emerging molecular diagnostic tests and therapies for melanoma

Emerging Molecular Diagnostic

Tests and Therapies for Melanoma::

From Research Benches to

Clinical TrenchesClinical Trenches

Aleodor (Doru) Andea, MD

Associate Professor of Pathology and Dermatology

Director of Dermatopathology Section

University of Alabama at Birmingham

Birmingham, USA

[email protected]

Disclosure information

I have no financial or industrial affiliation to

disclose.

Aleodor A Andea

Overview

1. Problems in the diagnosis

2. Molecular alterations (with implications

for diagnosis or treatment)

3. Diagnostic assays3. Diagnostic assays

4. New therapies

Melanoma diagnosis

1.Size

2.Symmetry

3.Circumscription

5.Nested architecture

NEVI MELANOMA

Small Large

Symmetric Asymmetric

Well-circumscribed Poorly-circumscribed

Predominantly nested Predominantly single cells

4.EpidermisNot affected Consumption, Ulceration

7.Pagetoid spread

8.Confluent growth at DEJ

10.Maturation

6.Nests uniformity

11.Cytologic atypia

12.Mitoses in dermis

Uniform Irregular

No Prominent

No Present

Present Absent

Absent Severe

Absent Present, Atypical

9.Dermal patternNested, Infiltrative Sheetlike, Expansile

• Age

• Site

• History of trauma

Melanoma diagnosis

Factors that influence interpretation of histologic

parameters

• History of trauma

2 y/o girl with lesion on the rt knee

Dermatopathology diagnosis

Spitz tumor of uncertain malignant

potential

Dermatopathology diagnosis

Spitz tumor of uncertain malignant

potential

Diagnosis 1 Diagnosis 2Diagnosis 1

Mitotically active

Spitz nevus

Diagnosis 2

Spitzoid melanoma

• ~1-2 million bx/ year in US to rule out

melanoma

• ~3%-6% of bx are melanomas

• In most cases dx can be made on • In most cases dx can be made on

histopathology but a small proportion

have ambiguous histology (Q.)

• 2.3%-25% of cases with diagnostic

discrepancies (McGinnin et al, Arch Dermatol 2002, Corona et al, J Clin Oncol 1996, Farmer et al, Hum Pathol 1996, Lodha et al J Cutan Pathol 2008)

• Ambiguous lesions with overlapping

criteria

– Severely atypical/ dysplastic nevus vs LM

or SSM

– Nevi of special site vs LM or SSM

– Spitz nevus vs spitzoid melanoma– Spitz nevus vs spitzoid melanoma

– Atypical blue nevi vs melanoma

– Proliferative nodules vs melanoma

– Nevoid melanoma

• Inter-observer variability

Zembowicz and Scolyer. Arch of Pathol and Lab Med. 2011; 135

57 borderline lesion with clinical follow-up

• 17-benign• 17-benign

• 26-melanoma

• 14-borderline

Panel performance:

• 73% sensitivity

• 47% specificity

Need for a better test

• Prevent under/overtreatment of patients

• Reduce medical costs associated with unnecessary treatmentunnecessary treatment

• Impact positively on patient care

• Most common reason for medical malpractice in pathology

Kornstein, et al. Arch Pathol and Lab Med, 2007

Gerami, et al, Am J Surg Pathol, 2009

Danube River, Romania

Molecular alterations in

melanocytic neoplasms

• MAP kinase alterations

• Replicative senescence in melanocytic

lesionslesions

GROWTH

FACTORS

RAS

RAF

CYTOPLASM

H-Ras, K-Ras, N-Ras

A-Raf, B-Raf, C-Raf1

MAP KinaseKIT, EGFR

MEK

ERK

PROLIFERATION

CYTOPLASM

NUCLEUS

MEK-1, MEK-2

ERK-1, ERK-2

CCND1

CDK4/6

p16

GROWTH

FACTORS

RAS

RAF

CYTOPLASM

KIT mutations in 14-19% of melanomas

MAP Kinase pathway activation in

melanocytic lesions

MEK

ERK

CYTOPLASM

NUCLEUS

Curtin JA et al. J. Clin. Oncol. 2006; 24; 4340–6

Beadling C et al. Clin Cancer Res 2008; 14:6821-8

PROLIFERATIONCCND1

CDK4/6

p16

GROWTH

FACTORS

NRAS

RAF

CYTOPLASM

10-22% of melanomas

80% large congenital nevi


melanocytic lesions

MEK

ERK

CYTOPLASM

NUCLEUS

Curtin JA, et al. N Engl J Med. 2005 Nov 17;353(20):2135-47

PROLIFERATIONCCND1

CDK4/6

p16

GROWTH

FACTORS

HRAS

RAF

CYTOPLASM

27-29% of Spitz’s nevi


melanocytic lesions

MEK

ERK

CYTOPLASM

NUCLEUS

Bastian B et al, Am J Pathol 2000

Van Dijk M et al. Am J Surg Pathol 2005; 29:1145-51

PROLIFERATIONCCND1

CDK4/6

p16

GROWTH

FACTORS

RAS

B-RAF

CYTOPLASM


melanocytic lesions

40-60% of melanomas (Q.)

80% of nevi

MEK

ERK

CYTOPLASM

NUCLEUS

Davies et al, Nature 2002, Marcia et al. Cancer Res 2002, Curtin et al, N Engl J Med 2005, Pollock et al., Nat Genet 2002

PROLIFERATIONCCND1

CDK4/6

p16

GROWTH

FACTORS

RAS

B-RAF

CYTOPLASM


melanocytic lesions

GNAQ

PKC

Guanine nucleotide-binding protein

G(q) subunit alpha

•Blue nevi: 80%

•Uveal melanoma: 50%

MEK

ERK

CYTOPLASM

NUCLEUS

Van Raamsdonk C et al. Nature 2009 Ian 29; 457:599-603

PROLIFERATIONCCND1

CDK4/6

p16

Nevus type KIT NRAS HRAS KRAS BRAF GNAQ

Congenital NA 70-81% 0 NA 0-

88%

0

Acquired NA NA 0 NA 70-

88%

0

Clark’s NA NA 0 NA 52-

80%

NA

Spitz NA 0% 27- NA 0% 0

Genetic alterations in nevi

Spitz NA 0% 27-

29%

NA 0% 0

Blue nevus NA 0-5% NA 0-15% 0-12% 40-

83%

Curtin JA, et al. N Engl J Med. 2005 Nov 17;353(20):2135-47, Maldonado JL et al. J Natl

Cancer Inst. 2003 Dec 17;95(24):1878-90.

Satzger I et al. Br J Cancer. 2008 Dec 16; 99(12):2065-9

Emley A et al. Hum Pathol. 2011 Jan; 42:136-40

Van Raamsdonk C et al. Nature 2009 Ian 29; 457:599-603

Yazdi et al. J Invest Dermatol. 2003 Nov; 121:1160-2


Saldanha et al. Int J Cancer 2004 Sep 20;111:705-10

Van Dijk M et al. Am J Surg Pathol 2005; 29:1145-51

Bauer et al. J Invest Dermatol 2007; 127:179-182

Melanoma type KIT NRAS HRAS KRAS BRAF GNAQ

MM on skin

without chronic

sun damage

0% 22% 0 0% 59% 0

MM on skin with

chronic sun

damage

16% 15% 0 0% 11% 15%

Genetic alterations in melanomas

damage

Acral 10-23% 10% 0 0% 23% 0%

Mucosal

melanoma

15-21% 5% 0 0% 11% 0

Uveal melanoma 0% 0% NA 0% 0% 50%

Malignant BN NA NA NA NA NA 46%

Curtin JA, et al. N Engl J Med. 2005 Nov 17;353(20):2135-47,

Curtin JA et al. J. Clin. Oncol. 2006; 24; 4340–6

Maldonado JL J Natl Cancer Inst. 2003 Dec 17;95(24):1878-90.

Satzger I, Br J Cancer. 2008 Dec 16;99(12):2065-9

Zuidervaart, W et al. Br J Cancer 2005 Jun 6; 92:2032-8

Beadling C et al. Clin Cancer Res 2008; 14:6821-8

HRAS NRAS BRAF

Spitzoid melanoma 0 19% 64%

AtypicalSpitz nevus 14% 0% 0%

Spitz nevus 29% 0% 0%

Practical implication for DX

van Dijk MC et al. Am J Surg Pathol. 2005 Sep;29:1145-51.

Orthodox Cathedral, Timisoara, Romania

Replicative senescence in

melanocytic lesions

• Cells exit cell cycle after a number of

divisions

• Chromosomes are protected by • Chromosomes are protected by

telomeres and the enzyme telomerase

Greider and Blackburn EH, Nature 1989

Szostak et al, Cell 1989

Chromosome

Chromosome

Chromosome

P53, RB

T T T T

T T T

T T

p53, RB,

p16

Mitosis

MitosisCheckpoint

Cell cycle arrest

(Senescence)

Chromosome

Chromosome

Chromosome

Chromosome

T T T T

T T T

T T

T

p53, RB,

p16

P53, RB

Mitosis

Mitosis

Mitosis

Chromosome

Chromosome

T

Mitosis

Chromosome

Chromosome

Chromosome

Chromosome

T T T T

T T T

T T

T

p53, RB,

p16

P53, RB

Mitosis

Mitosis

Mitosis

Chromosome

Chromosome Cell Crisis

DNA breaks, fusions

T

MitosisMitosis

•Gross chromosomal

abnormalities

•Increase apoptosis

Cell Death

Chromosome

Chromosome

Chromosome

T T T T

T T T

T T

p53, RB,

p16

Mitosis

Mitosis

Nevi MAPK

-B-RAF

-N-RAS

-H-RAS

-GNAQ

Cell cycle arrest

(“Oncogene-

Induced”

Senescence)

No chromosomal aberrations

Chromosome

Chromosome

Chromosome

Chromosome

T T T T

T T T

T T

T

p53, RB,

p16

P53, RB

Mitosis

Mitosis

Mitosis

Melanoma

Cell cycle arrest

(Senescence)

MAPK

-B-RAF

-N-RAS

-H-RAS

-GNAQ

p16 inactivation:

~50% of MM

Chromosome

Chromosome Cell Crisis

DNA breaks, fusions

T

MitosisMitosis

Cell Death

-Re-stabilize

telomeres

-growth advantage

Melanoma

Gross chromosomal abnormalities

Parliament House, Bucharest, Romania

Molecular tests differentiating

melanoma from nevi

• IHC

• CGH

• FISH

Ki-67Nevus

(<5%)

Melanoma (Q.)

(>5%)

HMB-45/ Cyclin D1

Nevus (Q.) Melanoma

HMB-45 problemsCombined Nevus Melanoma

p16

Nevus Melanoma

MAPK

activation

P16

inactivation

(and others)

PrecursorUnchecked

division/ growthCell crisis

Reactive p16

overexpresionNo p16

expresion

~50%

p16Spitz Nevus Melanoma

p16Spitz Nevus Melanoma

Stefanaki et al, J Am Acad Dermatol 2007

Positive in

100% of Spitz

nevi

Negative in 32-50%

of melanomas

Comparative Genomic

Hybridization

• Screens the entire genome for gains

and losses in DNA material in one

experiment (Q.)experiment (Q.)

• Variants:

– Conventional CGH

– Array based CGH

Conventional CGH

www.empiregenomics.com

Bauer, J. and Bastian, B. Dermatologic Therapy, Vol 19, 2006, 40-49.

The ratio of green:red signals along each chromosome is determined

Array CGH

• Arrays of genomic bacterial artificial

chromosome (BAC) clones or

oligonucleotides

CGH in Melanocytic lesions

• 54 benign nevi – 27 Spitz nevi

– 19 Blue nevi

– 7 Congenital nevi

• 132 MM – 22 Acral location

– 108 non-Acral


Gains: 6p Losses: Gains: 11p: 11% Losses: Gains: 6p

1q

7p

7q

8q

17q

20q

Losses:

9p

9q

10q

10p

6q

11q

Gains: 11p: 11%

7q: 2%

Losses:

0%

•The 7 cases were all Spitz

nevi (no progression to MM

at 7 yrs FU)


96% of cases

Conclusion

• CGH will show multiple gains and

losses of DNA material in the majority

of cases (Q.).

• Potential diagnostic test for ambiguous • Potential diagnostic test for ambiguous

melanocytic lesions.

• Sensitivity 96%

• Specificity 98%

Chromosomal Aberrations in

Melanocytic Lesions

• Spitz nevi:

– no abnormalities

– gains on 11p (12-18%)

• Congenital nevi: no abnormalities

• Proliferative nodules: no abnormalities or whole chromosomal gains or losses

• Cellular blue nevi: no abnormalitiesBastian B et al, Am J Pathol 2000

Bastian B et al, J Invest Pathol 1999

Bastian B et al, Am J Pathol 2002Maize Jr, JC et al, Am J Surg Pathol 2005

Disadvantages of CGH

• Requires 30-50% pure tumor cells

• Does not allow histologic correlation

• Cannot detect tumor subpopulations• Cannot detect tumor subpopulations

• 1q31 (COX2)

• 4q12 (KIT)

• 7q34 (BRAF)

• 6p35 (RREB1)

• 6q23 (MYB1)

• 6 cen

• 7 cen

• 9p31 (p16)

CGH date from

melanomas

• 9p31 (p16)

• 10 cen

• 11q13 (CCND1)

• 17q25 (TK1)

• 17q21 (RARA)

• 17 cen

• 20q13 (ZNF217)

Gerami et al. Am J Surg Pathol 2009

• Training cohort 301 melanocytic tumors

– 148 melanomas

– 153 nevi

• Validation cohort

– Unequivocal lesions:

• 83 melanomas

• 86 nevi

– 27 ambiguous cases with clinical follow-up

• 6 cases developed metastases

• 21 free of disease at > 5 years follow-up


11q13

CCND1

6p25

RREB1

6cen 6q23

MYB1

Gain Gain Loss

>38% nuclei

contain >2

signals for

11q13

>29% nuclei

contain >2

signals for 6p25

>55% nuclei

with >1 ratio of

6p25 / 6cen

>40% nuclei

with <1 ratio of

6q23 / 6cen

CCND1 RREB1 MYB1

OR OR

OR


• Validation cohort:

– 72 of 83 melanomas FISH positive

• 87% sensitivity

– 82 of 86 nevi FISH negative

• 95% specificity

• 4 nevi were FISH positive• 4 nevi were FISH positive


• 27 cases with indeterminate histology

– 6 developed metastases, all FISH positive

• 100% sensitivity

– 21 disease free at > 5 years, 15 FISH

negative

• 71% specificity


CCND1 –green

RREB –red

MYB –gold

CEP6 -aqua

20 Nevi 20 Melanomas

19/20 negative

Specificity 95%

18/20 positive

Sensitivity 90%

Morey et al, Pathology 2009

MYB lossNo alterations

Superficial

Spreading

N=70

Lentigo

Maligna

N=28

Nodular

N=22

Acral

lentiginous

N=3

DM

N=15

Sensitivity of FISH in melanoma subtypes

Sensitivity 81% 82% 91% 100% 47%

Most

common

alteration

6p25 gain

(RREB1)

73%

6p25 gain

(RREB1)

68%

6p25 gain

(RREB1)

82%

6p25 gain

(RREB1)

100%

6p25 gain

(RREB1)

86%

Gerami et al. Arch Dermatol 2010; 146:273-8

Gerami et al. J Cutan Pathol 2011; 38: 329–334

22 total lesions

- Sensitivity: 60%

FISH positive

- Sensitivity: 60%

- Specificity: 50%

12 ambiguous lesions

FISH worked in 8 cases

-5 malignant (3 FISH

positive)

-3 benign (1 FISH negative)

Summary of

sensitivity/specificity estimates

1 2 3 4 5 6 7

Study sample 169 233 40 43 10 324 324

Sensitivity 87% 83% 90% 85% 42% 84%

(75)

64%

Specificity 95% 94% 95% 90% 100% 95% 98%

1. Gerami et al. Am J Surg Pathol 2009

2. Gerami et al. Arch Dermatol 2010; 146:273-8

3. Morey et al, Pathology 2009

4. Vergier et al. Mod Pathol 2011;24:613-23

5. Gaiser et al, Mod Pathol 2010; 23:413-9

6. NeoGenomics validation set

7. NeoGenomics validation set with original cutoff criteria

Summary of sensitivity

estimates on ambiguous lesions

1 2 3

Study sample 27 113 8

Sensitivity 100% 43% 60%

Specificity 71% 80% 33%

1. Gerami et al. Am J Surg Pathol 2009

2. Vergier et al. Mod Pathol 2011;24:613-23

3. Gaiser et al, Mod Pathol 2010; 23:413-9

Reasons for False

Positives/Negative• Technical problems

• Lack of experience

• Tetraploidy in Spitz nevi (5-10%)

• Detection of non-significant clones • Detection of non-significant clones

• Signal cutoffsProbe Criterion Original Cutoff Neo

Genomics

Cutoff

RREB1 >2 RREB1 >29 >16

RREB1 RREB1>CEP6 >55 >53

MYB MYB<CEP6 >40 >42

CCND1 >2 CCND1 >38 >19

Other potential applications

• “Nevoid” melanomas vs “Mitotically

active” nevi

Mitotically active

nevus

N=10

Nevoid melanoma

N=10


10/10 cases FISH negative 10/10 cases FISH positive



active” nevi

• Intranodal nevus vs Metastatic

MelanomaMelanoma

Metastatic Nodal nevusMetastatic

melanoma

N=24

Nodal nevus

N=17

FISH status 20 cases FISH

positive

16 cases FISH

negative

Sensitivity

83%

Specificity

94%

Scott et al. Am J Surg Pathol 2010



active” nevi

• Intranodal nevus vs Metastatic

MelanomaMelanoma

• Blue nevus vs Blue nevus-like

metastatic melanoma

Blue nevus-like

metastatic

melanoma

N=10

Blue nevus

N=10

FISH status 9 cases FISH

positive

10 cases FISH

negative

Sensitivity

90%

Specificity

100%

Pouryazdanparast et al. Am J Surg Pathol 2009

6p25 gains

Conclusion

• FISH provide additional criteria to help

diagnose histologically ambiguous

cases

• This test should be performed in

conjunction with standard

histopathologic evaluation

CGH• Technically difficult

• Needs tumor enrichment (30-

50% tumor purity)

• Cannot be used in thin lesions

• No histologic correlations

FISH• Technically easier

• Does not need tumor

enrichment

• Can be used in thin lesions

• Allows some histologic

correlations

Vs.

• Can miss changes in

subpopulations (low sensitivity)

• Scans entire genome (improve

sensitivity)

• Positive aberrations are likely

significant (high specificity)

• Will detect focal changes (high

sensitivity)

• Evaluates only 3 loci (decrease

sensitivity)

• Higher probability of false

positive (lower specificity)– Lack of experience in counting

– Tetraploidy

– Different cutoff points

Ambiguous lesion

Favor benign Favor malignant

FISH - FISH + FISH - FISH +

MelanomaNevus Ambiguous

(20-25% false

negative)

R/O False

Positive

•Tetraploidy

•Focal changes

Yes No

Nevus Ambiguous

Is this final?

• Sensitivity not great

• Interpretation is labor intensive

• Technically challenging

• Expensive• Expensive

• More probes/cutoff points need to be

evaluated

“Vidraru” Dam, Romania

Melanoma treatment

FDA approved therapy

• High-dose interleukin-2

• Dacarbazine

• Response rates: 7-20%• Response rates: 7-20%

• No improvement in survival

New FDA approved drugs

• Ipilimumab (YERVOY)

– FDA approval: 3/25/2011

• Vemurafenib (ZELBORAF)• Vemurafenib (ZELBORAF)

– FDA approval: 8/17/2011

Ipilimumab

• Blocks cytotoxic T-lymphocyte–associated

antigen 4 (CTLA4) and promoted

antitumor immune response(Q.)

APC

MHC

Ag

TCR

T-cell

CD80/86

CD28

CD80/86

Melanoma

APC

MHC

Ag

TCR

T-cell

CD80/86

CTLA-4

CD80/86

Melanoma

APC

MHC

Ag

TCR

T-cell

CD80/86

CTLA-4

Melanoma

Anti

CTLA-4

AB

Ipilimumab phase 3 trial

Ipi alone: 10.1 months

Ipi+gp100: 10 months

gp100: 6.4 months

Med survival

Hodi et al. N Eng J Med, 2010,

Ipilimumab phase 3 trial

Ipi+dacarbazine: 11.2 months

Dacarbazine: 9.1 months

Med survival

Robert et al. N Eng J Med, 2011,

Vemurafenib

• BRAF V600E inhibitor (Q.)

GROWTH

FACTORS

RAS

B-RAF

CYTOPLASM


melanocytic lesions

40-60% of melanomas, 90% is V600E

MEK

ERK

PROLIFERATION

CYTOPLASM

NUCLEUS

GROWTH

FACTORS

RAS

B-RAF

CYTOPLASM


melanocytic lesions

Vemurafenib

MEK

ERK

PROLIFERATION

CYTOPLASM

NUCLEUS

PLX4032 (Vemurafenib)

• Phase III (BRIM3):

– 675 patients

– Vemurafenib vs Dacarbazine

– 63% reduction in risk of death

– 74% reduction in risk of death and disease

progression

Chapman PB. Et al. N Engl J Med 2011;364:2507-16.

Vemurafenib phase 3 trial

Vemurafenib: 84%

Dacarbazine: 64%

6-month survival

Side effects

• Rash

• Photosensitivity

• Hair loss

• Joint pain• Joint pain

• Liver problems

• Arrhythmias

• Allergic reactions

• Cutaneous SCC (26%) (Q.)

Cobas 4800 BRAF V600E

Mutation Test• FDA approved companion diagnostic test

• Detects BRAF V600E mutation

• Real time PCR (Q.)• Real time PCR (Q.)

Downtown, Timisoara, Romania

QUESTIONS?

ReferencesMolecular alterations in melanoma1. Albino AP, Nanus DM, Mentle IR, Cordon-Cardo C, McNutt NS, Bressler J et al. Analysis of ras oncogenes in

malignant melanoma and precursor lesions: correlation of point mutations with differentiation phenotype.

Oncogene 1989;4:1363-74.

2. Bastian BC, LeBoit PE , Pinkel D. Mutations and copy number increase of HRAS in Spitz nevi with distinctive

histopathological features. Am J Pathol 2000;157:967-72.

3. Curtin JA, Busam K, Pinkel D , Bastian BC. Somatic activation of KIT in distinct subtypes of melanoma. J Clin

Oncol 2006;24:4340-6.

4. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H et al. Distinct sets of genetic alterations in

melanoma. N Engl J Med 2005;353:2135-47.melanoma. N Engl J Med 2005;353:2135-47.

5. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. Mutations of the BRAF gene in human cancer.

Nature 2002;417:949-54.

6. Haqq C, Nosrati M, Sudilovsky D, Crothers J, Khodabakhsh D, Pulliam BL et al. The gene expression signatures

of melanoma progression. Proc Natl Acad Sci U S A 2005;102:6092-7.

7. Jaeger J, Koczan D, Thiesen HJ, Ibrahim SM, Gross G, Spang R et al. Gene expression signatures for tumor

progression, tumor subtype, and tumor thickness in laser-microdissected melanoma tissues. Clin Cancer Res

2007;13:806-15.

8. Kashani-Sabet M, Rangel J, Torabian S, Nosrati M, Simko J, Jablons DM et al. A multi-marker assay to distinguish

malignant melanomas from benign nevi. Proc Natl Acad Sci U S A 2009;106:6268-72.

9. Koh SS, Opel ML, Wei JP, Yau K, Shah R, Gorre ME et al. Molecular classification of melanomas and nevi using

gene expression microarray signatures and formalin-fixed and paraffin-embedded tissue. Mod Pathol

2009;22:538-46.

10. Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM et al. High frequency of BRAF mutations in

nevi. Nat Genet 2003;33:19-20.

ReferencesCGH/ FISH in melanoma

1. Bastian BC, LeBoit PE , Pinkel D. Mutations and copy number increase of HRAS in Spitz nevi with distinctive

histopathological features. Am J Pathol 2000;157:967-72.

2. Bastian BC, Olshen AB, LeBoit PE , Pinkel D. Classifying melanocytic tumors based on DNA copy number

changes. Am J Pathol 2003;163:1765-70.

3. Bastian BC, Wesselmann U, Pinkel D , Leboit PE. Molecular cytogenetic analysis of Spitz nevi shows clear

differences to melanoma. J Invest Dermatol 1999;113:1065-9.differences to melanoma. J Invest Dermatol 1999;113:1065-9.

4. Bastian BC, Xiong J, Frieden IJ, Williams ML, Chou P, Busam K et al. Genetic changes in neoplasms arising in

congenital melanocytic nevi: differences between nodular proliferations and melanomas. Am J Pathol

2002;161:1163-9.

5. Bauer J , Bastian BC. Distinguishing melanocytic nevi from melanoma by DNA copy number changes:

comparative genomic hybridization as a research and diagnostic tool. Dermatol Ther 2006;19:40-9.

6. Dalton SR, Gerami P, Kolaitis NA, Charzan S, Werling R, LeBoit PE et al. Use of fluorescence in situ hybridization

(FISH) to distinguish intranodal nevus from metastatic melanoma. Am J Surg Pathol 2010;34:231-7.

7. Gaiser T, Kutzner H, Palmedo G, Siegelin MD, Wiesner T, Bruckner T et al. Classifying ambiguous melanocytic

lesions with FISH and correlation with clinical long-term follow up. Mod Pathol 2010;23:413-9.

8. Gerami P, Jewell SS, Morrison LE, Blondin B, Schulz J, Ruffalo T et al. Fluorescence in situ hybridization (FISH)

as an ancillary diagnostic tool in the diagnosis of melanoma. Am J Surg Pathol 2009;33:1146-56.

9. Gerami P, Mafee M, Lurtsbarapa T, Guitart J, Haghighat Z , Newman M. Sensitivity of fluorescence in situ

hybridization for melanoma diagnosis using RREB1, MYB, Cep6, and 11q13 probes in melanoma subtypes. Arch

Dermatol 2010;146:273-8.

ReferencesCGH/ FISH in melanoma

10. Gerami P, Wass A, Mafee M, Fang Y, Pulitzer MP , Busam KJ. Fluorescence in situ hybridization for

distinguishing nevoid melanomas from mitotically active nevi. Am J Surg Pathol 2009;33:1783-8.

11. Maize JC, Jr., McCalmont TH, Carlson JA, Busam KJ, Kutzner H , Bastian BC. Genomic analysis of blue nevi and

related dermal melanocytic proliferations. Am J Surg Pathol 2005;29:1214-20.

12. Moore SR, Persons DL, Sosman JA, Bobadilla D, Bedell V, Smith DD et al. Detection of copy number alterations

in metastatic melanoma by a DNA fluorescence in situ hybridization probe panel and array comparative genomic in metastatic melanoma by a DNA fluorescence in situ hybridization probe panel and array comparative genomic

hybridization: a southwest oncology group study (S9431). Clin Cancer Res 2008;14:2927-35.

13. Morey AL, Murali R, McCarthy SW, Mann GJ , Scolyer RA. Diagnosis of cutaneous melanocytic tumours by four-

colour fluorescence in situ hybridisation. Pathology 2009;41:383-7.

14. Newman MD, Lertsburapa T, Mirzabeigi M, Mafee M, Guitart J , Gerami P. Fluorescence in situ hybridization as a

tool for microstaging in malignant melanoma. Mod Pathol 2009;22:989-95.

15. Newman MD, Mirzabeigi M , Gerami P. Chromosomal copy number changes supporting the classification of

lentiginous junctional melanoma of the elderly as a subtype of melanoma. Mod Pathol 2009;22:1258-62.

16. Pouryazdanparast P, Newman M, Mafee M, Haghighat Z, Guitart J , Gerami P. Distinguishing epithelioid blue

nevus from blue nevus-like cutaneous melanoma metastasis using fluorescence in situ hybridization. Am J Surg

Pathol 2009;33:1396-400.

References

Ipilimumab

1. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB et al.

Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J

Med 2010;363:711-23.

2. Robert C , Ghiringhelli F. What is the role of cytotoxic T lymphocyte-associated

antigen 4 blockade in patients with metastatic melanoma? Oncologist 2009;14:848-

61.

3. Robert C, Thomas L, Bondarenko I, O'Day S, M DJ, Garbe C et al. Ipilimumab plus

dacarbazine for previously untreated metastatic melanoma. N Engl J Med

2011;364:2517-26.

ReferencesVemurafenib1. Arkenau HT, Kefford R , Long GV. Targeting BRAF for patients with melanoma. Br J

Cancer 2011;104:392-8.

2. Capper D, Preusser M, Habel A, Sahm F, Ackermann U, Schindler G et al.

Assessment of BRAF V600E mutation status by immunohistochemistry with a

mutation-specific monoclonal antibody. Acta Neuropathol 2011;122:11-9.

3. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J et al.

Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N EnglImproved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl

J Med 2011;364:2507-16.

4. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA et al. Inhibition

of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010;363:809-19.

5. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H et al. Melanomas acquire

resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature

2010;468:973-7.

6. Ribas A, Kim KB, Schuchter LM, et al. BRIM-2: an open-label, multicenter phase II

study of vemurafenib in previousl treated patients with BRAFV600E mutationpositive

melanoma. J Clin Oncol 2011; 29:Suppl:8509. abstract.

7. Vultur A, Villanueva J , Herlyn M. Targeting BRAF in advanced melanoma: a first step

toward manageable disease. Clin Cancer Res 2011;17:1658-63.

ReferencesOther1. Corona R, Mele A, Amini M, De Rosa G, Coppola G, Piccardi P et al. Interobserver

variability on the histopathologic diagnosis of cutaneous melanoma and other

pigmented skin lesions. J Clin Oncol 1996;14:1218-23.

2. Farmer ER, Gonin R , Hanna MP. Discordance in the histopathologic diagnosis of

melanoma and melanocytic nevi between expert pathologists. Hum Pathol

1996;27:528-31.

3. Greider CW , Blackburn EH. A telomeric sequence in the RNA of Tetrahymena 3. Greider CW , Blackburn EH. A telomeric sequence in the RNA of Tetrahymena

telomerase required for telomere repeat synthesis. Nature 1989;337:331-7.

4. Kornstein MJ , Byrne SP. The medicolegal aspect of error in pathology: a search of

jury verdicts and settlements. Arch Pathol Lab Med 2007;131:615-8.

5. Lodha S, Saggar S, Celebi JT , Silvers DN. Discordance in the histopathologic

diagnosis of difficult melanocytic neoplasms in the clinical setting. J Cutan Pathol

2008;35:349-52.

6. Lundblad V , Szostak JW. A mutant with a defect in telomere elongation leads to

senescence in yeast. Cell 1989;57:633-43.

7. McGinnis KS, Lessin SR, Elder DE, Guerry Dt, Schuchter L, Ming M et al. Pathology

review of cases presenting to a multidisciplinary pigmented lesion clinic. Arch

Dermatol 2002;138:617-21.

emerging molecular diagnostic tests and therapies for melanoma

Documents