotherapy

(needs EGFR/ALK result)

EGFR therapy (Need EGFR +result)

Decide ALK therapy (needs ALK result)

? BRAF V600E

Day7-10

ROS1 FISHDecide ROS1 targeting

Day 10-14

50 gene panel

EGFRKRASERBB2BRAF

Day 10-14

Oncomine

EGFRKRASERBB2BRAF

MET ex14MET amplificationNTRK translocationsMEK1 mutationsNRG translocations

EARLY - standard of care and first line decisions LATER– subsequent therapy or clinical trials

EGFR - needed for immuno-oncology decision

14-21days

Whole exome seq

Tumor mutation burden

But does it end here…Clinician asked for it, maybe part of guideline, for clinical trial enrollment• Some PDL1 requests

• Oncotype prostate, colon

• Coloprint, ColDx

• Prolaris, Decipher, Promark

• Tumor mutation burden

• Many mutation tests

Guidelines adherence – Arch Pathol Lab Med 2015

• Reporting of 26 institutions from 2013• Colon, lung, melanoma

• Strict adherence – median of 71% (33‐90%)

• Loose adherence – median of 95% (57‐100%)

• Adequate tissue – median 98% (86‐100%)

• Highest for lung and in institutions with multidisciplinary conference

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How do we get there?

What happens next depends on what happened before!• No decalcification

• Grossing into separate blocks

• Pre‐cut unstained slides

• Cytology – use smears from onsite assessment or place majority of passes into cytolyte or formalin

• Cellblock

• Use cells or DNA in supernatants

Decalcification

• Bad for all molecular tests

• Cytologic preps are excellent

• Most bone core biopsies do not need decal• Separate soft from hard into different cassettes, including clot

How much is enough?

• We understand IHC – 1 slide – 1 test

• FISH testing – one slide, one analyte – second slide for modified protocols if first failure

• Molecular tests (DNA) – depends on test selected and laboratory

• Molecular tests (RNA) ‐ depends on test selected and laboratory

• Tumor mutation burden – more still?

Tissue optimization as economic model

• Expenses/costs = test demand on tissue

• Supply or revenue= specimen type

• Currency • Molecular lab = DNA or RNA

• Pathologist = slide sections for IHC, FISH, DNA or RNA• And do not forget actual diagnosis

• Budget• No deficit spending

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Supply side of budget

• TISSUES• Specimen type

• Size of material

• Utilization

• Stretching supply – eliminate waste

• Liquid biopsy

Sample types

• Cytology• Smears

• Thin prep

• Pellet/Cell block

• Tissue• Small biopsy/core

• Larger samples – wedge, resections

UtilizationMake cell block from fluid in vial

Cut sectionsUse smears

OptimizationIncrease cells in vial

More passesAir or fluid through needle

StandardizationChallenging

Are cells in fluid or clot?How to divide liquid between smears and vial?

SMEARS

REMOVE COVERSLIPSCRAPE OFF CELLS

LOSE RECORD OF SLIDESCANHAVE MULTIPLE SLIDES

Sample types

• Cytology• Smears

• Thin prep

• Pellet/Cell block

• Tissue• Small biopsy/core

• Larger samples – wedge, resections

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DiagnosisIncluding 4 IHC

DiagnosisIHC – 6 slides10 blanks upfront, tissue left

5‐8 unstained

15‐18 unstained

UtilizationLimit H&E sections, cut blanks up frontUse IHC judiciously

OptimizationMore than one blockOne core per block up to 3 blocksBased on H&E, select further useThree cores, total of 3.0 cm of core length

StandardizationTissue processing algorithms

Largest tissue in first blockOne core per block, at most 2 coresUpfront unstained slides, one core per slide

Why not cut all slides up front?

• Not all biopies contain malignancy

• Histotechnology limited resource• e.g. WCMC ‐ >600,000 slides per year are cut

• Target stability on slides vs paraffin block• ?dipping slides in paraffin for storage.

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Foundation one CDX

Guideline adherence

• Gutierrez et al (Clin Lung Cancer)• Examined test rates in 2017

• Found high rate untested

• Insufficient material cited at high percentage (13%)

• Justifies liquid biopsy• Two authors disclose relationship with Guardant

• Morris et al (PLoS One, 2018)• 6.4% rate of insufficient sample

• Low tumor percent

• Common problem

• Authors with relationship to company (Paradigm diagnostics)

Common confusion





• Tissue is inadequate at a high rate• Depends on triage and test choice


Summary

• Need tissue conservation protocols • Small samples and cytology need proper triage

• Still diverse platforms for existing testing

• Liquid biopsy may be only a partial solution

• Under ‐ utilization of molecular testing remains a problem• IHC platforms have better turn around time

• IHC platforms have better adherence

• New tests, more tissue demands

Slides, not DNA or RNA, are my currency

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H&E IHC FISH MutationMulti‐exon

Multigene/NGS EXOME/WES CLINICAL TRIAL

CELLS (MINIMUM) ~ 20 ~20 50 200‐500 500‐25K 20K‐120K ?

SLIDES 1‐2 7‐10 2 10 10‐20 10 10

EBUS/FNA/thin corePaucicellular30 slides YES YES usually

Maybe?sensitivity

False negatives

Unlikely No No

EBUS/FNA/thin coreCellular20‐30 slides YES YES YES YES Possible No

Yes, dependsOn triage

3 cores/3 blocksEach with tumorOne 50% tumor60‐90 slides

YES YES YES YES YES PossibleYes, depends on triage

Wedge/surgical> 90 slides YES YES YES YES YES YES YES

“Adequate”For what?

CELL BLOCK




SLIDES 1‐2 7‐10 2 10 10‐20 10 10

EBUS/FNA/thin coreAdequate for diagnosis

YES YES Unsure

Unsure?sensitivity

False negatives

Unlikely No No






Small sampleHard to predict, hard to triage

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SLIDES 1‐2 7‐10 2 10 10‐20 10 10


Maybe?sensitivity

False negatives

Unlikely No No






Plenty of coresSnatching defeat from the jaws of victory

Put cores in separate blocksCould get 30‐40 from each90‐120instead of 30‐40




SLIDES 1‐2 7‐10 2 10 10‐20 10 10


Maybe?sensitivity

False negatives

Unlikely No No






Liquid biopsy/blood biopsy

• cfDNA approaches

If slides are my currency, is liquid biopsy BITCOIN?

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More examples

<2 mm2 butenough for 50 gene panel and IHC, FISH

Transbronchial biopsy

Bone FNA

No decalcification

<4 mm2 tissue

Successful Hot spot panel ONCOMINE at Weill Cornell

DNA and RNA based, captures mutations and fusions

Oncomine successfulDNA and RNA

KRAS, p53SOX2 amplification

Cytology cell block

36 mm2But 5% tumor

Endobronchial biopsy

2.5 x 3.5 mm8.75 mm230 % tumor

Sufficient for OncomineDNA and RNA

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Tumor mutation Burden

• Concept• More mutations, more mutated proteins, more neoantigens for immune response.

• Need more regions of sequencing than hot spot or Oncomine v1• Tissue requirement is greater

• Still evolving

Transbronchial bx

20 mm2, 75% tumorEnough for OncomineLikely Foundation One and TMB

Core biopsyEnough for OncomineEnough for Foundation One CDx/TMB

6.4 x 5 mm32 mm210‐20% tumor

Will TMB break the bank?

Summary

• Demand is increasing

• Supply needs optimization• Sample processing

• Appropriate test selection

• Better understanding of “currency”• Liquid biopsy as alternate option

Molecular Testing Guideline in Lung CancerCAP/IASLC/AMP – 2018 – Repeat testing• Addressed suitable sample types – cell block, cytology, tissue

• Assays with sensitivity to as little as 20% cancer cells

• Recommended targets – EGFR, ROS1, ALK as minimum stand alone tests or as part of larger panels

• Larger panels could include KRAS, HER2, MET, BRAF and RET

• Unexpected, discordant, equivocal or low confidence – confirmed or resolved using an alternate method or sample

• EGFR mutated lung adenocarcinoma with progression after treatment• For detection of EGFR T790M• Including use of cfDNA methods

• Repeat testing for ALK was not recommended at time of document

Lindeman et al, Arch Pathol Lab Med, 142:321‐346, 2018

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Retesting – sample related

• Inadequate sample• Low cellularity

• Lab might reject it

• If lab does not reject it, result will be free of mutations – false negative

• Utility of KRAS result as part of panel ‐ if detected, suggests sample adequacy

• Testing failure• Low rate overall

Retesting – Test platform

• EGFR mutation specific IHC

• Incorrect analyte or alteration• ALK point mutation vs translocation

• EGFR amplification

• MET immunohistochemistry – use in place of amplification or MET exon 14 skipping detection is not established

• Targeted testing vs. larger panel• Inclusion of rarer variants

• NCCN guidelines “optional” or emerging

EGFR mutated adenocarcinoma

• Post – treatment• Testing for EGFR T790M

• Use of osimertinib after disease progression on first or second generation TKI

• Testing for MET amplification

• Morphologic evaluation – squamous or small cell histology

EGFR mutated adenocarcinoma – post treatment – T790M• “liquid biopsy” – blood sample based testing

• Repeat tissue sample

IASLC Statement paperRolfo et al, JTO 13:1248, 2018

EGFR mutated adenocarcinoma

• Use of osimertinib upfront may limit need for T790M testing

• HOWEVER – osimertinib resistance (Oxnard et al, JAMA Oncology 2018)

• EGFR C797S mutation with maintained T790M• Note L792 and L718 also reported by Yang et al, Clin Cancer Res, 2018

• PIK3CA mutation

• T790M loss• Small cell (or squamous)


• BRAF mutation

• Single examples of fusions and KRAS Q61K

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ALK translocation – crizotinib therapy

• Ceritinib, alectinib, brigatinib in crizotinib resistance• Ceritinib and alectinib can be used as initial therapy

• Alectinib (Peters et al, NEJM, 2017)

• Emergence of point mutations on crizotinib

• Emergence of point mutation on alectinib and ceritinib• ? More frequent?

Gainor et al, 2016 Gainor et al, Cancer Discovery, 2016

Start on crizotinib – develop resistance

Switch to which drug?Might pick drug with 0% for that alteration

So L1196M – might pick BrigatinibG1269A – might pick severalG1202R ‐ ?none

Common confusion



• ALK – translocation versus point mutation• Pick the right test for treated patient



Retesting for molecular alterations

• If initial test on low cellularity sample and without mutations

• Wrong test or desire for expanded panel (no driver detected)

• Post targeted therapy – targets differ

Goals

• Definitions• Test examples

• Analytes

• Reasons for testing

• Sample types and tissue triage• Maximizing supply

• Determining molecular adequacy

• Examples

• Retesting

Molecular Pathology –You happens next?...epithelioid sarcoma ini loss (smarcb1) extrarenal rhabdoid ini loss extraskeletal myxoid chondrosarcoma ewsr1‐nr4a3 rbp56‐nr4a3 tcf12‐nr4a3

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