molecular analysis of cutaneous neoplasia: a practicing ... · should i develop my own test...
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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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