Australasian Division of the International Academy of Pathology

Vincent McGovern Memorial Lecture Sydney June 2013

Richard A Scolyer MD FRCPA FRCPath Senior Staff Pathologist, Tissue Pathology & Diagnostic Oncology,

Royal Prince Alfred Hospital, Co-Director of Research, Melanoma Institute Australia,

Clinical Professor, Discipline of Pathology, Sydney Medical School, The University of Sydney, Sydney, Australia

Fighting Australia’s National Cancer: Progress to Date & Future Prospects

Founding President of Australasian Division IAP (1973-78)

Outline • “Australia’s National Cancer”

• Multipronged strategy for disease control

– Prevention

– Early detection, Dx & Rx

• New diagnostic techniques

• Accurate pathological dx

• Rx of primary tumour

– Rx of metastatic disease

Melanoma in Australia

• Melanoma is a major public health problem

• Australia has highest incidence worldwide

Melanoma incidence

0 10 20 30 40

Po

pu

lati

on

Incidence per 100,000

Hong Kong

S Thames

Florence

Denmark

Zurich

Norway

US Atlanta

NZ

Aus

62.9 / 51.3

59.9 / 37.4

31 / 24.4

QLD

NSW

VIC

Melanoma Incidence Rates (/100,000, M/F)

Melanoma in Australia: 1996-2009

Men Women

Incidence ↑22% ↑17%

Mortality ↑16% ↑6

Contrasts with ↓mortality rates for most other solid cancers in

Australia during this period

Melanoma: Dimensions of the Problem

• 3rd most common cancer in both men and women in Australia

• Overall life time risk of 5.8%

• >10,000 new cases annually

• 1,200 Australians die of melanoma each year

• “Australia’s National Cancer”

Melanoma: Dimensions of the Problem

• Commonest cancer in

– Men: 15-50 years

– Women: 15-35 years

• Therefore has a disproportionate impact on most productive years of life

Melanoma Disease Control

Prevention Early Diagnosis & Treatment

Treatment of Metastatic Disease

Outline • “Australia’s National Cancer”

• Multipronged strategy for disease control

– Prevention

– Early detection, Dx & Rx

• New diagnostic techniques

• Accurate pathological dx

• Rx of primary tumour

– Rx of metastatic disease

UV Damages DNA

Melanoma Risk Factors

High

Risk

Lower

Risk

Sun Smart Behaviour • Avoid outdoor activities in middle of day

• Physical barriers

– Shade protection

– Clothes

– Hat

• Sun screen (50+ BS)

• Need some exposure (Vit D)

1980 Public Health Campaign

Sunbed use causes 16% of melanomas in those aged 18-29 yr

Sunbed use causes 76% of all melanomas in 18-29 years who ever used a sunbed

Cust AE, et al. Int J Cancer. 2011 May 15;128(10):2425-35

States with sun bed bans

Outline • “Australia’s National Cancer”

• Multipronged strategy for disease control

– Prevention

– Early detection, Dx & Rx

• New diagnostic techniques

• Accurate pathological dx

• Rx of primary tumour

– Rx of metastatic disease

• Diagnostics:

Evolution of Dermoscopy Kerry Crotty Scott Menzies

Sequential digital dermoscopy imaging - time often tells

melanoma

3 months

Principle: any lesion that is changing over a 3 months

should be excised

99.6% of the lesions that do not change over 3 months

are not melanoma

Altamura D,. Arch. Dermatol. 144(4), 502–506 (2008).

benign

3 months

Total Body Photography

Patients with multiple

naevi or very high risk

for primary melanoma

Digital Images:

Reference for both

- clinician

- patient self monitoring

Reflectance Confocal Microscopy

Optical biopsy

Particularly useful in

assessing Lentigo Maligna

Pascale Guitera

200-250um max.

Horizontal section

i

w i

e

CONFOCAL HISTOLOGY

epidermis

dermis

In-Vivo Confocal

vs. H&E Histological

Horizontal sections

Melanin back scattered the light= bright cells

H&E (enface, greyscale)

• 58 yr female: • Lentigo Maligna Melanoma

• (Breslow 0.3mm)

• amelanotic insitu tumour extended to excision margins

Confocal Imaging: Lentigo Maligna

Extent of abnormal melanocytes by CM

Surgical margin indicated by CM Following repair

New pic macro & micro & quote results Judy’s study

Grogan J, Cooper CL, McCarthy SW, Menzies SW, Scolyer RA. Punch scoring improves

clinicopathologic correlation in evaluating pigmented lesions: a review of 45 cases (in preparation)

Outline • “Australia’s National Cancer”

• Multipronged strategy for disease control

– Prevention

– Early detection, Dx & Rx

• New diagnostic techniques

• Accurate pathological dx

• Rx of primary tumour

– Rx of metastatic disease

Gold standard = Pathology

Gold standard = Pathology

Gold standard = Clinical Behaviour

Goal of Pathological Assessment

• Predict clinical outcome/behaviour of tumour

– From histopathological features

– Small part of lesion

– Static point in time

Pathological Assessment

• Routine pathol: dx most melanocytic lesions

– Accurately

– Quickly

– Reproducibly

• Small subset cause diagnostic problems

Why Can Melanocytic Path Be Difficult ?

• Diagnostic criteria

– Architectural

– Cytological

– Host response

• Each criterion can occur in naevi & melanoma

• Diagnosis (opinion) rests on

– Balancing criteria

– Awareness of pitfalls

– Correlating with clinical data

McCarthy SW, Scolyer RA. Pitfalls and important issues in the diagnosis of melanocytic

tumours. The Oschner Journal; 2010: 10:66–74

Pathological Diagnosis is Therefore Subjective

Grey Zones / “Borderline Lesions” Superficial Atypical Melanocytic Proliferations

Deep Dermal Atypical Proliferations

Superficial Atypical Melanocytic Proliferations

• Nearly all patients cured by complete excision

• Wide local excision with narrow (0.5-1cm) margins usually minimal morbidity

• Resolve most by careful CPC

Deep Dermal Atypical Proliferations

• Prognosis naevus v melanoma vastly different

• Mx per melanoma? – Morbidity WLE?

– Sentinel node bx?

– Counselling re

• Prognosis

• Follow up

• More difficult to resolve

• New molecular techniques may be helpful

Ng JC, et al. Impact of partial biopsy on histopathologic diagnosis of melanoma. Archives Dermatol 2010 Mar;146(3):234-9

• Victoria Melanoma Service 1995-2006: 2470 pts

• ↑ odds ratio pathologic misdiagnosis – Punch bx 16.6 (p<0.001)

– Shave bx 2.6 (p=0.02)

• Punch bx: OR 20 (p<0.001) misdx + adverse outcome

Incomplete Biopsies of Melanocytic

Lesions: WARNINGS!

• Be careful when reporting incomplete biopsies

• Any atypical features: be cautious in report

• “Complete ex & reassess may be advisable”

Scolyer RA, Prieto VG. Melanoma pathology: important issues for clinicians involved in the

multidisciplinary care of melanoma patients. Surg Oncol Clin N Am. 2011 Jan;20(1):19-37

Diagnostic Pitfalls

Naevi Mimicking Melanoma

• Spitz naevus

• Deep penetrating naevus

• Acral naevus

• Combined naevus

Melanomas Prone to Misdiagnosis

• Desmoplastic melanoma

• Acral melanoma

• Spitzoid melanoma

• Naevoid melanoma

Thigh: DPN F35 Acral Naevus

Both this bx and

another 1 year

previously reported as

non-specific

inflammation

Desmoplastic

melanoma

Naevoid Melanoma: Architecture

• Nodular (or verrucous) with no RGP

• Circumscribed

• Basically symmetrical

• Pagetoid spread minimal or absent

• Long thin rete ridges

• Subtle asymmetry

• “Pseudomaturation”

• Expansile or sheet-like growth

Lower leg

Naevoid Melanoma

An Approach to Ambiguous Cases

• Give a preferred diagnosis but acknowledge degree of uncertainty

• Further opinions should be sought

• “Don’t sweep doubt under the carpet”

• Danger “UMP” becomes a wastebasket

• Complete excision probably mandatory

• Avoid SLNB

Scolyer RA, Murali R, McCarthy SW, Thompson JF. Histologically ambiguous ("borderline") primary

cutaneous melanocytic tumors: approaches to patient management, including the roles of molecular testing

and sentinel lymph node biopsy. Arch Pathol Lab Med. 2010 Dec;134(12):1770-7.

Tools for Resolving Diagnostic Uncertainty in Primary Tumours

• Molecular testing offers potential to identify tumour subgroups & predict their likely clinical course – CGH

– FISH

– PCR- BRAF, NRAS, HRAS

– Proteomics

– Immunochemistry

• Aim to find a molecular signature(s) to serve as diagnostic tool in differentiating melanoma from naevus

• OFTEN NOT DEFINITIVE MELANOMA

• WE STILL NEED BETTER TOOLS

Male 6yo

copy number gains at 6p25 homozygous deletions at 9p21

Developed distant metastases (including brain)

Importance of Histopathological Reporting

• Melanoma pathology report should

– Document the key diagnostic criteria on which the diagnosis was based

– Provide the pathological parameters important for prognosis & management

Cox regression analysis for 10,233

melanoma patients with localized melanoma

including mitotic rate

Variable Chi-square values

(1 d.f.) P HR

95%

CI

Tumor thickness 84.6 <0.0001 1.25 1.91 – 1.31

Mitotic Rate 79.1 <0.0001 1.26 1.20 – 1.32

Ulceration 47.2 <0.0001 1.56 1.38 – 1.78

Age 40.8 <0.0001 1.16 1.11 – 1.22

Gender 32.4 <0.0001 0.70 0.62 – 0.79

Site 29.1 <0.0001 1.38 1.23 – 1.54

Clark Level 8.2 0.0041 1.15 1.04 – 1.26

Melanoma of the Skin, 7th Edition AJCC Manual for Staging of Cancer –2010.

Cox regression analysis for 10,233

melanoma patients with localized melanoma

including mitotic rate

Variable Chi-square values

(1 d.f.) P HR

95%

CI

Tumor thickness 84.6 <0.0001 1.25 1.91 – 1.31

Mitotic Rate 79.1 <0.0001 1.26 1.20 – 1.32

Ulceration 47.2 <0.0001 1.56 1.38 – 1.78

Age 40.8 <0.0001 1.16 1.11 – 1.22

Gender 32.4 <0.0001 0.70 0.62 – 0.79

Site 29.1 <0.0001 1.38 1.23 – 1.54

Clark Level 8.2 0.0041 1.15 1.04 – 1.26

Melanoma of the Skin, 7th Edition AJCC Manual for Staging of Cancer –2010.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

SURVIVAL (years)

0 (n=807)

PR

OP

OR

TIO

N O

F S

UR

VIV

ING

1- 4 (n=1828)

5-10 (n=715)

(>=11 (n=311)

P value

Overall <.0001

(1) vs (2) <.0001

(1) vs (3) <.0001

(1) vs (4) <.0001

(2) vs (3) <.0001

(2) vs (4) <.0001

(3) vs (4) .0009

Results Mitotic Rate

Phase 2: Retrospective Analysis of Vince McGovern’s Work

• Performed the 1st detailed analyses assessing the prognostic importance of histopathologic features of melanomas

• Reviewed a very large number of melanomas during his career

Vincent McGovern 1915-1983

Francken et al. Ann Surg Oncol 2004; 11: 426-33.

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25

PR

OP

OR

TIO

N S

UR

VIV

ING

Mitosis 0/5 HPF (n=581)

Mitosis 1-4/5 HPF (n=455)

Mitosis 5 or more/5 HPF (n=281)

P-valueOverall <0.0001

SURVIVAL TIME (years)

Results

Phase 2: Methods

• VJMcG followed recommendations of 1972 International Pigment Cell Conference in determining TMR

• Average no. mitoses in at least 10 HPF determines and expressed as no. mitoses/5HPF

• Coded as – 1: 0 mitoses /5HPF

– 2: 1-4 mitoses / 5HPF

– 3: >=5 mitoses /5HPF

Phase

• Problem in converting his TMR grade to TMR per mm2

• His papers state he used magnification of X 320

• Need to determine HPF area of the microscope he used

Pathologic

feature

Whole group MIA

Pathologists

Non-MIA

Pathologists

Breslow thickness 0.96 0.95 0.94

Tumor Mitotic

Rate

0.76 0.72 0.80

Clark level 0.60 0.56 0.59

Ulceration 0.83 0.91 0.73

Pathologic

feature

Whole group MIA

Pathologists

Non-MIA

Pathologists

Breslow thickness 0.96 0.95 0.94

Tumor Mitotic

Rate

0.76 0.72 0.80

Clark level 0.60 0.56 0.59

Ulceration 0.83 0.91 0.73

AJCC: Recommended Method for Mitotic Rate Enumeration

• Find area with most mitoses (“hot spot”)

• Extend to adjacent HPF until 1mm2 assessed

• If no “hot spot”: start in field with a mitosis

• Express MR as no./mm2

Early Regression= TILs

Intermediate Regression= Angiofibroplasia

Late Regresssion

Multipanel Figure Illustrating TIL Grades

TIL Grade 0 TIL Grade 1

TIL Grade 2 TIL Grade 3

TIL Grade 2 TIL Grade 3

Sentinel

Node

Status

TIL grade

0 1 2 3 Total P value

Negative 299 406 147 34 886 0.0001

Positive 111 95 29 2 237 % Positive 27.1 19.0 16.5 5.6 100

Sentinel Lymph Node Biopsy

Performed in 1123 (60%) patients

Scolyer RA*, Azimi F*, Moncrieff M, Rumcheva P, Murali R, McCarthy SW, Saw RP, Thompson JF. Tumor-infiltrating lymphocyte grade (TIL grade) is an independent predictor of sentinel lymph

node status and survival in cutaneous melanoma patients. J Clin Oncol July 2012

Scolyer RA*, Azimi F*, et al. J Clin Oncol July 2012

Melanoma-specific survival Recurrence-free survival

Factors predicting melanoma-specific survival

Variable

Hazard

Ratio

95% Confidence

Intervals

Test

Statistic p value

Melanoma-Specific

Survival

Mitotic Rate 1.02 1.01-1.04 9.13 0.003

Satellitosis 2.70 1.64-4.43 15.38 <0.0001

Ulceration 2.38 1.80-3.14 37.42 <0.0001

Gender * 1.45 1.09-1.92 6.65 0.01

Breslow Thickness 1.10 1.07-1.12 74.90 <0.0001

TIL Grade ** 0.66 0.55-0.79 20.58 <0.0001

Age at Diagnosis - - - NS * Female as reference value

** TIL grade 0 as reference value

Survival in SLNB Patients

Variable Hazard Ratio 95% Confidence

Intervals Wald Statistic p value

Melanoma-Specific Survival* Sentinel node positive 3.62 2.56-5.12 53.25 <0.0001 Satellitosis 2.65 1.43-4.93 9.49 0.002 Ulceration 2.38 1.88-3.80 29.78 <0.0001 Breslow Thickness 1.08 1.04-1.11 18.20 <0.0001 TIL Grade 0.74 0.58-0.94 6.26 0.012 Recurrence-Free Survival**

Sentinel node positive 3.51 2.66-4.63 78.73 <0.0001 Satellitosis 2.10 1.24-3.55 7.57 0.006 Ulceration 1.98 1.51-2.61 23.70 <0.0001 Age at Diagnosis 1.02 1.01-1.03 14.66 0.0001 Breslow Thickness 1.06 1.03-1.09 15.37 <0.0001 TIL Grade 0.82 0.68-0.99 4.24 0.04

* Mitotic rate, age at diagnosis and gender were not significant ** Mitotic rate and gender were not significant

P<0.0001

Melanoma-specific survival N (% of ulcerated cases)

5-year survival 10-year survival

Ulceration

Absent 4126 91.30% 81.10%

Present 519 77.60% 62.40%

Extent of ulceration categorized as diameter 519 (100%)

≤ 5.00 mm 386 (74.4%) 82.70% 69.10%

> 5.00 mm 133 (25.6%) 59.30% 33.00%

Extent of ulceration categorized as percentage 216 (100%)

≤ 70% 162 (75.0%) 80.40% 67.30%

>70% 54 (25.0%) 66.40% 37.90%

Melanoma-specific Survival Rates

ICCR Primary Invasive Melanoma Protocol • Required elements

– Breslow thickness

– Ulceration

– Mitotic rate

– Lymphovascular invasion

– Satellites

– Desmoplastic component

– Neurotropism

– Margins

• Insitu

• Invasive (peripheral & deep)

– AJCC staging • pT

• pN

• Recommended elements

– Melanoma subtype

– Extent of ulceration

– Clark level

– TILs

– Regression (intermediate & late)

– Associated melanocytic lesion

DATASET FOR PATHOLOGY REPORTING OF CUTANEOUS INVASIVE MELANOMA: RECOMMENDATIONS FROM

THE INTERNATIONAL COLLABORATION OF CANCER REPORTING (ICCR)

Richard A. Scolyer MD, FRCPA, FRCPath1,2,4, Meagan J. Judge BSc, DipEd6, Alan Evans BMedBiol, MD, FRCPath7, David

P. Frishberg MD8, Victor G. Prieto MD, PhD9, John F. Thompson MD, FRACS, FACS1,3,5, Martin J. Trotter MD, PhD,

FRCPC10, Maureen Y. Walsh MB, FRCPath11, Noreen M.G. Walsh MD, FRCPC, FRCPath12, David W. Ellis MBBS, FRCPA13

Am J Surg Pathol (in press)

Outline • “Australia’s National Cancer”

• Multipronged strategy for disease control

– Prevention

– Early detection, Dx & Rx

• New diagnostic techniques

• Accurate pathological dx

• Rx of primary tumour

– Rx of metastatic disease

http://www.nhmrc.gov.au/guidelines/publications/cp68

RECOMMENDED EXCISION MARGINS

• Melanoma insitu: 5mm

• Melanoma BT <1.0mm: 1cm

• Melanoma BT 1-2mm: 1-2cm

• Melanoma BT >2mm: 2-3cm

What about SLN biopsy?

SLN status • Strongest predictor of outcome

• Most accurate staging method available

Breslow % + SLN

<0.75 <5%

0.75-1.0 5-10%

1.0-4.0 15-25%

>4.0 30-40%

Recommendations for SLNB

• ANZ melanoma guidelines: discuss BT >1mm

• MIA:

– Offered BT >1mm

– BT 0.75-1.0mm if ulcerated, MR>2/mm2, (<45yo)

– Histological ambiguous tumours: avoid SLNB

Picture naevus cells in capsule and mm

Naevus Cells in Capsule Melanoma Cells

HMB45 HMB45

SNB: Problems in Interpretation

• Occas single MelanA or HMB45+ cells- signif?

• Suggest be careful not to over interpret them as melanoma if no corroborative evidence

MelanA

HMB45

3 Important Questions Unanswered

• significance of very small SN deposits?

• Do all SN+ patients require CLND?

• Optimal protocol for SLNB

• Long term follow-up & MSLT-II & Minitub

Outline • “Australia’s National Cancer”

• Multipronged strategy for disease control

– Prevention

– Early detection, Dx & Rx

• New diagnostic techniques

• Accurate pathological dx

• Rx of primary tumour

– Rx of metastatic disease

Systemic Treatments

• Major advances in Rx advanced stage disease since 2009

• 2 classes of effective therapy

1. Targeted therapies

2. Immunotherapies

Traditional Clinicopathological Classification

• Current (2006) WHO classification of melanoma lists 4 main melanoma subtypes – Superficial spreading melanoma

– Nodular melanoma

– Lentigo maligna melanoma

– Acral lentiginous melanoma

• 1972 Sydney classification – Based on work McGovern, Clark, Mihm, Reed, Cochran & others

McGovern VJ, Mihm MC, Bailly C, Cochran A, et al. The classification of

malignant melanoma and its histologic reporting. Cancer 1973;32:1446-1457.

Melanoma Subtypes: Traditional Clinicopathologic Classification

SUPERFICIAL SPREADING MELANOMA - most common

• Mean age 40s

• Large number of melanocytic naevi & more than a few dysplastic naevi strong risk factors

• Linked to intermittent UV exposure & sunburns

NODULAR MELANOMA – 15%

• Older people, esp. men

• More common on head & neck

• > 50% amelanotic (red or pink)

LENTIGO MALIGNA MELANOMA – 10–15%

• Older people, esp. outdoor workers

• More common on head & neck

• Linked to large cumulative doses of UV exposure

• Hutchinson’s melanotic freckle = lentigo maligna = premalignant

ACRAL LENTIGINOUS MELANOMA – 1–3%

• Equally common in people with dark skin

• Acral skin of palms & soles

• May have no relationship with UV exposure

Thompson JF, Scolyer RA, Kefford RF. Cutaneous melanoma. The Lancet 2005; 365: 687-701

Melanoma Classification in the 21st Century

• Move towards a molecular-based classification

• Recent discovery of critical somatic mutations

• >80% MAPK pathway

• Exploitation of mutations as therapeutic targets

• Revolutionizing patient management

Boris Bastian

Benign

Borderline

Malignant

Site

Epithelium associated

High UV

CSD Desmopl.

melanoma

Glabrous Mucosa

Acral melanoma

Mucosal melanoma

Low UV

Acquired nevus

Dysplastic nevus

Non-CSD melanoma

Spitz nevus

Atypical Spitz

tumor

Spitzoid melanoma

BRAF

NRAS

HRAS

KIT

Benign

Borderline

Malignant

Site

Non-epithelium associated

Skin

Congenital nevus

Melanoma in CN

Blue nevus

Atyical blue nevus

Blue nevus like melanoma

Internal organs

Melanoma

Melano-cytoma

Eye

Uveal melanoma

Uveal nevus

GNAQ

GNA11

NRAS

Image courtesy of Grant McArthur, Peter MacCallum Cancer

Institute, Melbourne

Day 1 Day 15

FDG-PET response to vemurafenib V600E+ melanoma

Flaherty KT, et al N Engl J Med 363:809, 2010

RAF

P

P

MEK

P

ERK

RTK

Cell Responses

SOS

GRB2

P P P

ATP

P

Normal Cell - MAPK Pathway

RAS

Cellular Proliferation, Survival, Migration

PI3K

P

Akt

P

mTOR

P

RAS

RAF

P P

P

MEK

ERK

P

RTK

Cell Responses

SOS

GRB2

Mutated BRAF

Cellular Proliferation, Survival, Migration

P

BRAF inhibitors vemurafenib

dabrafenib

LGX 818

BRAF Mutations

• In about 50% of primary melanomas

• Assoc with

– Young patient age

– Low cumulative solar damage

– Trunk or limb primary site

– Characteristic pathological features

Bauer J, Büttner P, Murali R, Okamoto I, Kolaitis NA, Landi MT, Scolyer RA, Bastian BC. BRAF mutations in

cutaneous melanoma are independently associated with age, anatomic site of the primary tumor and the degree

of solar elastosis at the primary tumor site. Pigment Cell Melanoma Res. 2011 Apr;24(2):345-51.

BRAFoma

“BRAFoma”

Long GV, Menzies AM, Nagrial AM, Haydu LE, Hamilton AL, Mann GJ, Hughes TM, Thompson JF, Scolyer RA,

Kefford RF. Prognostic and Clinicopathologic Associations of Oncogenic BRAF in Metastatic Melanoma. J Clin

Oncol. 2011 Apr 1;29(10):1239-46.

48% metastatic

melanoma carry

BRAF mutation

No specific metastatic

phenotype, other

than youth

Poorer prognosis from

diagnosis met mel,

not prognostic for

primary

LYS VAL/V THR ALA SER

598 599 600 601 602

G T G G C T A A G A C C T C T

LYS GLU/E THR ALA SER

G A G G C T A A G A C C T C T

Mutation = V600E

LYS VAL/V THR ALA SER

598 599 600 601 602

G T G G C T A A G A C C T C T

LYS LYS/K THR ALA SER

A A G G C T A A G A C C T C T

Mutation = V600K

BRAF Mutation N=308

V600E

73%

V600K

19%

Other BRAF

genotypes

8%

BRAF WT

54%

BRAF Mut

46%

BRAF Mut

46%

Cutaneous 86%

Occult 10%

Mucosal 2%

Acral 2%

A. M. Menzies, L. E. Haydu, L. Visintin, M. S. Carlino, J. R. Howle, J. F. Thompson, R. F. Kefford, R. A. Scolyer, and G. V. Long CCR 2012

BRAF Rate by Decade N=308

0

25

50

75

100

20-29 30-39 40-49 50-59 60-69 >70

90% V600E 55% V600E

20% V600K

Menzies AM, Haydu LE, Visintin L, Carlino MS, Howle JR, Thompson JF, Kefford RF, Scolyer RA, Long GV. Age and Chronic Sun

Damage Predict BRAF Genotype in BRAF-mutant Metastatic Melanoma. Clin Cancer Res. 2012 Jun 15;18(12):3242-9

Dabrafenib Best unconfirmed response : V600K (BREAK-2)

-50

-40

-30

-20

-10

0

10

20

30

40

Max

imu

m %

red

uct

ion

fro

m b

ase

line

M1c M1a M1 M1b

Scans unavailable for 1 patient

M-Stage at screening Missing

Trefzer U et al SMR 2011

Mutation Testing: Issues for Discussion

1. When should a BRAF test be performed?

2. Which specimen to test (primary v metastasis)?

3. What type of tissue is required?

4. Which technique for mutation testing?

1. When should a BRAF test be performed?

• Planning treatment for metastatic disease

– Unresectable AJCC stage III

– AJCC stage IV

2. Which specimen to test? (primary v metastasis)

• BRAF concordance primary v’s metastasis

– Yes: 80%-96% (Colombino et al J Clin Oncol 2012)

best

• distant metastatic tissue (most recent)

then

• locoregional/in-transit metastasis

last

• primary melanoma

3. What type of specimen is required?

• Formalin-fixed paraffin-embedded tissue ok (don’t require fresh tissue)

• Biopsy with high % tumour cell content best

• Core biopsies & FNA cell blocks often ok

X X

Sensitivity of Mutation Tests

Technical Sensitivity

Diagnostic Sensitivity /

Comprehensiveness

HIGH

Detect mutations

<1% tumour

(Risk false +)

LOW

eg Need >25% tumour

(Risk false negaives)

HIGH All mutations

(rare mutations of

unknown significance)

LOW Targeted

common

mutations only

(Risk false

negatives)

Diagnostic Sensitivity /

Comprehensiveness

4. The Ideal Mutation Assay

Next Generation Sequencing • Sensitive and comprehensive • Advantages

– Fully sequence many genes (100s-1000s) in a single test – Simultaneously detect translocations, base substitutions,

deletions, insertions, copy no. changes in cancer-related genes

• Challenges – $ Infrastructure cost – Bioinformatics – loads of data – Interpretation – TAT ~7-14d for multiplex (>100-gene) cancer genome

sequence , but expected to improve++

Wilmott et al Molecular Cancer Therapeutic 2012 Dec; 11:2704-8

pERK

Ki67

Cyclin D1

V600E Antibody - VE1

BRAF

wt

BRAF

V600K

BRAF

V600E

Long GV, Wilmott JS, Capper D, Preusser M, Thompson JF, Kefford RF, von Deimling A, Scolyer RA.

Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in

melanoma. Am J Surg Pathol 2013 Jan;37(1):61-5

BRAF V600E

37

97 melanoma metastases

DNA mutation testing HRM Sanger sequence

Non-V600E

10

BRAF WT

50

VE1 IHC +

35

VE1 IHC -

2

VE1 IHC -

47

VE1 IHC +

3

All

VE1 IHC -

Re-sequenced

K601Q

1

FNAB

1

PCR-mass spec

V600E

2

Lymph node

isolated VE1

1

Long, Wilmott, Capper, Preusser Zhang, Thompson, Kefford,

von Deimling, Scolyer, Am J of Surg Path, 2013

V600E Antibody - VE1

Isolated staining within LN

Molecular testing BRAFwt

Molecular testing initially

BRAF WT

FNAB – BRAF V600E

By molecular methods

Long – Wilmott & Scolyer et al AJSP 2013 Jan;37(1):61-5

BRAF Antibody • Detects V600E BRAF mutations

– Accurate

– Rapid

– Cost effective method

• Facilitates rapid triage into Rx pathways

• Does not detect non-V600E mutations (Cobas)

• MIA: all patients undergoing mutation testing

BRAFi Cutaneous Reactions

• Grover’s disease

• Hyperkeratotic lesions

• KA/SCC

Cellular Proliferation, Survival, Migration

BRAF I

RAS

BRAFwt Keratinocyte

CRAF

MHC

TCR

Blocking CTLA-4 ligation

enhances T-cell responses

Ipilimumab

T cell

CTLA-4

APC

T-cell activation

B7

CD28

Immunopotentiating Agents

Ipilimumab (anti-CTLA-4) & PD-1 inhibitors stimulate immune system to destroy melanoma cells

Anti-PD1

Anti-PD-L

Biopsy BEFORE Treat with trial drug Biopsy AFTER 7days Biopsy PROGRESSION

Examine for biomarkers of response and resistance

PRE POST PROG

“TEAM” Protocol Treat Excise Analyse Melanoma

J Wilmott, G V Long & R A Scolyer CCR 2012

CD8 CD4 Lymphocytes

PRE

EDT - Responding Day 7

Progression

Cellular Proliferation, Survival, Migration

PI3K

Akt

mTOR

MEK inhibitors

ERK inhibitor

BRAF inhibitors PI3K inhibitors

AKT inhibitors

mTOR inhibitors

PD-1

Ipilimumab

Cell cycle inhibitors

Tumor microenvironment inhibit

Outline • “Australia’s National Cancer”

• Multipronged strategy for disease control

– Prevention

– Early detection, Dx & Rx

• New diagnostic techniques

• Accurate pathological dx

• Rx of primary tumour

– Rx of metastatic disease

Conclusions

• Pathology remains critical to melanoma disease control

• Despite recent advances in Rx

• Metastatic melanoma remains a bad disease

• Must continue to raise the bar higher

Vincent McGovern 1915-1983

Stan McCarthy John Thompson

Vincent McGovern

Kerry Crotty Bill McCarthy

Gerry Milton

Acknowledgements

• Tissue Pathology, Royal Prince Alfred Hospital (alphabetic order)

– Lyndal Anderson – Caroline Cooper – Wendy Cooper – Oana Crainic – Ruta Gupta – Rooshdiya Karim – James Kench – Soon Lee – Annabelle Mahar – Stan McCarthy – Catriona McKenzie – Paul McKenzie – Sandra O’Toole – Elizabeth Robbins – Geoff Watson

– Julia Pagliuso – Others

• Melanoma Institute Australia – John Thompson – Jon Stretch – Georgina Long – Rick Kefford – Others

• Others – Kerry Crotty – Bill McCarthy – Scott Menzies

• Research Funding Support – Cancer Institute NSW – NHMRC – Melanoma Foundation of the

University of Sydney

Cameron Fellowship in Melanoma Pathology

• Based in Tissue Pathology at RPAH

• Aims

– Additional experience in skin pathology reporting

– Participation in translational research

• Applications

– Close: 7th September 2013

• Australian Speakers • Haematopathology: Benhur Amanuel

• Soft Tissue Pathology: Irene Low

• Neuropathology: Peter Robbins

• GI pathology: James Kench

• Gynaecological pathology: Lyndal Anderson

• Skin pathology: Richard Scolyer

• Other RPAH & PathWest staff

• MD Anderson speakers • Jeff Medeiros - Hematopathology

• Jae Ro - Genitourinary - Prostate

• Dipen Maru - Gastrointestinal

• Victor Prieto - Dermatopathology

• Jeanne Meis - Soft Tissue

• Neda Kalhor/ Cesar Moran

Lecture and case-based format

Strictly limited to 200 registrants

Friday 14th to Sunday 16th Feb 2014

Convenors – Cesar Moran

– Dominic Spagnolo

– Richard Scolyer

Registrations forms: http://www.sswahs.nsw.gov.au/RPA/AnatPathology/

Fri 14th - Sun 16th Feb 2014