Association of Molecular Pathologyaz9194.vo.msecnd.net/pdfs/130401/3.pdf•13q14.3 (Retinoblastoma gene) •11q13 (MEN gene) •1q25-31 (HPRT2 gene) •Fractional allelic loss . Syndromic

The Association for Molecular Pathology Education. Innovation and Improved Patient Care. Advocacy.

www.amp.org

Molecular Oncology

Jennifer L. Hunt, MD, MEd Aubrey J. Hough Jr, MD, Endowed Professor of Pathology

Chair of Pathology and Laboratory Medicine University of Arkansas for Medical Sciences

April 2013

Disclosure(s)

In accordance with ACCME guidelines, any individual in a position to influence and/or control the content of this ASCP CME activity has disclosed all relevant financial relationships within the past 12 months with commercial interests that provide products and/or services related to the content of this CME activity.

The individual below has responded that he/she has no relevant financial relationship(s) with commercial interest(s) to disclose:

Jennifer L. Hunt, MD, MEd

2

Agenda

• Tumor Suppressor Genes

• Parathyroid carcinoma

• Oligodendroglioma (Samowitz)

• Oncogenes

• Lung cancer

• Thyroid cancer

• Melanoma (Samowitz)

• Translocations in solid tumors

• Viruses

• Other types of tumor analysis

3

Types of Molecular Assays

4

• DNA based assays

– Single gene mutation analysis

– Quantitative assays for copy number

• Gene amplification

• Gene deletion (loss of heterozygosity)

• Comparative genomic hybridization arrays (copy number)

– Translocation analysis

• RNA based assays

– Expression microarray

– RT-PCR assays

Knudson’s Hypothesis

5

• Retinoblastoma observations

–Led to “Two-Hit Theory”

–Tumor suppressor genes

Family history No family history

Younger Older

Bilateral Unilateral

Multifocal Unifocal

Knudson A; PNAS, 68 (4): 820-3, 1971

Tumor Suppressor Gene Inactivation

Examples: Tumor Suppressor Genes

7

Gene Location

P53 17q13

Rb 13q14.1

APC 5q23

VHL 3p26-25

Tumor Suppressor Genes

8

• Inactivation events

• Point mutation

• Genetic loss

• Methylation

Tumor Suppressor Analysis

Loss of Heterozygosity FISH

Sequencing IHC

PCR-based Analysis

Requirement

Discrimination of two

copies of the gene

Vocabulary

11

• DNA polymorphisms

• Short tandem repeats (STRs)

• Microsatellites

• Variable nucleotide tandem repeats (VNTRs)

• Single nucleotide polymorphisms (SNPs)

DNA Polymorphisms

12

• Short tandem repeats

• 2 to 7 basepairs in length

• Dinucleotide, Trinucleotide, Tetranucleotide…

• Repeated a variable number of times

ATCG

ATCG ATCG ATCG ATCG ATCG ATCG

Tumor suppressor gene

13

TSG

TSG STR

STR

PCR Analysis

14

Normal Loss of heterozygosity

Loss of 1p: LOH Analysis

15

Normal

Parathyroid tumor

Allele 2 Allele 1

Allele 2 Allele 1

Loss of Genetic Material

• Loss of heterozygosity

• In situ hybridization (FISH or CISH)

• Comparative genomic hybridization

17

Parathyroid Carcinoma

• Clinical clues

• High serum calcium (>14)

• Mean in one series was 12

• Parathyroid hormone > 5 x normal

• Palpable mass

• Bone and cardiac symptoms

• Adherent gland intra-operatively

18

Sporadic Parathyroid Disease

19

• Implicated genes

• Loss of 1p

• 13q14.3 (Retinoblastoma gene)

• 11q13 (MEN gene)

• 1q25-31 (HPRT2 gene)

• Fractional allelic loss

Syndromic Hyperparathyroidism

20

• MEN 1

– Multiglandular hyperplasia (high percentage)

• MEN 2A

– Hyperplasia or adenoma (

HPT Jaw Tumor Syndrome

21

• HPRT2 gene

– Tumor suppressor gene

– Loss in tumors

– Point mutations in syndromic cases

HRPT2 Gene

22

• Tumor specificity – Parathyroid Carcinomas

– Fibro-osseous lesions

– Renal tumors

• Tumor Suppressor Gene – Chromosome 1q25-31

– Classic tumor suppressor gene • Point mutation

• Loss of second copy

Parafibromin Immunohistochemistry

23

Adenoma Carcinoma

Courtesy of Drs. Weinreb and Asa

Agenda




• Oncogenes

• Lung cancer

• Thyroid cancer



• Viruses


24

Companion Diagnostics

25

26

Rudolph Virchow Chronic Myelogenous Leukemia, 1847

//upload.wikimedia.org/wikipedia/commons/7/75/Hypolobated_small_megakaryocyte.jpg

27

Peter Nowell Chronic Myelogenous Leukemia, 1960

28

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Oncogenes

29

Normal Cell Cancer Cell

Proto-oncogene Oncogene

Oncogenes

30

• Proto-oncogene

• Function

– Encode proteins

• Cell growth

• Differentiation

• Apoptosis

• Signal transduction

Oncogene Activation

31

Oncogenes

32

Type Gene Location

Growth factor Basic FGF 4q26

Tyrosine kinase Her-2-neu 17q11.2

GTP-ase K-Ras 12p12

Serine-threonine kinase

BRAF 7q34

Transcription factor Myc 8q24

Oncogene Mutations

33

• Structural alteration

• Point mutations

• Increased concentration

• Amplification

• Inappropriate activation

• Translocations

Oncogene Detection Techniques

34

• PCR and detection methods

• Heteroduplex formation

• Allele specific PCR

• Full sequencing methods

• Sanger sequencing

• Single base extension

• Pyrosequencing

• Next generation sequencing

Agenda




• Oncogenes

• Lung cancer

• Thyroid cancer



• Viruses


35

Classification

36

Lung Carcinoma

Small Cell Carcinoma

Non-Small Cell Carcinoma

Adenocarcinoma Squamous cell carcinoma

Epidermal Growth Factor Receptor

37

• Altered in many tumors

– Protein Expression

– Gene copy number

– Gene sequence

Epidermal Growth Factor Receptor

38

• Chromosome 7p12

– Member of the ErbB family

• ErbB1: Her1 (EGFR)

• ErbB2: Her2/neu

• Trans-membrane receptor tyrosine kinase

– Signaling through:

• RAS-RAF-MAP

• PI3K-AKT

39

AKT

EG

FR

PI3K RAS

RAF

MEK

MAP Kinase mTOR

40 H&E p16 IHC HPV ISH

EGFR Mutation

41

Demographics Rate

Never-Smokers 51%

Smokers 9%

Female 38%

Male 13%

Adenocarcinoma 31%

Other 2%

East-Asian 29%

Other 8%

Pao W. et al. JCO 2005; 23(11); 2556-68.

Mutations in Lung Tumors

42

0%

20%

40%

60%

80%

EML4-ALK EGFR KRAS HER2

Never/light

Smoker

Sasaki T, Eur J Cancer 46:1773, 2010

EGFR Mutations

43

2 5 7 13 16 18-21 22-24 28

EGF binding EGF binding TM Tyrosine Kinase Autophosphorylation

Exon 18 Exon 19 Exon 20 Exon 21

Deletions Mutations Mutations Mutations

Distribution of EGFR Mutations

44 Sasaki T, Eur J Cancer 46:1773, 2010 Sharma S, Nature rev/Cancer 7:169, 2007

Deletion Mutation

Point Mutation

Targeting EGFR with Therapy

45

Generic name Trade name & company

Target of the drug Biomarker

Erlotinib Tarceva ®

OSI, Roche,

Genetech

EGFR tyrosine

kinase

EGFR mutations

Gefininib Iressa ®

Astra Zeneca

EGFR tyrosine

kinase

EGFR mutations

Cetuximab Erbitux ®

Merck

EGFR extracellular

domain

EGFR mutation,

possibly

amplification

Panitumimab Vectibix ®

Amgen

EGFR extracellular

domain

EGFR mutation,

possibly

amplification

46

RT

K

RAS

RAF P

MEK

MAP kinase

GTP

Monoclonal antibodies

Tyrosine kinase inhibitors

AKT

EG

FR

PI3K RAS

RAF

MEK

MAP Kinase

AL

K

RO

S1

RE

T

ME

T

mTOR

EML4-ALK Translocation

48

• Anaplastic lymphoma kinase (ALK)

– Chromosome 2p23

– Encodes for trans-membrane receptor tyrosine kinase

• Signaling through RAS-RAF-MAP and PI3K-AKT

• Echinoderm microtubule-associated protein-like 4 gene (EML4)

– Chromosome 2p21


49

• Clinical

– Demographics: similar to EGFR mutations

• More common in Asians, non-smokers, women, adenocarcinoma

– Incidence: 2 to 7% of lung cancers

• Tumor histology and type

– Adenocarcinoma

– Cells with intracellular mucin

– Solid, acinar growth

– Patients present at high stage

Targeting ALK with Therapy

50

Generic name Tradename &

company

Target of the drug Biomarker

Crizotinib Xalkori ®

(Pfizer)

Chimeric ALK

tyrosine kinase

EML4-ALK

translocation

ALK over-

expression by IHC

62%

6

26

EML4

ALK 20 24 28 22 16 18

8 10 12 14 16 18

27 21 25 29 23 19

9 11 13 15 17 19

17

7

20 – 29 1-6

1-13 20 – 29

1-14 20 – 29

1-15 20 – 29

1-18 20 – 29

1-20 20 – 29

EML4-ALK Detection: FISH

52

Gene Specific Issues

• RET-PTC and EML4-ALK

– Intrachromosomal rearrangements

53

EML4

ALK


55

ALK Immunohistochemistry

56

Tumor ALK antibody D5F3 antibody

Anaplastic large

cell lymphoma 10/19 11/19

Lung

Adenocarcinoma

(translocation +)

16/22 22/22

Sensitivity

Specificity

67%

97%

100%

99%

Mino-Kenudson M. Clin Cancer Res 16(5):1562, 2010.

RET Translocation

• Rearranged during transfection (RET)

• Chromosome 10q21

• Encodes for receptor tyrosine kinase

• Signaling through RAS-RAF-MAP and PI3K-AKT

• KIF5b

• Chromosome 10p11.2

• Incidence:

ROS1 Translocation

• ROS1

• Chromosome 6q21

• Encodes for receptor tyrosine kinase

• Fused in Glioblastoma (FIG)

• Chromosome 6q

• Incidence:

Resistance Mutations

60

• Primary and acquired resistance mutations

– EGFR mutations

– MET amplification and mutations

– KRAS mutations

– BRAF mutations

Exon 20

T790M

resistance

EGFR Resistance Mutations Exon 18, 5%

Exon 19, 45%

Exon 20, 5%

Exon 21, 45%

Deletion Mutation

Point Mutation

Exon 19

D761Y

resistance

MET Resistance Mutations

• Oncogene • Activating point mutations and amplification

• Lung cancer • ~5% in primary tumors

• ~20% of tumors resistant to EGFR inhibitor

BRAF Resistance Mutations

• Oncogene • Activating mutation in exons 15 and 11

• Lung cancer • ~3% of primary tumors

• V600E mutation accounts for ~40%

RAS Resistance Mutations

• Oncogene • KRAS, NRAS, HRAS

• Activating point mutations in codons 12, 13, 61

• Lung Cancer • ~20% of primary tumors

• Almost all are KRAS

AKT

mTOR

EG

FR

PI3K

LY294002

Worfmannin

PX-866

Perifosine

PIAs

API-2

Rapamycin

RAS

RAF P

MEK

GTP Farnesylation

Inhibitors

RAF Inhibitors

Monoclonal antibodies

Tyrosine kinase inhibitors

MAP Kinase

Agenda




• Oncogenes

• Lung cancer

• Thyroid cancer



• Viruses


67

Dissecting “Indeterminants”

69

• Thyroid nodule management

• “Indeterminants”: Benign vs. Malignant

–Follicular variant of PTC

–Follicular carcinoma

–Molecular testing

“In pursuit of the low grade malignancy”

Benign

Indeterminant

Malignant

Molecular Mutations in Thyroid Cancer

71

• Papillary carcinoma

• BRAF gene mutations

• RET/PTC translocations

• RAS mutations

• Follicular carcinomas

• RAS mutations

• PPARγ/PAX8 translocations

BRAF Gene Mutations

72

• Oncogene • Activating mutation in exon 15

• Thyroid cancer

• T1799A (Nucleotide: T > A)

• V600E (Codon: Valine > glutamate)

• Also seen in other tumors • Colon cancer (nonhereditary MSI cancers)

• Melanomas

BRAF Gene Mutations

73

RAS Gene Mutations

74

• Oncogene –Activating mutations

–NRAS and HRAS: Codon 61

–KRAS: codons 12, 13, 61

• Also seen in other tumors –Colon carcinoma

–Lung carcinoma

–Others

RAS Gene Mutations

75

Translocations

76

• RET/PTC Translocations

• Up to 15 different partner genes

• ELE1 and H4 most common

• Fusion protein in cytoplasm

• Relatively specific to papillary carcinoma

• PAX8-PPARγ Translocation

• Relatively specific to follicular carcinoma

RET-PTC Translocation

77

H4 Exon 1 H4 Exon 2

RET Exon 11 RET Exon 12

Intron 1

Intron 11

RET-PTC

RET-PTC

78

0%

5%

10%

15%

20%

25%

30%

35%

FVPTCa PTCa FCa FAFVPTCa FCa PTCa FA

35%

25%

15%

5%

Molecular Testing in Cytology

79

• BRAF testing adds to sensitivity of FNA alone

• Cost effective utilization needs more study

– Atypical and suspicious will be highest yield

– Adding molecular for benign and malignant may have limited value

• Assay method selection is important to consider

80

Sensitivity of FNA + BRAF Testing

0%

20%

40%

60%

80%

100%

Marchetti Zatelli Kim SW Pellizzo

FNA (s)

FNA+BRAF (s)

BRAF Positives in FNAs

81

0%

20%

40%

60%

80%

100%

Atypical Suspicious Follicular neoplasm

Kim SY, JCEM 96:658, 2011

N=141

N=54

N=10

82

BRAF Mutations in Thyroid Lesions

0%

10%

20%

30%

40%

50%

60%

70%

80%

FVPTC Conv TCV

BRAF as Prognostic Tool

83

• Association with other high risk factors

• Few studies have assessed survival

Study LN Mets Extension Recurrence Survival

Kebebew (2007) Yes Yes Yes No

Yasuhiro (2008) No No No No

Lupi (2007) Yes Yes N/A N/A

Lee (2007) No Yes N/A N/A

Different Approaches

84

• Commercial Testing

–Expression array testing

–Panel based approach

• Lab Developed Testing

–Panel based approach

Expression Array Assay

85

• Marketed as Affirma ® by Veracyte

• Proprietary assay: 142 gene expression markers are not published

• Published studies are all industry sponsored

• Performance characteristics

Benign Histo Malignant Histo

Benign MDX 93 7

Suspicious MDX 87 78

Panel Based Testing

86

• Marketed as MiRInform ® by Assuragen

• Laboratory Developed Tests can also be obtained

Risk of

Cancer in

each group:

AUS/FLUS Follicular

neoplasm

Suspicious for

malignancy

Mutation

positive

88% 87% 95%

Mutation

negative

6% 14% 28%

BRAF Companion Diagnostic

87

Drug Link

in Drugs@FDA Therapeutic Area Biomarker

Label Sections

with

Pharmacogenomic

Information

Vemurafenib1 Oncology BRAF

Indications and

Usage

Warning and

Precautions

Clinical

Pharmacology

Clinical Studies

Patient Counseling

Information

http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm?fuseaction=Search.SearchAction&SearchTerm=202429&SearchType=BasicSearch

Choice, Choice, Choices!

88

• BRAF Gene mutation testing

• Traditional Sanger sequencing

• Pyrosequencing

• Allele specific PCR

• Quantitative PCR

• Kit based testing

• Commercial vendors

• New immunohistochemical stain

90

To promote patient safety and high quality care, AMP respectfully asks FDA to specify that diagnostics be described by the biological description of the gene or mutation on drug labels and that identification of recommended diagnostic testing not be by brand name.

Agenda




• Oncogenes

• Lung cancer

• Thyroid cancer



• Viruses


92

Differential Diagnosis

• Clear cell mucoepidermoid carcinoma

• Clear cell carcinoma

• Biphasic tumor

• Unusual salivary gland tumors

94

Mucoepidermoid Carcinoma

• Incidence

• Most common malignant salivary gland tumor (children and adults)

• Major and minor salivary glands

• Peak incidence 5th to 6th decades

• Clinical

• Mass lesion

• Surgical treatment with margins

95

96 Mucus cells and Cysts

Mucoepidermoid Translocation

97

• t(11;19)(q21;p13)

• MECT1-MAML2

• MECT1: also known as CRTC1, TORC1, WAMTP1

• cAMP response element binding protein (CREB) regulated transcriptional coactivator

• MAML2: Notch coactivator

• Translocation activates Notch target genes independent of Notch ligands

MECT-MAML2 Translocation

98

Courtesy of Dr. Sanja Dacic

University of Pittsburgh

Translocation Analysis in MEC

99

Fehr, Genes Chrom Canc 47:203, 2008

Behboudi, Genes Chrom Canc 45:470, 2006

Okabe, Clin Cancer Res 12:3902, 2006

Martins, J Molec Diag 6:205, 2004

Seethala, AJSP 34:1106, 2010

69/97 28/40

5/39

71% 70%

13%

11/24 46%

High Grade

Clear Cell Carcinoma

• Incidence

• Rare

• > major

• 21% in parotid

• Peak in 6th to 8th decades

100

101 Clear cell carcinoma

Clear Cell Carcinoma

102

• t(12;22)

• EWSR1-ATF1 Translocation

• Similar to the translocation seen in clear cell sarcomas of tendons and aponeuroses, angiomatoid fibrous hystioctyoma, and clear-cell sarcomas of the gastrointestinal tract

Adenoid Cystic Carcinoma

• Incidence

• Relatively common

• Any salivary gland location

• Clinical

• Mass lesion

• Nerve palsies

• Surgical treatment with margins

103

Adenoid cystic carcinoma, cribriform

Adenoid Cystic Translocation

105

• t(6;9) (q22-23; p23-24)

• MYB-NFIB

– MYB

• Transcription factor with an important role in cell proliferation, apoptosis, and differentiation

• Highly expressed in immature proliferating cells, and down-regulated as cells become more differentiated

– NFIB: nuclear factor 1B

• Deregulation mechanism is not completely understood

Translocation in ACC

106 West R, Am J Surg Pathol 2011;35:92–99

Immunohistochemistry for Myb

107

• Myb protein expression by IHC

• Strong nuclear expression

• Cells with basal phenotype (myoepithelial cells)

• Absent in the luminal epithelial cells

• Some other salivary tumors had weak staining

Comparison of FISH with IHC

108

78% myb

IHC (+)

67% myb IHC (+)

46%

myb IHC

(+)

West R, Am J Surg Pathol 2011;35:92–99

Mammary Analogue Secretory Carcinoma

109

• Clinical

• Rare tumor, but not well described yet

• Mean age 45

• Males > Females

• 69% in major salivary glands

• 22% with nodal metastasis

• 92 month disease free survival

Skalova A, AJSP. May 2010;34(5):599-608. Connor A, AJSP. Jan 2012;36(1):27-34. Chiosea SI, Histopathology. Feb 28 2012.

110 Mammary Analogue Secretory Carcinoma

Mammary Analogue Secretory Carcinoma

• ETV6-NTRK3

• t(12;15)

111

Other Examples of Solid Tumors with Translocations

112

• Synovial sarcoma

• Alveolar rhabdomyosarcoma

• Ewing’s Sarcoma

• Clear cell sarcoma

• Desmoplastic small round cell tumor

• Myxoid/round cell liposarcoma

• Extraskeletal myxoid chondrosarcoma

• Prostate cancer

• Renal cell carcinoma

Agenda

• Tumor Suppressor Genes • Parathyroid carcinoma


• Oncogenes • Lung cancer

• Thyroid cancer



• Viruses


113

Squamous Cell Carcinoma

114

• Carcinomas that do not progress through dysplastic precursors

• Basaloid squamous cell carcinoma

• Nonkeratinizing squamous carcinoma

• Lymphoepithelial carcinoma

Lymphoepithelial Carcinoma

115

• Confusing terminology

• Lymphoepithelial-like carcinoma

• Transitional

• Undifferentiated carcinoma

• Anaplastic carcinoma with lymphoid component

• Lymphoepithelioma

• Schmincke & Regaud types


116

• Clinical

• Rare in Western hemisphere (1/100,000)

• 30 times higher incidence in SE Asia

• Terminology

• Nasopharyngeal Undifferentiated

• Lymphoepithelial carcinoma

• Undifferentiated carcinoma

• Lymphoepithelioma

• Schmincke & Regaud types


117

• Risk Factors

• Exposure to carcinogens

• Tobacco and alcohol

• HLA subtypes

• Food with nitrates

• Epstein Barr Virus

• Particularly in Asia & North Africa

118 Lymphoepithelial Carcinoma

Lymphoepithelial Carcinoma, EBV ISH

120

Integrated Virus Episomal Virus

121 HPV ISH integrated HPV ISH episomal

Oncogenesis of HPV

122

E6

p53 MDM2

ARF

E2F Rb

E7

Cyclin D1 p16 S-phase

Apoptosis

p21

Human Papillomavirus

123

• Relationship between SCC and HPV

• Increasing incidence of HPV-related tumors

• Based on SEER data, annual percentage increase is ~3%

• ~25% of all HNSCC

• Oropharynx: >50% of tumors are positive for HPV

• Up to 95% are HPV-16

Ryerson AB, Cancer 113:2901, 2008

Syrjanen S, J Clin Virol, 32:S59, 2005

HPV and Tumor Characteristics

124

HPV Positive HPV Negative

Demographics •5 years younger

•Non-smokers/non-drinkers

•Typical ages

•Tobacco and alcohol

Site Tonsil & Tongue base All locations

Histology Poorly differentiated, non-

keratinizing, basaloid

Keratinizing SCC

Genetics •p53 inactivated by E6

•Rb inactivated by E7

•p16 over-expressed

•p53 inactivated by mutation

•Rb inactivated by cyclin D1 amplification

•Inactivation of p16

Virus Detection: HPV

125

0%

20%

40%

60%

80%

Q-PCRp16 IHC

in situ

Kuo KT, Mod Path, 21:376, 2008

p16 Immunohistochemistry

126

• Excellent surrogate marker for HPV

• Need for direct viral detection is limited

• p16 staining should be strong and diffuse

127 Tonsil, H&E p16 IHC HPV 16, ISH

Why Test for HPV?

128

• Epidemiologic

• Diagnostic

• Prognostic

• Therapeutic

Metastasis & Unknown Primary

129

• Histology

• Ribbon like epithelium

• Thickness of tonsillar type epithelium

• Can have endophytic or exophytic areas

• Cytology

• Moderate N:C ratio

• No maturation

• Bland appearance

130 Cystic Metastatic Squamous Carcinoma

131 Cystic Metastatic Squamous Carcinoma

Unknown Primary

132

Site Percentage

Tonsil/tongue base 63%

Nasopharynx 8%

Other 10%

None found 20%

HPV as a Diagnostic Tool

133

• HPV in cystic lymph node metastases

–Between 50 and 80% will be positive when originating from an oropharyngeal site

Desai PC, Exp Mol Pathol, 87:94, 2009

Goldenberg D, Head Neck, 30:898, 2008

Why Test for HPV?

134

• Epidemiologic

• Diagnostic

• Prognostic

• Therapeutic

Virus Detection: HPV

135

0%

20%

40%

60%

80%

Q-PCRp16 IHC

in situ

Kuo KT, Mod Path, 21:376, 2008

Better prognosis

Cumulative Incidence Relapse

136 Licitra, J Clin Onc, 24:5630, 2006.

Survival Rates

137 Licitra, J Clin Onc, 24:5630, 2006.

EGFR In HNSCC

138

• Over-expressed in >90% of tumors

• Very few cases with identified cause

• No somatic point mutaions

• 20-40% with EGFRvIII (truncated protein)

• 10% with amplification

• Anti-EGFR therapy is mainstay of therapy used today

Other Tumor Genes

139

• AKT1 (Rare)

• BRAF (Rare)

• EGFR (Rare)

• KRAS (Rare)

• PIK3CA (10-15%)

• NRAS (Rare)

• NOTCH 1 (~15%)

• p53 (>90%)

Agenda




• Thyroid cancer



• Viruses


140

Breast Cancer

141

• Incidence

•1 of 9 women (lifetime)

Country Incidence/100,000 Deaths/100,000

USA 69.6 14.5

SEER Data, 2000-2004

Breast Cancer

142

• Incidence

• 5-10% of breast cancer is hereditary

• 25-40% of cases under 35

• Remainder is sporadic

Types of Molecular Assays

143

• DNA based assays

• Single gene mutation analysis

• Quantitative assays for copy number

• Loss of heterozygosity (deletion detection)

• Comparative genomic hybridization arrays (deletion and amplification)

• Translocation analysis

• RNA based assays

• Expression microarray

• RT-PCR assays

Understanding Pathogenesis

144

• Expression Array Profiling • Variability and inconsistency across studies

• But, subtyping has stood up across studies

Sorlie T, PNAS 98(19), 2001: 10869

Breast Cancer Subtypes

145

• Luminal

• Luminal A

• Luminal B

• HER2

• Basal-like

146

Luminal HER2 Basal-Like

Expression Patterns

•High hormone receptors

(Lum A > Lum B)

•High HER2

•Low ER

•High basal epithelial genes, basal CKs

•Low ER & HER2

Clinical Features

•~70% of breast cancers

•ER/PR positive

•Lum B higher grade than Lum A

•Some express HER2 (Lum B)


•ER/PR negative

•More likely high grade and node+


•Most ER/PR/HER2 (-)

•BRCA1 dysfunction (germline, sporadic)

Treatment Response and Outcome

•Respond to endocrine therapy

•Response to chemo variable (Lum B > Lum A)

•Prognosis better for Lum A than Lum B

•Respond to trastuzumab

•Respond to anthracycline-based chemo

•Generally poor prognosis

•No response to endocrine therapy or trastuzumab

•May be sensitive to platinum/PARP inhibitors

•Generally poor prognosis

Today: Expression Array Testing

147

• Not currently used in pathology

• Given current treatment options, practical application of molecular sub-classification is debatable

• But, cost effective sub-typing could be done routinely: ER, PR, HER2, CK, EGFR, mitotic rate

Predicting Prognosis & Therapy Response

148

• Age

• Lymph node status

• Tumor size

• Histologic features • type, grade, LVI

• ER/PR status

• HER2 status

149

NEJM 2004

•668 ER+/N- patients (NSABP B14 •RT-PCR assay on paraffin sections (21 genes)

RS = +0.47 x HER2 group score -0.34 x ER group score +1.04 x proliferation group score +0.10 x invasion group score +0.05 x CD68 -0.08 x GSMT1 -0.07 x BAG1

31 High

OncotypeDx

(Genomic Health, Inc.)

Today: OncotypeDX

150

• Oncotype being used clinically with increasing frequency

• Recurrence score resulted in change in treatment recommendation by medical oncologist in 31.5% of patients (Shelly S, JCO, 2010)

• Most common use: to identify patients who could be spared cytotoxic therapy (i.e., with low recurrence score)

Today: OncotypeDX

151

• Is OncotypeDX really better than using a combination of clinical and pathologic factors supplemented by appropriate biomarkers detected by IHC (e.g., ER, PR, HER2 and Ki67)?

Today: Immunohistochemistry

152

• 1,125 pts with ER+ breast cancer from the ATAC trial

• ER, PR, HER2 and Ki67 assessed by IHC

• Combined “IHC4 Score” provided similar prognostic information as OncotypeDX Recurrence Score

Cuzick J, JCO, 29:4273, 2011

Other Issues: OncotypeDX

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GHI -

Equivocal

GHI –

Negative

GHI –

Positive

FISH/IHC Equivocal 0 23 0

FISH/IHC Negative 5 779 0

FISH/IHC Positive 12 14 10

Dabbs DJ, JCO, 29:1, 2011

Summary




• Thyroid cancer



• Viruses


154

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Association of Molecular Pathologyaz9194.vo.msecnd.net/pdfs/130401/3.pdf•13q14.3 (Retinoblastoma gene) •11q13 (MEN gene) •1q25-31 (HPRT2 gene) •Fractional allelic loss . Syndromic