From Morphology to Molecular Pathology: A Practical Approach for Cytopathologists Part 2 – Molecular Pathology Mary Lowery Nordberg, PhD Feist-WeillerCancer Center LSUHSC-S
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?