From Morphology to Molecular

Pathology: A Practical Approach for

Cytopathologists
Part 1-Cytomorphology

Songlin Zhang, MD, PhD

LSUHSC-Shreveport
I have no Conflict of Interest.
FNA on Lymphoproliferative
Disorders
 Why FNA for Lymphoproliferative
Disorders?
Initial diagnosis-less invasive than open biopsy.
Confirming recurrent lymphomas.
Unexpected lymphoma during routine work-up to
rule-out metastatic lesions.
New FNA techniques for deeply located lymph
nodes, such as EUS and EBUS FNA.
Available ancillary tests such as flow cytometry
and immunocytochemistry for phenotyping and
molecular testing.
 Diagnosis of Lymphoproliferative
Disorders
Diagnosis: reactive vs lymphoma
Classification: non-Hodgkin B cell lymphoma,
Hodgkin lymphoma, NK/T cell lymphoma and
others.
Subclassification: diffuse large B cell lymphoma,
follicular lymphoma, mantle cell lymphoma,
marginal zone lymphoma, Burkitt lymphoma,
and different T-cell lymphomas.
Grade: follicular lymphoma (low grade 1 and 2;
high grade 3A and 3B).
 Classic Flow Cytometry Phenotyping
for Small B-cell Lymphoma
CD CD19/ CD5 CD10 CD23
Lymphoma CD20
Small + + - +
lymphocytic
lymphoma
Follicular + - + -
lymphoma
Mantle cell + + - -
lymphoma
Nodal + - - -
marginal zone
lymphoma
 Case #1
25 year-old female with a 5.0 cm
axillary lymph node and diffuse
lymphadenopathy on CT.
Your Diagnosis?
 Flow Cytometry
Monoclonal B-cells positive for:
CD19, 20, 10, 79b and lambda light chain
restriction.

Negative for:
CD5 and 23.
Excisional biopsy, case #1
 Burkitt Lymphoma
Cytomorphology: monotonous intermediate
rounded lymphocytes, nuclei with 2-5 distinct
nucleoli, dense blue cytoplasm with lipid
vacuoles, many tingible body macrophages.
Immunophenotype: monoclonal B-cells with
positive CD19, 20, 10, 22, BCL-6 and negative
BCL-2.
Cytogenetic abnormalities: most cases with
MYC translocation [t(8;14); t(2;8); t(8;22)]; highly
characteristic but not specific for Burkitt
lymphoma.
 Case #2
53 year-old male with diffuse
lymphadenopathy and right
pleural effusion. FNA of right
cervical lymph node.
Your Diagnosis?
 Flow Cytometry
Monoclonal B-cells positive for:
CD19, 20, 10, 79B, BCL-2 and kappa light
chain.

B-cells negative for:

CD5 and 23.
Excisional biopsy case #2
 Follicular Lymphoma
Cytomorphology: heterogenous lymphocytes,
small to medium centrocytes with irregular
nuclei, large centroblasts with few nucleoli,
visible cell aggregates (follicular centers), and
often no tingible body macrophages.
Immunophenotype: monoclonal B cells positive
for CD19, 20, 22, 10, BCL-2 and BCL-6.
Cytogenetic abnormalities: t(14;18) up to 90%
grade 1-2 follicular lymphoma.
Grading follicular lymphoma on cytology: using
centroblast count.
 Case #3
51 year-old female with right neck
mass for 8 months.
Your Diagnosis?
 Flow Cytometry
Mixed population of T and B cells.
No evidence of light chain restriction
(normal kappa/lambda ratio).
Normal CD4/CD8 ratio.
Small population of B cells with lambda
predominance.
Flow diagnosis-atypical but non-
diagnostic.
Excisional biopsy, case #3
 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.
Smears from a recent case of DLBCL
 Case #4
43 year-old female with left neck
lymphadenopathy.
Your Diagnosis?
 Flow Cytometry
Mixed T and B cells.
No light chain restriction on B cells.
A population of CD45 (+) cells positive for
CD2, 4, bright 25 and 52, but negative for
CD3, 8, 5 and 7.
Flow cytometry interpretation: atypical T
lymphocytes, suspicious for T-cell
lymphoma.
Excisional biopsy, case #4
 Adult T-cell Leukemia/Lymphoma
Cytomorphology: a broad spectrum of
cytological features, typically medium-sized to
large cells, pronounced nuclear pleomorphism,
coarsely clumped chromatin, and sparse
background inflammation.
Immunophenotype: usually positive for CD2, 3,
5, but lack CD7; most CD4+; CD25 strongly
positive in all cases.
Cytogenetic: clonal rearrangement of T-cell
receptor; monoclonal integration of HTLV-1.
 Cytomorphologic Summary of
Lymphoproliferative Disorders
Monotonous lymphocytic population:
Small: SLL, MCL, atrophic nodes
Medium: Burkitt, lymphoblastic and MCL
Large: DLBCL, T cell lymphoma
Heterogenous:
Mainly small: reactive, FL, marginal zone
Large: reactive, FL, DLBCL, T cell and Hodgkin
lymphoma
Pleomorphic: ALCL, Hodgkin, and histiocytic
sarcoma.
 Flow Chart of Using FNA for Diagnosis
of Lymphoproliferative Disorders

Flow cytometry

Cytology-positive or Cytology-positive or
Cytology-reactive Atypical atypical
Flow cytometry-neg Flow cytometry- Flow cytometry-positive
inconclusive for clonal

PCR or other
Reactive LN FISH or PCR for
molecular testing
Clinical correlation subclassification
for clonality
Urine Cytology
Bladder Washing-Diagnosis?
Renal pelvic washing-Diagnosis?
 Case #1

67 year-old male with hematuria.

Bladder washing-Diagnosis?
Case #1. Bladder biopsy
 Case #2

63 year-old man with microscopic

hematuria.
Bladder washing
Ureteral washing
Your Diagnosis?
Case #2. Ureteral biopsy.
 Urine Cytology and UroVysion
Urine cytology is quite sensitive for detecting
high-grade urothelial carcinoma and carcinoma
in-situ; The sensitivity for low-grade urothelial
tumors is very poor.
UroVysion is a FDA-approved FISH test
performed on voided urine and targets
chromosomes 3, 7, 17 and 9p21; Not all
urothelial carcinomas are positive for UroVysion
(especially low-grade tumors) and there is
significant false positive rate in reactive
urothelial cells.
FNA on Soft Tissue Tumors
 Case #1
23 year-old male with back pain
and a large soft tissue mass (12.0
cm) at T12 on CT scan.
Vimentin
Your Diagnosis?
 Immunohistochemistry Results

CD 99 (-)
NSE and chromogranin (-)
Neurofilament protein (-)
S-100 (-)
Pancytokeratin (-)
Positive for Myo D1, myogenin and
desmin.
 Diagnosis
Alveolar rhabdomyosarcoma.

Which molecular test can be used to

confirm this diagnosis?
 Case #2

57 year-old female with left neck 2.0

cm nodule and history of resection
clear cell sarcoma 6 months ago.
Your Diagnosis?
 FNA Diagnosis
High-grade malignant neoplasm with
positive immunocytochemistry for S-100
and HMB-45.

Metastatic clear cell sarcoma? Metastatic

malignant melanoma?
 S-100

Mart-1
 Diagnosis
What is your diagnosis?

What molecular tests may help you to

differentiate between clear cell sarcoma
and melanoma?
 FNA Cytology on Soft Tissue Tumors
Cytomorphology:
Myxoid: myxoma, myxoid liposarcoma, myxoid MFH
Spindle: fibromatosis, Schwannoma, fibrosarcoma,
leiomyosarcoma, GIST
Pleomorphic: MFH, pleomorphic liposarcoma,
pleomorphic leiomyosarcoma
Round: rhabdomyosarcoma, Ewings/PNET, DPSCL
Polygonal (epithelioid): epithelioid sarcoma, alveolar soft
part sarcoma, granular cell tumor
Chromosomal translocations:
Ewings/PNET: t(11;22)(q24;q12), t(21;22), t(7;22)
DPSCL: t(11;22)(q13;q12)
Clear cell sarcoma: t(12;22)
Alveolar rhabdomyosarcoma: t(2;13), t(1;13)
Thank You!