Molecular analysis of cutaneous neoplasia: a practicing pathologist’s perspective Jane L. Messina

January 31, 2019

• I have no conflicts of interest to disclose

• Disclaimers: I am NOT a molecular pathologist; for questions about methodology, please refer to:– https://www.karger.com/Article/FullText/480089 (NGS)

MIGHTY WOMEN?

Goals

• Review current best practices• Billing and coding considerations• Routinely available tests and indications

Cl inical grade molecular test ing

Comprehensive genomic profiling of tumor

(e.g. Illumina MiSeq,Ion Torrent, Foundation Medicine)

Fluorescence in situ

hybridization

Melanoma-specificmutation screening

(e.g. MelaCarta)

Comparative genomic

hybridization

Specific mutation screening, e.g. BRAF,

NRAS, c-KIT

Gene expression profiling(Decision Dx, myPATH)

Genomic profiling of circulating DNA (“liquid

biopsy”, e.g. Guardant360)

Gene rearrangement for lymphoid and soft tissue tumors Carcinoma of

unknown primary panels

Molecular test ing pract ices among dermatopathologists

• Survey of ASDP membership, 2017

• 62% order >12 tests/year• More frequent testing

associated with training, pathology residency, academic practice, higher volume, onsite laboratory

• Barriers cited: coverage, cost, lack of evidence-based guidelines, availability

Torre K et al. J Cutan Pathol 2018; 45(6): 398-394

Evidence-based guidel ines: appropriate use cr iter ia

• Combine scientific evidence with expert panel rating to assess appropriateness of testing in specific scenarios

• 2015, ASDP AUC task force: 12 ancillary tests rated with 89% consensus– Lymphoid: TCR β and γ, IgH rearrangement– Melanocytic: FISH, CGH, qRT-PCR– Others: HPV, MMR, t(17;22) for DFSP, EWSR1 for clear cell

sarcoma

Vidal et al; J Am Acad Dermatol 2019; 80:189-207.

Evidence-based guidel ines: appropriate use cr iter ia

• Combine scientific evidence with expert panel rating to assess appropriateness of testing in specific scenarios

• 2015, ASDP AUC task force: 12 ancillary tests rated with 89% consensus– Lymphoid: TCR β and γ, IgH rearrangement– Melanocytic: FISH, CGH, qRT-PCR– Others: HPV, MMR, t(17;22) for DFSP, EWSR1 for clear cell

sarcoma

Vidal et al; J Am Acad Dermatol 2019; 80:189-207.

Pract ical issues

• How much tissue?– Most tests use equivalent of 5-10 5µ recuts, or a 1.5 mm

core of FFPE tissue

• Is the test covered by insurance?– Ordering physician should be familiar with national and

local coverage determinations developed by CMS– Patient may be billed if test not covered– Test must be ordered by physician (for a pathologist,

signature on the requisition form)

FDA approved companion diagnost ic tests for melanoma

• Foundation Medicine cDX• THXID BRAF kit (bioMerieux, Inc) and cobas 4800

BRAF V600 mutation test (Roche Molecular Systems, Inc.)

Pract ical issues: coding and reimbursement

Code Test Coverage in Florida?

MedicareReimbursement

88381 Microdissection Yes $150.26

81210 BRAF mutation Yes $175.40

81210-81479

Other genes (NRAS, KRAS, KIT) Yes* $200-350

81445 Targeted gene sequencing 5-50 genes

Yes, with provisions*

$597.91

81450 Targeted gene sequencing >51 genes

Yes, if meets NGS NCD

$759.93

G0452 Pathologist interpretation of results

Yes** $18.63

*must have five actionable genes to bill panel**no more than 6 G codes per panel allowed

Coding and reimbursement

Code Test Reimbursement?

88377 or 88374

FISH $300-400

81228 or 81229

Copy number variation testing i.e. CGH Sometimes (Aetna)

81599 Decision Dx pendingUnder

considerationmyPath

Under consideration

PLA (pigmented lesion assay)

86152/3 Circulating tumor cells Not covered

Should I deve lop my own test ( lab-developed/LDT) or use an FDA-approved test?

• Requires validation per CLIA requirements• The FDA was considering taking over approval of

more complex tests (2014), but still no final rule• Theoretically, reimbursement should be same as

FDA-approved tests

When to test?

Molecular testing Consider prognostic testing

Mutation testing or mutation profiling

Mutation profile or next-gen sequencing

Reassess tumor profile

Treatment Surgery SurgeryAdjuvant therapy

Molecular-based drug strategy

Molecular-based drug strategy

Stage I/II Stage III Stage IV Resistance

Avai lable molecular tests for melanoma

• Diagnostic aid: FISH, CGH, Myriad myPath• Prognostic testing: Decision Dx• Direct course of treatment: mutation testing

Diagnost ic molecular test ing

• Frequently used in the evaluation of diagnostically challenging melanocytic proliferations– Atypical Spitzoid proliferations (AST)– Atypical blue nevus-like proliferations– Nevoid melanoma

• NCCN: “consider the use of molecular testing for histologically equivocal lesions”

• ASDP: FISH and CGH usually appropriate in MM v all benign nevus variants, inappropriate in definitive cases

NCCN guidelines Cutaneous Melanoma 1.2019

10 year old with changing scalp les ion

Old assay6q23 CEP6 6p25, 11q13MYB RREB1 CCND1

Fluorescence in s i tu hybr idizat ion

• Availability:– NeoSITE® melanoma, Neogenomics Laboratories (Ft.

Myers, FL), $1500– Inform Diagnostics, Mayo Reference Lab

• What about borderline lesions such as this?

Sensitivity: 87%Specificity: 95%

New assay8q24 9p21 CEP9MYC CDKN2A

94%98%

Unequivocalmelanoma

Performance of F ISH in var ious melanocyt ic prol i ferat ions

• Blue nevus-like melanoma vs epithelioid blue nevus– 90% sensitive, 100% specific in 10 cases– Heavy melanin pigmentation can obscure stain result

• Nevoid melanoma– performance comparable to all melanomas– 6p25 in 49%; 8q24 in 27%-especially amelanotic lesions

• Acral melanoma: – performance comparable to all melanomas

Yélamos O et al. J Cutan Pathol. 2015 Nov;42(11):796-806. Pouryazdanparast P et al. Am J Surg Pathol. 2009 Sep;33(9):1396-400. Su J et al. Pathology. 2017 Dec;49(7):740-749.

FISH in histological ly ambiguous Spitzoidles ions (AST)

Probe set AST w/orecurrence

“AST” with poor outcome(melanoma)

Typical Spitz

OLD sensitivity 50-100% 6,1,4

OLD specificity 57-91% 4,1 75-100%5,1

NEW sensitivity 50-100%3,2

NEW specificity 74-87%2,3

1 Demarchis EH et al, Pediatr Dermatol. 2014; 31(5) 561-92 Gerami et al, Am J Surg Pathol Feb 20133 Tetzlaff et al, Am J Surg Pathol. Dec 20134 Massi et al. J Am Acad Dermatol 2011;64:919-355 Dika et al Mel Research 2015; 25(4): 295-3016 Vergier et al. Modern Pathology 2011; 24: 613-623

FISH performance on ambiguous les ions]

• Tetzlaff et al., 34 ambiguous lesions tested with newer assay– “Benign” favored on histology: 3/24 FISH+– “Malignant” favored on histology: 5/10 FISH+– Sensitivity 50% , specificity 87%– In setting of a predominantly benign histology, negative FISH is

reassuring

Gerami et al, Am J Surg Pathol, Feb 2013Tetzlaff et al, Am J Surg Pathol, Dec 2013

Back to our case…

• IHC:– P16 retained– Ki-67: 10% proliferation index (4 mitoses noted)

• FISH– Could not be performed due to heavy melanin

pigmentation

• Next step: comparative genomic hybridization

Comparat ive genomic hybr idizat ion• Assesses chromosomal copy number changes across

tissue, not individual cell abnormalities• 95% of melanomas harbor numerous chromosomal

gains and losses • Nevi rarely show aberrations• Exceptions:

– 12-26% of Spitz nevi (esp. recurrent) have 11p or 7q gain– Some AST have abnormal CGH (45%, most common gain of

1p)• Does not predict SLN involvement• Proliferative nodules in congenital nevi may have whole

chromosomal gains

Bastian BC et al. J Invest Dermatol. 1999;113:1065–1069.Bastian BC et al. Am J Pathol. 2003;163:1765–1770Raskin et al. Am J Surg Pathol 2011;35:243–252

Comparat ive genomic hybr idizat ion

• Most commonly used to distinguish pediatric atypical Spitz tumor from melanoma

• Not widely available, insurance coverage varies• Typical out-of-pocket cost $2500

Back to our case

• CGH shows gains involving chromosome 4, 6p, interstitial 17q, 20q

• Losses involving 6q, 9q• Plan: perform sentinel node biopsy

Sentinel node 1

Diagnosis: animal-type (pigment synthesizing melanoma, 1 of 2 SLN positive

Exhibit 2:19 year old female with cyst-like lesion on scalp

Molecular test ing

• FISH– 6p25: 30% nuclei have extra copies (>29% high stringency

cutoff)– 8q24(cMYC), CDKN2a, and CCND1 probes WNL

• GNAQ/GNA11 testing– mutation in q209P of GNAQ gene

• CGH– 4 whole chromosomal gains: 6, 8, 9, and 18

CONCLUSION: Probable melanoma arising in blue nevus

Myriad MyPath-diagnost ic a id for equivocal les ions • 23-gene expression signature that can help differentiate nevi from

melanoma- utilizes 5 recut slides from paraffin block• Training and validation cohorts of 900+ unequivocal melanoma and nevi:

sensitivity 90% specificity 91%• Currently undergoing large-scale testing of clinical utility with melEvalPRO

Clarke et al. J Cutan Pathol 2015; 42:244-252

Superficial spreading>nodular>lentigo

maligna

Compound and dermal

nevi

Independent val idat ion of myPath

• 1400 lesions independently reviewed by 3 experts– Triple concordance: 993 lesions (70.9%) – Excluded indeterminate scores: 860 lesions (24%

malignant, 76% benign)– Excluded lesions with <10% tumor volume: 763 lesions

• Sensitivity 91.5% -- Specificity 92.5% – False negative risk-lentigo maligna and desmoplastic

melanoma variants– False positive risk-dysplastic nevi

Clarke et al., Cancer, October 2016Clarke LE et al. Human Pathol, 2017 Dec; 70: 113-20

29 year old pregnant female with changing lesion on back

Junctional dysplastic nevus with moderate to severe atypia

MyPath and FISH for diagnost ical ly chal lenging les ions (1)

• 39 unequivocal lesions: 62% sensitivity, 95% specificity

• 78 challenging lesions with expert consensus (27 favor malignant, 30 favor benign, 21 ambiguous)

• myPath score agreed with histology in 64%, agreed with FISH 70% of time– Limited by lack of clinical outcome

SUMMARY: myPath score can increase diagnostic certainty and influence treatment recommendations, BUT performance not proven in ambiguous lesions

Minca et al, Modern Pathology, August 2016, 29:832-843.

MyPath and FISH for diagnost ical ly chal lenging les ions (2)

• 198 unequivocal and 70 morphologically ambiguous cases

• Performance for unambiguous cases similar to original studies

• 69% agreement between FISH and myPath• Ambiguous cases using histology as gold standard:

– FISH 61% sensitive, 100% specific– MyPath 50% sensitive, 93% specific

Reimann JDR et al. Mod Pathol 2018 Nov; 31(11):1733-1743

PLA assay (pigmented les ion assay)Dermtech

• Gene expression assay from stratum corneum harvested by adhesive– Expression of LINC00518 and PRAME

• Performance on unequivocal lesions:– 81% sensitive, 55% specific

• Potential utility for selection of lesions for biopsy

Gerami P, et al, J Am Acad Dermatol 2017; 76:114-120Ferris LK, et al, JAMA Dermatol. 2017 Jul 1;153(7):675-680Childs MV: JAMA Dermatology, Dec. 2017 (Published Online)

TERT promoter mutat ions

• Most common non-coding cancer mutation, found in 60-80% of melanoma

• Presence in early-stage tumors suggest early role in tumorigenesis

• Identified in subsets of atypical Spitz tumors with aggressive behavior (distant metastases, death)

• Is part of some large cancer gene panels, but as a single test is only available in research setting currently

Hayward et al. Nature 2017; 545(7653): 175-180Lee et al. Sci Rep 2015; 5 (1-7).

Molecular tests for melanoma diagnosisSummary

• FISH and myPath sensitivity and specificity ~90% in unequivocal melanomas and nevi

• FISH sensitivity drops to 50-100% and specificity to 74-87% in most commonly used scenario: the atypical Spitz tumor

• A stepwise approach utilizing FISH, then CGH in equivocal cases may be considered

• CGH can aid in atypical cases with negative FISH, but hampered by limited availability and reimbursement

Uses for molecular test ing in melanoma

• Diagnostic aid• Describe distinct prognostic classes• Direct course of treatment• Predict response to treatment

Decis ion-Dx prognost ic test ing (Cast le Biosc iences)

• 31-gene expression profile predicts outcome• Training/validation set of 268 Stage I-IV patients

– Multivariate analysis: superior to and independent of Breslow depth, ulceration, MR and AJCC stage in predicting metastatic disease

Gerami et al. Clin Cancer Res 2015; 21(1): 175-83.

n=104p<0.0001

Training set n=164 Validation set n=107

91%

25%

97%

31%

DMFS OSDFSClass 1/SLN- Class 1/SLN-

Class 1/SLN-

Class 1/SLN+ Class 1/SLN+

Class 1/SLN+

Class 2/SLN- Class 2/SLN-

Class 2/SLN-

Class 2/SLN+Class 2/SLN+ Class 2/SLN+

Gerami et al. J Am Acad Dermatol 2015;72:780-5. 40

Dec is ion-Dx + SLNB resu l ts may improve prognost ic pred ict ion

• Convenience sample of 217 patients from 7 institutions-not representative of typical population undergoing SLNB

Decis ion Dx- independent val idat ion in 532 pat ients

• 40% are Class 2• 5 year RFS Class 1=88%, Class 2=52%• Test identified 70% of patients who developed

metastatic disease-improved sensitivity over SLNB• Utility of testing unclear

– Adjuvant therapy trial?– Enhanced follow up regimens?– Significant number of Class 2 did NOT recur-anxiety?

Zager JS et al. BMC Cancer. 2018 Feb 5;18(1):130

Decis ion-Dx pract ice guidel ines

• AAD: lack of surgical or therapeutic trials in context of GEP discourages their use for prognostication or management decisions

• NCCN: routine (baseline) prognostic genetic testing of primary cutaneous melanomas not recommended outside of a clinical study (trial)

Swetter et al. J Am Acad Dermatol 2019: 80(1):208-250NCCN Guidelines Cutaneous Melanoma 1.2019

Uses for molecular test ing in melanoma

• Diagnostic aid• Describe distinct prognostic classes• Direct course of treatment• Predict response to treatment

Molecular a lterat ions direct targeted therapy

• Oncogenes– BRAFv600E/K: ~50%

• Dabrafenib/trametinib• Vemurafenib/cobimetinib

– NRAS: 20-30%• Binimetinib

– c-KIT: 2-5%• Imatinib

• Tumor suppressors– PTEN: 10-50%– CDKN2a/CDK4: 30-70%

• Clinical trials ongoing

SCF EGF C-KIT EGFR

RAS

PI-3K

HGF αMSHcMET MC1R

BRAF

MEK

ERK

CCND1/CDK4/6

mTOR

AKT

ProliferationSurvival

p16

PTEN

Palmieri et al. Front Oncol 2015;5:183

What panel do I use?

• Laboratory-developed tests: next generation sequencing using pre-determined panels (e.g. Illumina Trusight, Agena Melacarta)

• Commercially available tests: – Foundation One-324 genes (cost $5800, no balance billing)– Genoptix-15 genes– MSK-IMPACT-468 genes– Academic reference labs, e.g. Washington University,

ARUP, Mayo Medical labs

Which gene panel/platform?

• Genoptix Melanoma Molecular Profile: AKT3, BRAF, CCND1, CDK4, ERBB4, GNA11, GNAQ, KIT, MPA2K1, MAP3K0, MITF, NF1, NRAS, PIK3CA, PTEN

Ion AmpliSeq

Agena Melacarta

COMMON GENES:BRAF, NRAS, GNAQ, KIT,

PIK3CA, but most can be customized

Test for one versus mult iple alterat ions?

• NCCN: Consider broader genomic profiling if the test results might guide future treatment decisions or eligibility for participation in a clinical trial

NCCN guidelines Cutaneous Melanoma 1.2018

Whi le you are wai t ing for your test resu l ts…ident i f y ing melanoma mutat ions by immunohistochemistr y

• VE1 antibody: identifies v600E mutant melanoma by IHC- sensitivity 97%, specificity 98%

• SP174: NRAS Q61R-sensitivity and specificity 100%• 26193: NRAS Q61L-sensitivity 82%, specificity 96%• Initial reports note heterogeneity of staining within

patients and individual tumors in up to 18% of cases

Capper et al. Acta Neuropathol 2012 Feb;123(20):223-33Long et al. Am J Surg Pathol 2012 (epubahead of print).Kakavand H et al, Histopathology 2016: 69:680-686

c-KIT immunohistochemistry

• Immunohistochemical expression does not correlate with sensitivity to inhibitor

• IHC stain useful in screening tumor before testing

• 181 cases: 85% had >15% cells positive by IHC• no cases with <10% cells positive by IHC were

mutation positive

Torres-Cabala CA et al. Modern Pathol 22:1446-1456, 2009.

28% BAP-1 loss

15%NTRK fusion

11% HRAS mutation

9.5% ALK-1 fusion

6.1% ROSBRAFRET fusion

34Incidence (%)

unknown

Van-Engen-van Grunsven et al. Am J Surg Pathol 2010;34:1436-41Wiesner et al. Nat Commun 2014;5:3116

Other uses of genomic test ing: k inase fus ions frequent in Spitzoid les ions

RET

• Potentially useful if metastatic disease arises

Other potent ial ly targetable genes

• NF1 mutation (14% of TCGA set): preclinical studies suggest resistance to BRAF inhibition, sensitivity to MEK inhibitors

• ALK fusion found in 2.4% primary/metastatic non-Spitzoid melanomas: one reported partial response to crizotinib

• MET fusions identified in 0.24% of tumors: potential response to cabozantinib

Maertens et al, 2013, Whittaker et al, 2013Busam et al, Am J Surg Pathol Feb 11 2016 epub of printYeh et al. Nat Commun 2015 May 27; 6:7174

Molecular testing-negative results?

• Case study: 45 year old male with recurrent soft tissue mass distal arm, originally diagnosed as melanoma

• BRAF, NRAS, c-KIT negative

• EWS/ATF translocation: positive• Diagnosis: clear cell sarcoma

L iquid biopsy

• Detects alterations in 73 genes using circulating tumor DNA (~0.4% of total DNA in blood)

• Indications: insufficient tumor, follow patients on therapy, or detect rarely tested mutations

• Guardant360: in 400 advanced cancer patients, ctDNA and tumor results matched 87% of time; 98% when within 6 months– Uncovered additional mutations not in tumor samples– Little experience in melanoma (n=1)

Zill et al. J Clin Oncol 34, 2016 (suppl; abstr LBA11501)

Circulat ing tumor DNA

• Short nucleic acid fragments (~166 bp) in plasma, released by tumor apoptosis/necrosis

• BRAF, cKIT, NRAS, TERT, PIK3CA have all been investigated

• Pre-treatment levels correlate with worse outcome• Sequential levels parallel disease course, may even

precede radiologic changes– May distinguish pseudoprogression from true progression

Calpare L et al. Cancer Letters

Molecular test ing in other cutaneous neoplas ia

68 year old woman with 6-month history of forehead mass

CK7Ck5/6 p63

First and second round of IHC

• Panel of organ-specific markers (NKX3.1, TTF-1, napsin, GCDFP, mammaglobin, WT-1, PAX8, ER, CA-125, mesothelin, CDX2) negative

Carc inoma of unknown pr imary

• Median survival 2-4 months• Workup expensive, time-consuming• 56 patients presenting to MCC with CUP:

– Primary site found in 7%– 20% survived >1 year (mean 8 months)– Average cost of diagnosis $17,973

Schapira and Garrett Arch Int Med 1995 Oct 23; 155(19): 2050-4

Workup of CUP: NCCN guidel ines

• H&P, blood panel, CT chest/abdomen/pelvis, site-directed endoscopy, biopsy

• Biopsy– Immunohistochemical analysis-”exhaustive panels have

not been shown to increase diagnostic accuracy”• Tier 1: tissue lineage (carcinoma v sarcoma v lymphoma v

melanoma)• Tier 2: putative primary site• Tier 3: biomarkers (e.g. RAS, HER2, ALK)

– “Gene signature profiling for tissue of origin is not recommended for standard management at this time”

Molecular prof i les to assess tumor or ig in

• DNA-based gene expression profiling (Cancer TYPE ID) and miRNA profile (Rosetta Cancer Origin test) commercially available, accuracy in validation sets ~85%

• Accuracy of blinded testing in patients with latent/known primary– GEP: ~75%– IHC: ~65%

Hainsworth JD and Greco FA. Virchows Arch (2014) 464:393-402

GEP v IHC in assess ing CUP

• In five series of 117 patients, IHC predicts single site of origin in ~55% of cases– GEP prediction identical in 78% of these

• Directing treatment with GEP diagnosis: no evidence of better survival compared to IHC in a single prospective study

Greco et al. 2013 J Natl Cancer Inst. Doi: 10.1093/jnci/djt099Hainsworth JD and Greco FA. Virchows Arch (2014) 464:393-402

One last test

CD34

Dermatof ibrosarcoma protuberans

• When to test for t(17:22)?• ASDP: test with CD34+

tumor with atypical histology for DFSP, or considering treatment

• Routine testing of unequivocal tumors negative

CD34-negative DFSP with fibrosarcomatous transformation

Putting it al l together: molecular test ing

• Testing for diagnostically controversial lesions common, may augment histologic impression, know pitfalls

• Utility of prognostic GEP needs further investigation• Genomic testing of melanoma directs therapy• EWSR1 in patients with dermal tumors with

melanoma markers• ctDNA shows great promise• Carcinoma of unknown primary tests do not exceed

IHC in accuracy or significantly add to patient care

…molecular analysis—allowing more efficient use of healthcare dollars?

Thank you

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Thank you