From Morphology to Molecular Pathology: A

Practical Approach for Cytopathologists

Part 2 – Molecular PathologyPart 2 – Molecular Pathology

Mary Lowery Nordberg, PhD

Feist-Weiller Cancer Center

LSUHSC-S

I have no Conflict of InterestI have no Conflict of Interest

Acknowledgements

• Questions adapted from:

– Lefkowitch 2006: Anatomic

Pathology Board Review,

SaundersSaunders

– Mais and Nordberg, 2008:

Quick Compendium of

Molecular Pathology, ASCP

Press.

OBJECTIVES

• Integrate the various genetic test results with morphologic, clinical, and

other relevant laboratory data to arrive at a single interpretation for

diagnostic and prognostic purposes.

• Review how laboratories are using molecular diagnostics, particularly

fluorescence in situ hybridization (FISH) technologies and outline key

advantages and challenges of oncologic molecular testing relative to advantages and challenges of oncologic molecular testing relative to

more traditional diagnostic methods.

• Recognize the advantages and limitations of the different testing

modalities such that they are able to help physicians to prioritize test

requests and eliminate redundant testing.

• Acquire knowledge of how to access and utilize published literature and

databases to be able to continue to integrate new findings in the

genetics field into daily clinical practice.

FNA ON LYMPHOPROLIFERATIVE

DISORDERSDISORDERS

Flow Chart of Using FNA for Diagnosis of

Lymphoproliferative Disorders

Flow cytometry

Cytology-reactive

Flow cytometry-neg

Cytology-positive or

Atypical

Flow cytometry-

inconclusive

Cytology-positive or

atypical

Flow cytometry-positive

for clonal

Reactive LN

Clinical correlation

PCR or other

molecular testing

for clonality

FISH or PCR for

subclassification

Genomic Disease Management

Various technologies can detect abnormalities at all levels

IHC FISH PCR/FISH Sequencing

Centromeric

Enumerating Probe

CEP

Locus Specific

Identifier

LSI

Whole Chomosome

Paint

WCP

FISH Probe Types

Counting

Chromosomes

Amplification/Deletion/Translocation

Counting Genes Translocation

Chromosome View of CEP ProbeChromosome View of CEP Probe

Normal 2 copiesOne chromosome

Deleted

Three copies

Chromosome View of LSI HybridizationChromosome View of LSI Hybridization

Normal Deleted Amplification

FISH Technology

Break Apart Probes II

Dual Color, Dual Fusion

Case #1

25 year-old female with a 5.0 cm 25 year-old female with a 5.0 cm

axillary lymph node and diffuse

lymphadenopathy on CT.

•t(8;14): 75-85%

•t(2;8): 5%

• t(8;22): 10%;

•c-myc: IgH @ 14q32

Burkitt Lymphoma

•c-myc: IgH @ 14q32

•IgH @ 14q32

•IgK @ 2p12

•IgL @ 22q11

c-myc@8q24

Fusion IgH/CMYC

Burkitt Lymphoma

IgH@14q32

c-myc@8q24

Case #2

53 year-old male with diffuse 53 year-old male with diffuse

lymphadenopathy and right pleural

effusion. FNA of right cervical lymph

node.

Question 1

The corresponding

figure shows a

karyotype of a CD20,

CD10, PAX5 and

BCL6 positive lymphoid BCL6 positive lymphoid

neoplasm. Which of the

following statements

about this type of tumor

is TRUE?

(A) This tumor harbors a t(14;18)(q32;q21) translocation which causes overexpression of cyclin D1.

(B) Polymerase chain reaction (PCR) detects this translocation in a greater proportion of cases than do fluorescence in situ hybridization (FISH) or Southern blotting.

(C) Immunostaining for BCL2 in B cells is diagnostic of follicular lymphoma.diagnostic of follicular lymphoma.

(D) Overexpression of BCL2 is associated with reduced apoptosis and resistance to chemotherapy.

(E) Detection of the BCL2 translocation by PCR is diagnostic of follicular lymphoma.

BCL2 protein expression – non-diagnostic

(although not present in non-neoplastic follicular center cells)

• Flow cytometry

• Immunohistochemistry

– Normal mantle cells

– B-cells in hyperplastic marginal

zones

– Normal plasma cells

– T-cells

– Follicular lymphomas

– MALT lymphomas

– Mantle cell lymphomas

– Lymphocytic leukemias

– ALL

Anti-

apoptotic

BCL2 FISH

t(11;14)(q13;q32) BCL1/CCND1

• Diagnostic of mantle cell

lymphoma

Oncogene

t(11;14)(q13;q32)

BCL2

• Historically, BCL2/JH t(14;18) Translocation Assays have been used to: Distinguish lymphoma from benign lymphoid hyperplasiahyperplasia

• Distinguish follicular lymphoma from other B-cell lymphomas that may have a similar appearance

• Monitor and evaluate disease recurrence and detect residual disease

PCR for BCL2/IGH

• Wide variability of BCL2

breakpoints

• Standard primers for MBR and

mbr

– Detects only 35-65% of cases

• Can be seen at a low level in

some reactive lymphoid

proliferations

Case #3

51 year-old female with right neck mass 51 year-old female with right neck mass

for 8 months.

Smears from a recent case of DLBCL

Diffuse Large B Cell Lymphoma, NOS

• Cytomorphology: diverse cytology with three

common variants-centroblastic, immunoblastic and

anaplastic.

• Immunophenotype: monoclonal B cells positive for

CD19, 20, 22 and 79a; germinal center (GC)-like

DLBCL with >30% cell CD10+ or CD10-, BCL-6+,

IRF4/MUM1-; all others non-GC type.

• Cytogenetic abnormalities: 30% BCL6 translocation,

20-30% BCL2 translocation.

Case #4

43 year-old female with left neck 43 year-old female with left neck

lymphadenopathy.

Flow Cytometry

• Mixed T and B cells.

• No light chain restriction on B cells.

• A population of CD45 (+) cells positive for CD2,

4, 25 and 52, but negative for CD3, 5 and 7.4, 25 and 52, but negative for CD3, 5 and 7.

• Flow cytometry interpretation: atypical T

lymphocytes, suspicious for T-cell lymphoma.

Cytomorphologic Summary of

Lymphoproliferative Disorders

• Monotonous lymphocytic population:

Small: SLL, MCL, atrophic nodes

Medium: Burkitt, lymphoblastic and MCL

Large: DLBCL, T cell lymphoma

• Heterogenous:• Heterogenous:

Mainly small: reactive, FL, marginal zone

Large: reactive, FL, DLBCL, T cell and Hodgkin lymphoma

• Pleomorphic: ALCL, Hodgkin, and histiocytic sarcoma.

Question 3

� This polyacrylamide gel

electrophoresis image shows

a clonal control and a

polyclonal sample product

obtained after PCR with

immunoglobulin heavy-chain immunoglobulin heavy-chain

joining region and framework

region 3 primers. Which of

the following statements

about antigen receptor gene

rearrangement analysis for

detection of lymphoid

clonality is TRUE?

(A) PCR detects a larger proportion of rearrangements than does Southern blotting.

(B) Unlike Southern blotting, which can be performed on paraffin-embedded tissue, PCR requires frozen tissue, because formalin inhibits DNA polymerases.

(C) PCR is especially sensitive for the detection of clonal rearrangements among follicular lymphomas.lymphomas.

(D) Use of PCR- either standard PCR or sequence-specific PCR- to detect residual disease in ALL is complicated by ongoing rearrangements.

(E) If a sample fails to show a clonal rearrangement by PCR, then Southern blotting will be of little value.

VDJ/VJ rearrangements of antigen receptor genes

• Normal response for

immunologic competency

• Clonal rearrangements

targeted for lymphoid

proliferations

PCR for clonality (IGH/JH)

• Somatic hypermutations

• Deletions

• Failure of “consensus” primers

to amplify different V families

– Detects on 50-75% of all – Detects on 50-75% of all

rearrangements

• Good for minimal residual

disease

Southern blot for B-cell clonality

• Targets broader region of

genome

• Laborious

• Requires more tissue

• Unreliable with paraffin• Unreliable with paraffin

URINE CYTOLOGY

Bladder washing-Diagnosis?

Case #1. Bladder biopsy

Question 15

• Which of the following

statements about FISH,

using the Urovysion™

assay to detect bladder

cancer, is TRUE?

(A) FISH is more specific but less sensitive than cytologic examination.

(B) Loss of at least one copy of any two of chromosomes 3, 7 or 17 identified by centromeric probes, and amplification of chromosome 9p21 with a sequence-specific probe, are the most important markers of urothelial carcinoma.

(C) The probe set is chosen to evaluate for polysomy of chromosome 3, 7 and 17, and/or polysomy of chromosome 3, 7 and 17, and/or homozygous loss of 9p21.

(D) Evaluation of 100 successive cells is more sensitive than evaluating 25 morphologically abnormal cells.

(E) Papillary urothelial carcinoma is more likely to be FISH positive than is carcinoma in situ.

(p16 gene)

UroVysion

(p16 gene)

Normal CellNormal Cell Malignant Cell

OligoFISHTM Bladder Test vs UroVysion

67

OligoFISHOligoFISHOligoFISHOligoFISHOligoFISHOligoFISHOligoFISHOligoFISHTMTMTMTMTMTMTMTM Bladder Test Bladder Test Bladder Test Bladder Test Bladder Test Bladder Test Bladder Test Bladder Test vsvsvsvsvsvsvsvs UroVysionUroVysionUroVysionUroVysionUroVysionUroVysionUroVysionUroVysion

(3, 6, 7, 20 (3, 6, 7, 20 (3, 6, 7, 20 (3, 6, 7, 20 (3, 6, 7, 20 (3, 6, 7, 20 (3, 6, 7, 20 (3, 6, 7, 20 vsvsvsvsvsvsvsvs 3, 7, LSI 9p21, 17)3, 7, LSI 9p21, 17)3, 7, LSI 9p21, 17)3, 7, LSI 9p21, 17)3, 7, LSI 9p21, 17)3, 7, LSI 9p21, 17)3, 7, LSI 9p21, 17)3, 7, LSI 9p21, 17)

UroVysionUroVysionUroVysionUroVysionUroVysionUroVysionUroVysionUroVysion

97% concordance

68

PositivePositivePositivePositive NegativeNegativeNegativeNegative Total

OligoFISHOligoFISHOligoFISHOligoFISHOligoFISHOligoFISHOligoFISHOligoFISHTMTMTMTMTMTMTMTM

TestTestTestTestTestTestTestTest3, 6, 7, 203, 6, 7, 203, 6, 7, 203, 6, 7, 203, 6, 7, 203, 6, 7, 203, 6, 7, 203, 6, 7, 20

PositivePositivePositivePositive 101101101101 4444 105105

NegativeNegativeNegativeNegative 1111 86868686 8787

Total 102102 9090 192

FISH Time Comparison

Traditional FISHTraditional FISHTraditional FISHTraditional FISH OligoFISHOligoFISHOligoFISHOligoFISH

DenaturationDenaturationDenaturationDenaturation 2-5 min 2-5 min

HybridizationHybridizationHybridizationHybridization Overnight (16h) 5-10 min

69

HybridizationHybridizationHybridizationHybridization Overnight (16h) 5-10 min

WashesWashesWashesWashes 20 min 5 min

Total timeTotal timeTotal timeTotal time ~16h 25min~16h 25min~16h 25min~16h 25min 15151515----20 min20 min20 min20 min

SOFT TISSUE TUMORS

Case #1

23 yo male with back pain and a large 23 yo male with back pain and a large

soft tissue mass

Case #2

57 year-old female with left neck 2.0 cm 57 year-old female with left neck 2.0 cm

nodule and history of resection clear

cell sarcoma 6 months ago.

The role of RAS-RAF proteins in the

RAS-RAF pathway

RAS

BRAF

RAF proteins play a role

in the regulation of essential

biologic functions1-3

� Cell growth

� Cell proliferation

801. Wong. Recent Pat Anticancer Drug Discov. 2009;4:28-35. 2. Wan et al. Cell. 2004;116:855-867. 3. McCubrey et al. Adv Enzyme Regul. 2006;46:249-279.

ERK

MEK

� Cell proliferation

� Cell differentiation

Oncogenic BRAF� BRAF mutations at position 600

(BRAFV600) can lead to overactive BRAF signaling1

� The most common BRAF mutation, BRAFV600E, is implicated in:

� ~50% of melanoma tumors1

1. McCubrey et al. Adv Enzyme Regul. 2006;46:249-279. 2. Pritchard et al. Biochem Soc Trans. 2007;35:1329-1333. 3. Cho et al. Int J Cancer. 2006;119:1858-1862.

V600E mutation

� ~50% of melanoma tumors1

� ~40% of papillary thyroid tumors1,2

� ~30% of serous ovarian tumors2

� ~10% of colorectal tumors3

� ~10% of prostate tumors3

81

Overview of metastatic melanoma

disease state� Median overall survival

of ~8 months1

� 1-year survival rate of ~25%2

� ~50% of metastatic melanoma

tumors have the BRAFV600 mutation3

821. Bhatia et al. Oncology. 2009;23:488-496. 2. Korn et al. J Clin Oncol. 2008;26:527-534. 3. McCubrey et al. Adv Enzyme Regul. 2006;46:249-279. 4. Skarin, ed. Atlas of Diagnostic Oncology. 4th ed. Philadelphia, PA: Mosby, Inc; 2010:467.

Metastatic melanoma is an important area

for continued research and development of

strategies for patient management

Reprinted with permission from Elsevier.4

Molecularly targeted therapy

Question 4

� Direct epithelial growth factor

receptor gene sequencing of

paraffin-embedded lung

tumor showed a 15-bp, in-

frame deletion in exon 19

(shown here), which leads to (shown here), which leads to

a five amino acid deletion

within the catalytic kinase

domain of EGFR (delE746-

A750). Which of the

following statements about

EGFR and tumors of this type

is TRUE?

(A) Given the function of EGFR- a tyrosine kinase-this is likely to be an inactivating mutation and associated with sensitivity to gefitinib (Iressa), an EGFR inhibitor.

(B) This lung tumor is likely to be a small cell carcinoma.

(C) The patient from whom this sample was obtained is most likely a smoker.

(D) This carcinoma is likely to have an activating k-rasmutation.mutation.

(E) This lung tumor is most likely an adenocarcinoma, and the patient more likely to be a nonsmoker and female.

Clinical management and EGFR status

FISH analysis for EGFR copy

number

Question 9

� This small round blue cell tumor (A) in the chest wall of a 14-year-old child showed strong immunohistochemicalstaining for CD99 and Fli-1, and was negative for desmin, smooth muscle actin, HHF-35, cytokeratin, CD45, CD3, CD20, cytokeratin, CD45, CD3, CD20, MyoD1, myogenin, S-100, WT1 and epithelial membrane antigen. Classic cytogenetics (B) showed a t(11;22)(q24;q12), which was confirmed by FISH (C). Which of the following statements is FALSE?

(A) This tumor belongs to the Ewing’s sarcoma/primitive

neuroectodermal tumor (ES/PNET) family.

(B) RT-PCR can be performed on paraffin-embedded tissue, and in

this tumor would be likely to demonstrate an EWS-FLI1 fusion

(C) When cytogenetics cannot be performed, a negative result for

the EWS-FLI1 and EWS-ERG, using a properly designed RT-PCR

assay to include all EWS-FLI1 fusion variants, rules out a diagnosis

of ES/PNET.

(D) Demonstration of “split” signals with a “break-apart” EWS probe

would have strong evidence in favor of a diagnosis of ES/PNET in

this case, had karyotyping been unsuccessful.

(E) In addition to ES/PNET, other tumors with fusion transcripts

involving the EWS gene include clear cell sarcoma (“malignant

melanoma”) of soft parts, desmoplastic small round cell tumors,

extraskeletal myxoid chrondrosarcoma and, rarely, myxoid

liposarcoma.

Question 10

• This tumor from the leg of

an 8-year-old girl was

positive for HHF-35,

MyoD1 and myogenin. The

tumor was negative for

cytokeratin, CD45, S-100 cytokeratin, CD45, S-100

and epithelial membrane

antigen. Classic

cytogenetics showed a

t(2;13) (q35;q14). The

following are true EXCEPT:

(A)This tumor is an alveolar rhabdomyosarcoma (ARMS).

(B)RT-PCR is likely to show a fusion transcript involving the 5’ portion of PAX3and the 3’ portion of FOXOA1 (FKHR).

(C)A more common translocation in alveolar rhabdomyosarcoma is t(1;13) (p36;q14), which results in a PAX7-FOXOA1 fusion. FOXOA1 fusion.

(D)This transcript is associated with a higher rate of bone marrow involvement than the PAX7-FOXOA1 transcript.

(E)The PAX7-FOXOA1 translocation is more likely to be amplified as double minutes (dmin).

THANK YOU. QUESTIONS?