An Overview on Molecular Cytopathology
Manuel Salto-Tellez
Professor and Chair of Molecular Pathology
Queen’s University Belfast
“The Cytology sample, in the context of appropriate
laboratory validations, should not
be treated differently to any other sample for
molecular testing"
“The ideal sample for molecular testing is the
first one available which, often, is the cytology
sample"
DIAGNOSTIC &
CLINICAL
APPLICATIONS
Association for Molecular Pathology (AMP)
Diagnostic Molecular Cytopathologyis the
application of molecular diagnosis to those
samples that are firstly analysed by
cytopathologists:
FNAs, effusion and exfoliative cytology
Cell Blocks
Diagnostic MolecularCytopathology
Why Molecular Cytopathology?
CELL BLOCK (X20)
1.5 mm
SMEAR (X20)
The right DNA/RNA protocol:
1. Proven for the sampling method
2. Good DNA/RNA quality checks
The right materials:
1. Enough malignant cells
2. Enough malignant/benign ratio
The right (RT-)PCR protocol:
1. Designed for FFPE materials
2. Good internal and external
controls
The increasing importance of
Molecular Cytopathology
DIAGNOSIS?
Architecture +++
Cytology +++
IHC quality +++
Usually more
challenging to obtain
Architecture +
Cytology +++
IHC quality +
Easier to obtain
Tissue biopsy?
FNA cytology?
M
DISCORDANCE RATE
EGFR = 16.2%
Han HS. ClinLung Cancer. 2011 May 20.
KRAS = 5%
Mariani P. Anticancer Res. 2010 Oct;30(10):4229-35.
HER2 = 5.5%
Houssami N. Breast Cancer Res Treat. 2011 Oct;129(3):659-74.
PRIMARY
TUMOUR
METASTATIC
TUMOUR
HER2 = 32.0%
Mittendorf EA. Clin Cancer Res. 2009 Dec 1;15(23):7381-8.
PRETREATMENT
POSTTREATMENT
Translocations in sarcomas
Translocations in sarcomas
Tumor
Ewing’s sarcoma
Peripheral PNET
Myxoid liposarcoma
Translocation
t(11;22)
t(21;22)
t(12;16)
t(12;22)
Alveolar Rhabdomyosarcoma
t(2;13)
t(1;13)
Clear cell sarcoma
t(12;22)
Desmoplastic small round
t(11;22)
cell tumor
Synovial sarcoma
t(X;18)
Myxoid chondrosarcoma
t(9;22)
Dermatofibrosarcoma
t(17;22)
protuberans
Infantile fibrosarcoma
t(12;15)
Fusion Product
EWS-FL11
EWS-ERG
TLS-CHOP
EWS-CHOP
PAX3-FKHR
PAX7-FKHR
EWS-ATF1
EWS-WT1
SYT-SSX1/ SYT-SSX2
EWS-CHN
COL1A1-PDGFB
STRONG DIAGNOSTIC VALUE
ETV6-NTRK3
Synovial Sarcoma
DiagnCytopathol. 2003 Dec;29(6):341-3.
Sequence confirmation of the EWS-WT1 fusion gene
transcript in the peritoneal effusion of a patient with
desmoplastic small round cell tumor.
Chiu LL, Koay ES, Chan NH, Salto-Tellez M.
We present a case of a 17 year-old male with
disseminated peritoneal disease and
peritoneal effusion.
a
b
c
a) PAP
b) Cam 5.2
c) AE1/3
d) CD99
e) Desmin
f) NSE
d
e
g
1 2 3 M
M
310 bp
281/271 bp
234 bp
194 bp
189 bp
Phosphoglycerte kinase (PGK)
E
W
S
j
i
f
W
T1
W
T1
E
W
S
1
2
3
h
310 bp
281/271 bp
234 bp
194 bp
118 bp
EWS exon 7 / WT1 exon 8
Reverse
Forward
a
b
c
a) PAP
b) Cam 5.2
c) AE1/3
d) CD99
e) Desmin
f) NSE
d
e
g
1 2 3 M
M
310 bp
281/271 bp
234 bp
194 bp
189 bp
Phosphoglycerte kinase (PGK)
E
W
S
j
i
f
W
T1
W
T1
E
W
S
1
2
3
h
310 bp
281/271 bp
234 bp
194 bp
118 bp
EWS exon 7 / WT1 exon 8
Reverse
Forward
Translocations in sarcomas
Tumor
Ewing’s sarcoma
Peripheral PNET
Myxoid liposarcoma
Translocation
t(11;22)
t(21;22)
t(12;16)
t(12;22)
Alveolar Rhabdomyosarcoma
t(2;13)
t(1;13)
Clear cell sarcoma
t(12;22)
Desmoplastic small round
t(11;22)
cell tumor
Synovial sarcoma
t(X;18)
Myxoid chondrosarcoma
t(9;22)
Dermatofibrosarcoma
t(17;22)
protuberans
Infantile fibrosarcoma
t(12;15)
Fusion Product
EWS-FL11
EWS-ERG
TLS-CHOP
EWS-CHOP
PAX3-FKHR
PAX7-FKHR
EWS-ATF1
EWS-WT1
SYT-SSX1/ SYT-SSX2
EWS-CHN
COL1A1-PDGFB
STRONG DIAGNOSTIC VALUE
ETV6-NTRK3
a
b
c
a) PAP
b) Cam 5.2
c) AE1/3
d) CD99
e) Desmin
f) NSE
d
e
g
1 2 3 M
M
310 bp
281/271 bp
234 bp
194 bp
189 bp
Phosphoglycerte kinase (PGK)
E
W
S
j
i
f
W
T1
W
T1
E
W
S
1
2
3
h
310 bp
281/271 bp
234 bp
194 bp
118 bp
EWS exon 7 / WT1 exon 8
Reverse
Forward
Diagnostic Cytopathology, 2003 Dec; 29(6): 341-3.
a
b
c
a) PAP
b) Cam 5.2
c) AE1/3
d) CD99
e) Desmin
f) NSE
d
e
g
1 2 3 M
M
310 bp
281/271 bp
234 bp
194 bp
189 bp
Phosphoglycerte kinase (PGK)
E
W
S
j
i
f
W
T1
W
T1
E
W
S
1
2
3
h
310 bp
281/271 bp
234 bp
194 bp
118 bp
EWS exon 7 / WT1 exon 8
Reverse
Forward
Haemato-oncology
Lymphomas
CLONALITY STUDIES
All lymphomas
IgH receptor and TCR gene rearrangement
TRANSLOCATIONS
c-ski (1q23); c-ets
(11q23-24); bcr-abl
Precursor B-ALL
t(1;19), t(4;11), del(6q), t(9;22)
B-CLL / SLL
del(13); trisomy 12
Mantle cell lymphoma
t(11;14)
Cyclin D1
Follicular lymphoma
t(14;18)
Bcl-2
Extranodal marginal zone B-cell lymphoma
t(11;18)
API2/ML1
Splenic Marginal zone B cell lymphoma
del(7), del(10)
Lymphoblastic lymphoma (immunocytoma)
t(9;14)
PAX-5
Diffuse large B-cell lymphoma
t(3;14) & t(14;18)
Bcl-6 & Bcl-2
Burkitt lymphoma
t(8;14), t(2;8), t(8;22)
c-myc
Plasmacytoma
t(4;14) & t (6;14)
FGFR3 & MUM1/IRF 4
T-cell prolymphocytic leukemia
Inv(14)
TCL-1
Angioimmunoblastic T cell lymphoma
+3, +5, +X
Anaplastic large cell lymphoma
t(2;5)
Hepatosplenic γδ
i(7)
(adapted from Ng, Lee and Salto-Tellez EOMD 2008)
NMP/ALK
Clinical history
• 53 year old male
• 2 months of left neck and
facial swelling
• CT: well-demarcated
homogenous tumour in
the left parotid gland,
measuring up to 55 x 50
x 50 mm.
FNAC Cytomorphology
Discohesive lymphoid
population
Cells with irregular nuclear
membrane and small
nucleoli, reminiscent of
centrocytes.
Fewer medium to large size
and show prominent
nucleoli.
Paucity of tingible body
macrophages.
Immunohistochemistry on cell block
CD 20
Molecular findings
Cell block unstained sections
sent for FISH
for the 14:18 FL
translocation
Many tumour cells showed
fusion signals of less than
one signal diameter
separation
Final diagnosis:
Follicular Lymphoma
CLONALITY STUDIES
All lymphomas
IgH receptor and TCR gene rearrangement
TRANSLOCATIONS
c-ski (1q23); c-ets
(11q23-24); bcr-abl
Precursor B-ALL
t(1;19), t(4;11), del(6q), t(9;22)
B-CLL / SLL
del(13); trisomy 12
Mantle cell lymphoma
t(11;14)
Cyclin D1
Follicular lymphoma
t(14;18)
Bcl-2
Extranodal marginal zone B-cell lymphoma
t(11;18)
API2/ML1
Splenic Marginal zone B cell lymphoma
del(7), del(10)
Lymphoblastic lymphoma (immunocytoma)
t(9;14)
PAX-5
Diffuse large B-cell lymphoma
t(3;14) & t(14;18)
Bcl-6 & Bcl-2
Burkitt lymphoma
t(8;14), t(2;8), t(8;22)
c-myc
Plasmacytoma
t(4;14) & t (6;14)
FGFR3 & MUM1/IRF 4
T-cell prolymphocytic leukemia
Inv(14)
TCL-1
Angioimmunoblastic T cell lymphoma
+3, +5, +X
Anaplastic large cell lymphoma
t(2;5)
Hepatosplenic γδ
i(7)
(adapted from Ng, Lee and Salto-Tellez EOMD 2008)
NMP/ALK
Follicular lymphoma and diffuse
large B-cell lymphoma
transformation.
Diff-Quik smear (A) and
cell block preparation (B)
IHC CD20 (C )
IHC bcl2 (E)
IHC CD3 (D)
FISH IGH/BCL2 Dual Color,
Dual Fusion Translocation
Probe Set detected two
orange/green (yellow) fusion
signals in the cells, confirming
the presence of t(14;18)
(q32;q21) and hence the
diagnosis of follicular
lymphoma (F).
Thyroid Pathology
Nikiforov YE et al. J Clin Endocrinol Metab. 2011 Aug 31.
Impact of Mutational Testing on the Diagnosis and Management of Patients with
Cytologically Indeterminate Thyroid Nodules:
A Prospective Analysis of 1056 FNA Samples.
Molecular diagnostics and
personalised / predictive medicine
Gastrointestinal stromal tumour
Br J Cancer
2007;96:776–82
Cytopathology
2009;20:297–303
Mod Pathol 2003;16:79–85
Hum Pathol 2003;34:362–8
J Clin Pathol2010;63:839–42
J Clin Pathol 2011; July 14th
Arch Pathol Lab Med 2011; 135(6):693-5
C-kit mutations
Imatinib
HER2-neu FISH
Breast
cancer
Trastuzumab
Lapatinib
Gastric
cancer
Lung cancer
Clin Chem
2007;53:62–70
J Thorac Oncol
2011, on line
EGFR
mutations
EML4-ALK
Colon cancer
J Mol Diagn 2009;11:543–52
Pathology 2008;40:295–8
Cytopathology 2010; Oct 4th
Int J Colorectal Dis 2010; Dec 3th
Erlotinib
Gefitinib
ALK inhibitor
PF-02341066
Cetuximab
K-Ras mutations
Pathology 2008;40:295–8
Cytopathology 2010; Oct 4th
B-Raf
mutations
Malignant Melanoma
GSK2118436
PLX4032
Modern pathology must be a synergy of morphology, IHC and molecular Dx
Gastrointestinal stromal tumor
Br J Cancer
2007;96:776–82
Cytopathology.
2009; 20:297-303
c-kit Mutations
C-kit mutations
Imatinib
Cytopathology. 2009 Oct;20(5):297-303.
Comparative validation of c-kit exon 11 mutation analysis on cytology
samples and corresponding surgical resections of gastrointestinal stromal
tumours
Pang NK, Chin SY, Nga ME, Chang AR, Ismail TM, Omar SS, Charlton A, Salto-Tellez M.
Primary Extragastrointestinal Stromal Tumor of the Pleura:
Report of a Unique Case With Genetic Confirmation.
Long KB, et al. Am J SurgPathol. 2010 May 3
Molecular analysis of c-Kit and PDGFRA in GISTs diagnosed
by EUS. Gomes AL, Bardales RH, Milanezi F, Reis RM,
Schmitt F. Am J ClinPathol. 2007 Jan;127(1):89-96
Fine-needle aspiration biopsy diagnosis of gastrointestinal
stromal tumors using morphology, immunocytochemistry,
and mutational analysis of c-kit. Rader AE, Avery A, Wait CL,
McGreeveyLS, Faigel D, Heinrich MC. Cancer. 2001 Aug
25;93(4):269-75
Colon cancer
J Mol Diagn. 2009; 11:543-52
Pathology 2008;40:295–8
Cytopathology 2010, Oct 4, in press
Int J Colorectal Dis. 2010 Dec 3.
KRAS mutations
B-Raf
mutations
K-Ras mutations
Cetuximab
KRAS and BRAF mutation analysis
can be reliably performed on
aspirated cytological specimens of
metastatic colorectal carcinoma
NKB Pang, ME Nga, SY Chin, TM
Ismail, GL Lim, R Soong and M
Salto-Tellez
Lung cancer
Clin Chem
2007;53:62–70
JTO
2011, accepted
EGFR mutations
EGFR
mutations
EML4-ALK
Erlotinib
Gefitinib
ALK inhibitor
PF-02341066
Sharma et al. Nat Rev Cancer 2007
Lung cancer
Clin Chem
2007;53:62–70
J Thorac Oncol
2011; accepted
EGFR
mutations
Gefitinib
Erlotinib
Colon cancer
EGFR mutation testing – clinical materials
Obtaining lung tumour samples:
what are the challenges?
Small Sample Revolution
In samples that are getting smaller,
pathologists need to generate
more meaningful information
Diagnostic / Therapeutic
Chin et al. Clin Chem 2007
66-year-old female, non-smoker,
asymptomatic, finding on routine X-ray left
upper lobe lung tumour, 4×4.5 cm
CELL BLOCK (X20)
CELL BLOCK (X600)
Wild-type
1.5 mm
RESECTION (MACRO)
719B: G>C
RESECTION (X20)
719B: G>A
719B: G>T
Negative
Chin et al. Clin Chem 2007
EGFR mutation testing of cytology samples:
experience from NUHS Singapore
A review of EGFR Mutational Analysis on Non Small
Cell Lung Cancer (NSCLC) Cytology Specimens
A Dhewar, B Pang, ME Nga,
Q Ahmed and M Salto- Tellez
Joint BSCC-NAC Annual Scientific Meeting 13-16 July
2011 Keele University
EGFR - Unsatisfactory rate 2011
13.3%
Excised during
surgery
9.1%
Bronchoscopic
biopsy (for central
lesions)
9.1%
Guided needle biopsy
(for peripheral
lesions)
5.3%
FNA cytology and
serous effusions (for
peripheral lesions)
EGFR mutation testing of cytology samples:
experience from NUHS Singapore
A comparative study of surgical and cytological
specimens for EGFR mutation testing- review of
data from two major tertiary hospitals in Singapore
S Gupta, JE Seet,M Salto-Tellez
Joint BSCC-NAC Annual Scientific Meeting 13-16 July
2011 Keele University
Diagnostic and therapeutic recommendations
Tumour sampling
‘Diagnostic’ sampling
Not adequate for
diagnosis
Adequate for diagnosis
Adequate for therapeutic
decision making?
Salto-Tellez et al. J Thorac Oncol 2011,
in press
Diagnostic
opinion
Yes
Therapeutic
decision
No
Pathology sequence – reflex testing
Diagnostic material (bronchoscopic, needle core or cytology)
SCLC
NSCLC
EGFR exons 18–21 mutation detection
EGFR mut
EGFR wt
KRAS Ex 2 & 3
KRAS mut?
KRAS wt?
EML4-ALK
EGFR-TKI
treatment
KRAS-related
treatment?
EML4-ALK AMP
EML4-ALK not AMP
ALK inhibitors?
Other treatments
Other treatments
Fujii T et al.
Molecular testing and Cytopathology of Pleural
Effusions in NSCLC(EGFR/KRAS/AML4-ALK).
O-73
Molecular Diagnostics and the
Specific Weight of Morphology
SQUAMOUS CELL CARCINOMA
ADENOCARCINOMA
NON-SMALL-CELL CARCINOMA
SMALL-CELL CARCINOMA
Non-small cell carcinoma
continuum
ADENOCA
NSCLC,
FAVOUR ADENOCA
YES
YES
+++
++(+)
NSCLC, NOS
(YES)
+
NSCLC,
FAVOUR SCC
SCC
(NO)
NO
(-)
-
Who should be tested for EGFR mutations?
What are the chances of mutation detection?
Mod Pathol 2003;16:79–85
Hum Pathol 2003;34:362–8
JCP2010;63(9):839-42
JCP 2011, accepted
Archives Path Lab Med 2001, in press
HER2-neu FISH
Breast
cancer
Gastric
cancer
Her-2/neu amplification
Trastuzumab
Lapatinib
Clinical history provided
53 year old female
Hep B carrier, sAg+ve on FU
Elevated AFP (37.4ug/L).
History of previous gastrectomy in another hospital
?diagnosis
CT abdomen showed no liver lesions
Left adrenal nodule
Approximately 2.0 x
2.6 cm in size
Occult HCC
metastasis?
EUS-FNAC done
Cytomorphology
Many papilleroid clusters of
cohesive tumour cells
Branching pattern
Very cellular smears
•
•
•
•
Cell block
Adenocarcinoma
Papillary pattern
Focal CK 20 +
Negative for CK 7, AFP &
TTF-1
Morphological diagnosis: Metastatic adenocarcinoma
Excerpt from conversation with the managing
oncologist:
“Did the patient have a gastric cancer?”
“Yes, please do a Her-2 scoring on the metastasis… both
FISH and IHC.”
“In the cytology sample?”
“Yes, the original materials a re in another hospital….., by
the way, it was intestinal type adenocarcinoma of the
stomach…”
•
•
•
•
Her 2 IHC
Heterogeneity of staining
Baso lateral staining pattern
No complete staining
Approximately 10 % showed
++ intensity in the cell block.
Chromogenic ISH
Fluorescent ISH
Kapila K et al.
FNA in Breast Cancer: FISH, CISH and IHC Comparison
P2-068
Beraki E et al.
Her-2 Status by DuoSISH
O-74
66 | 1.1 Topic goes here | Project number | 14.12.08
Copyright © 2008 National University Health System
Molecular diagnostics and
personalised / predictive medicine
Gastrointestinal stromal tumour
Br J Cancer
2007;96:776–82
Cytopathology
2009;20:297–303
Mod Pathol 2003;16:79–85
Hum Pathol 2003;34:362–8
J Clin Pathol2010;63:839–42
J Clin Pathol 2011; July 14th
Arch Pathol Lab Med 2011; 135(6):693-5
C-kit mutations
Imatinib
HER2-neu FISH
Breast
cancer
Trastuzumab
Lapatinib
Gastric
cancer
Lung cancer
Clin Chem
2007;53:62–70
J Thorac Oncol
2011, on line
EGFR
mutations
EML4-ALK
Colon cancer
J Mol Diagn 2009;11:543–52
Pathology 2008;40:295–8
Cytopathology 2010; Oct 4th
Int J Colorectal Dis 2010; Dec 3th
Erlotinib
Gefitinib
ALK inhibitor
PF-02341066
Cetuximab
K-Ras mutations
Pathology 2008;40:295–8
Cytopathology 2010; Oct 4th
B-Raf
mutations
Malignant Melanoma
GSK2118436
PLX4032
67 | 1.1 Topic goes here | Project number | 14.12.08
© 2008 National University Health System
Modern
pathology must be a synergy of morphology, Copyright
IHC and
molecular Dx
“The Cytology sample, in the context of appropriate
laboratory validations, should not
be treated differently to any other sample for
molecular testing"
“The ideal sample for molecular testing is the
first one available which, often, is the cytology
sample"
DIAGNOSTIC &
CLINICAL
APPLICATIONS