Best Pathology Practice
• Tissue specimens should be managed not only
for diagnosis but also to maximize the amount of
tissue available for molecular studies
• To guide therapy for patients with advanced lung
adenocarcinoma, each institution should
develop a multidisciplinary team that
coordinates the optimal approach to obtaining
and processing biopsy/cytology specimens to
provide expeditious diagnostic and molecular
results
International association for the study of lung cancer/American thoracic society/European
respiratory society international multidisciplinary classification of lung adenocarcinoma.
Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin
JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP,
Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton
P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E,
Tsao M, Yankelewitz D. J Thorac Oncol. 2011 Feb;6(2):244-85.
Best Pathology Practice
• When paired cytology and biopsy specimens exist,
they should be reviewed together to achieve the
most specific and nondiscordant diagnoses
• Cell blocks should be prepared from all cytology
samples including pleural fluids
International association for the study of lung cancer/American thoracic society/European
respiratory society international multidisciplinary classification of lung adenocarcinoma.
Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin
JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP,
Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton
P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E,
Tsao M, Yankelewitz D. J Thorac Oncol. 2011 Feb;6(2):244-85.
Criteria for Morphologic Diagnosis of ADC and SQC
on H&E Stained Slides
• Morphologic features of glandular (adenomatous)
differentiation:
– Gland formation
– Mucin production
• Morphologic features of squamous differentiation:
– Intercellular bridges
– Single cell keratinization
– Keratin production (keratin pearl formation)
Large Cell Carcinoma
• This diagnosis should not be made on small samples
(biopsies or cytology)
• The diagnosis should be NSCLC NOS
What Is The Rate Of NSCLC NOS?
• Study of California Cancer Registry: 175, 298 lung
cancer patients diagnosed from 1989 to 2006
• On histology: 22.1% (may be higher)
• On cytology: 37%
• The rate increases with decrease in sample size
• The rate has risen over time
Ou, S and Zell, J (2009) J Thorac Oncol 4(10): 1202.
Reproducibility of Pathologic
Diagnosis in NSCLC
• Digitalized H&E from 94 surgically resected specimens
were reviewed by 24 Pathologists (12 lung expert
Pathologists, 12 community Pathologists)
• Inter-rater agreement on differentiating SQC vs. non-SQC
• Kappa 0.55 for all pathologists
• Kappa 0.64 among lung expert pathologists
• Kappa 0.41 among non-expert community
pathologists
Grilley-Olson, J et al (2009) ASCO Meeting Abstracts 27(15S): 8008.
IHC as a Diagnostic Tool in NSCLC
Review of Literature
• No marker is 100% sensitive or specific
• Most frequently cited stains are:
– For ADC
TTF-1, mucin histochemical stains
– For SQC
(MA903)
p63, CK5/6, HMWK 34bE12
Travis, W et al (2010) J Thorac Oncol 5(4): 4.
IHC as a Diagnostic Tool in NSCLC
Mucin Histochemical Stains in Lung ADC
PAS
Mucicarmine
Alcian Blue
IHC as a Diagnostic Tool in NSCLC
TTF-1 in Lung ADC
Nuclear staining
• Sensitivity 54%*
• Specificity 97%*
• Positive PV 88%*
• Negative PV83%*
Loo, P et al (2010) J Thorac Oncol 5(4): 442.
IHC as a Diagnostic Tool in NSCLC
TTF-1 in Lung ADC
• Cytoplasmic staining should
be interpreted as
NEGATIVE
False Positive Immunostaining —TTF-1
200x
Type II Pneumocytes
Summary—Napsin A and TTF-1 IHC Staining (I)
Tumours
Napsin A
(% positive)
TTF-1
(% positive)
Adenocarcinoma
79/95 (83%)
69/95 (73%)
Well differentiated
42/47 (89%)
38/47 (81%)
Moderately differentiated
27/32 (84%)
24/32 (75%)
Poorly differentiated
11/16 (69%)
7/16 (44%)
Squamous cell carcinoma
0/46 (0%)
0/48 (0%)
Large cell carcinoma
3/9 (33%)
4/9 (44%)
5/12 (41.7%)
3/12 (25%)
Clear cell
14/41 (34%)
0/41 (0%)
Papillary
34/43 (79%)
0/43 (0%)
1/34 (3%)
0/34 (0%)
Papillary carcinoma
2/38 (5%)
37/38 (97%)
Follicular carcinoma
0/15 (0%)
15/15 (100%)
Follicular adenoma
0/28 (0%)
28/28 (100%)
“Non-small cell carcinoma,”
Not otherwise specified
Renal cell carcinomas
Chromophobe
• Sensitivity seems ≥ TTF-1
• Often positive in renal
neoplasms
• Rarely positive in thyroid
neoplasms
• Complementary to TTF-1?
• Not ready for prime time
Thyroid lesions
Bishop, J et al (2010) Hum Pathol 41(1): 20.
IHC as a Diagnostic Tool in NSCLC
p63 in Lung SQC
Nuclear staining
• Sensitivity
92%*
• Specificity
74%*
• Positive PV 82%*
• Negative PV 88%*
Loo, P et al (2010) J Thorac Oncol 5(4): 442.
IHC as a Diagnostic Tool in NSCLC
CK5/6 in Lung SQC
Cytoplasmic staining
• Sensitivity
84%*
• Specificity
79%*
• Positive PV 84%*
• Negative PV 79%*
Loo, P et al (2010) J Thorac Oncol 5(4): 442.
Modern Pathology (2012) 25, 405–415
IHC as a Diagnostic Tool in
NSCLC—Review of Literature
S100 A7
CK 5/6
Proportion
Proportion
0
1
2
3
0
0
1
2
3
• TTF-1
•
Moderate > 50% or strong >
10%
•
Proportion
Proportion
1
2
3
0
1
2
Any droplet
1
0
3
• AB/PAS
3
2
p63
Intensity
Moderate or strong > 10%
2
1
HMWCK
0
Intensity
•
3
3
0
• CK5/6 and p63
2
0
Intensity
Intensity
Predictive scoring cutoffs
1
1
2
3
Predictor of adenocarcinoma:
TTF1
Proportion
0
2
3
Not predictive
0
Intensity
Proportion
1 : 1-10%
2 : 10-50%
3 : > 50%
1
1
Predictive
2
3
Loo, P et al (2010) J Thorac Oncol 5(4): 442.
No data
Malignant bronchial biopsy
(NSCLC)
Diagnostic Algorithm for Classification
of NSCLC in Small Biopsy Samples
H&E stain
75% subtyped
(87% accuracy-SQC; 80% accuracy-ADC)
IHC and AB/PAS
18% have subtype predicted
(86% accuracy)
7% not typed
“Null” IHC on biopsy
“Null” Case
Resection histology
14% Squamous cell
(1% total)
43% Adenocarcinoma
(3% total)
43% Large cell
(3% total)
Loo, P et al (2010) J Thorac Oncol 5(4): 442.
IHC as a Diagnostic Tool in NSCLC
Review of Literature
Large cell carcinoma of the lung: an endangered species?
Pardo J, Martinez-Peñuela AM, Sola JJ, Panizo A,
Gúrpide A, Martinez-Peñuela JM, Lozano MD.
Appl Immunohistochem Mol Morphol. 2009 Oct;17(5):383.
IHC as a Diagnostic Tool in NSCLC
Review of Literature
• 101 LCC TMA, including 82 « classic » LCC
• 31 monoclonal antibodies
• Reclassification of 82 « classic » LCC
– 33% ADC
TTF1(+) CK7(+) CK19(+) p63(-)
– 37% SQC
34bE12(+) p63(+) TM(+)
CD44v6(+)
– 20% AS*
Hybrid AC-SQC
immunophenotype
•
Panel of 7 useful antibodies
– TTF-1, CK7, CK19, p63, 34bE12, TM, CD44v6
ADC: Adenocarcinoma; SQC: Squamous cell carcinoma; LCC: Large cell carcinoma;
ASC: Adenosquamous
carcinoma;
NOS: Not Otherwise
– < 10%
of residual
LCC Specified; IHC: Immunohistochemistry
al (2009) Appl Immunohistochem
– > Pardo,
90%J etreclassified
ADC, SQCMolorMorphol
ASC17(5): 383.
Reporting Small Lung Samples
• The term NSCLC-NOS should be used as little as
possible, and be applied only when a more
specific diagnosis is not possible by morphology
and/or special stains
• The term large cell carcinoma should not be
used for diagnosis in small biopsy or cytology
specimens and should be restricted to resection
specimens where the tumor is thoroughly
International association for the study of lung cancer/American thoracic society/European
respiratory society international multidisciplinary classification of lung adenocarcinoma.
sampled to exclude a differentiated component
Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K, Austin
JH, Asamura H, Rusch VW, Hirsch FR, Scagliotti G, Mitsudomi T, Huber RM, Ishikawa Y, Jett J, Sanchez-Cespedes M, Sculier JP,
Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton
P, Henderson D, Johnson B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R, Saijo N, Thunnissen E,
Tsao M, Yankelewitz D. J Thorac Oncol. 2011 Feb;6(2):244-85.
Algorithm for adenocarcinoma
diagnosis
in small biopsies and/or cytology
STEP 1
POSITIVE BIOPSY
(FOB, TBBx, Core, SLBx)
POSITIVE CYTOLOGY
(effusion, aspirate, washings, brushings)
Histology: Lepidic, papillary, and/or
acinar architecture(s)
Cytology: 3-D arrangements, delicate
foamy/vacuolated (translucent)
cytoplasm,
Fine nuclear chromatin and often
prominent nucleoli
Nuclei are often eccentrically situated
Classic morphology: ADC
NE morphology, large cells,
NE IHC+
NE morphology, small cells, no
nucleoli, NE IHC+, TTF-1 +/-, CK+
Keratinization, pearls and/or
intercellular bridges
NSCLC,
?LCNEC
SCLC
Classic Morphology: SQCC
No clear ADC or SQCC
morphology: NSCLC-NOS
STEP 2
Apply ancillary panel of
One SQCC and one ADC marker
+/OR Mucin
Algorithm for adenocarcinoma
diagnosis
in small biopsies and/or cytology
NSCLC, favor SQCC
STEP 2
Apply ancillary panel of
One SQCC and one ADC marker
+/OR Mucin
SQCC marker +ve
ADC marker -ve/or
Mucin -ve
ADC marker and/or Mucin +ve;
SQCC marker -ve (or weak in
same cells)
IHC -ve and
Mucin -ve
ADC, marker or Mucin +ve;
as well as SQCC marker +ve
in different cells
NSCLC, favor ADC
NSCLC NOS
NSCLC, NOS,
possible adenosquamous ca
Algorithm for adenocarcinoma
diagnosis
in small biopsies and/or cytology
Classic morphology: ADC
NSCLC, favor ADC
NSCLC NOS
Molecular analysis:
e.g. EGFR mutation†
NSCLC, NOS,
possible adenosquamous ca
STEP 3
If tumor tissue inadequate for
molecular testing, discuss need
for further sampling – back to
STEP 1
Identification of the transforming EML4-ALK fusion gene in non-small-cell
lung cancer.
Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S,
Watanabe H, Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y,
Aburatani H, Niki T, Sohara Y, Sugiyama Y, Mano H.
Nature. 2007 Aug 2;448(7153):561.
Oncogenic
Chr. 2p23-22
Mano, H (2008) Cancer Sci 99(12): 2349.
Perner, S et al. (2008) Neoplasia 10(3): 298.
ALK Break Apart FISH
e20
ALK
~12 Mb
EML4
Maximum change in tumour size (%)
Updated Phase I-II Trial of an Oral Cmet and ALK Inhibitor Crizotinib (PF–
02341066)
60
Progressive disease
Stable disease
Confirmed partial response
Confirmed complete response
40
20
0
-20
-30%
-40
-60
ONLY 3-5% OF NSCLC
-80
Overall Response Rate = 57%
Disease Control Rate (CR+PR+SD) at 8 weeks= 87%
-100
* Partial response patients with 100% change have non-target disease present
Bang, Y et al. (2010). ASCO Meeting Abstracts 28(18_suppl):
(EML4)-ALK Fusion Gene Tumors Occur Mainly
in Adenocarcinoma
First author
Adeno
(total no.)
Squamous
(total no.)
Others
(total no.)
Imamura (2008)
3.4% (149)
0% (48)
0 (24)
Shinamura (2008)
4% (50)
0% (20)
0% (7)
Takeuchi (2008)
4.3% (253)
0% (71)
0% (19)
Koivunen (2008)
3.8% (208)
0% (88)
0 (100)
Rodig (2009)
5.6% (358)
-
-
Martlelli (2009)
4.8% (63)
8.3% (48)
22.2% (9)
Wong (2010)
5.3% (209)
0% (34)
8.7% (23)
Salido (2010)
4.3% (69)
0% (30)
0% (8)
Jokoji (2010)
3.1% (254)
-
-
Takahashi (2010)
2.4% (211)
0% (75)
0% (27)
Paik (2011)
6.3% (423)
0% (163)
3.7% (27)
Tumors studied
2247
577
244
Histological Features of Adenocarcinoma
Associated with EML4-ALK Aberration
No. Studied
ALK +ve
Acinar
Papillary
Solid
BAC
Others
Rodig
(N. America)
Inamura
(Japan)
342
253
20 (5.8%)
11 (4.3%)
4/124 (3.2%)
6/34 (17.6%)
0/46 (0%)
5/206 (2.4%)
11/134 (8.2%)
0/5 (0%)
1/22 (4.5%)
0/7 (0%)
0
0/1 (0%)
Signet ring
None
≤ 10%
>10%
Tend to be
mucin producing
2/21 (10%)
and TTF1+
12/26 (46%)
3/295 (1%)
Rodig SJ, et al. CCR 2009;15:5216-23; Inamura K, et al. Modern Pathol 2009;22:508-15
CRIBRIFORM
SIGNET RING
ALK +
(n=54)
ALK –
(n=100)
P value
Any lepidic
24%
84%
<0.0001
Any acinar
87%
49%
<0.0001
Any papillary
67%
70%
0.7170
Any solid
65%
33%
0.0002
Cribriform
80%
16%
<0.0001
Signet ring cells
65%
1%
<0.0001
Am J Pathol 2011;35:1226-34
ALK1 antibody with ADVANCED
detection system (DAKO)
3+
2+
1+
0
J Thorac Oncol. 2011;6: 466–472
2+/3+ IHC: Sensitivity 90%
Specificity 97.8%
J Thoracic Oncol 2011;6:466-72
Lung Cancer 2012 (Epub)
A novel, highly sensitive antibody allows for the routine detection of ALKrearranged lung adenocarcinomas by standard immunohistochemistry.
Mino-Kenudson M, Chirieac LR, Law K, Hornick JL, Lindeman N,
Mark EJ, Cohen DW, Johnson BE, Jänne PA, Lafrate AJ, Rodig SJ.
Clin Cancer Res. 2010 Mar 1;16(5):1561.
Antibody
D5F3
ALK1
Sensitivity
100%
67%
Specificity
99%
97%
Conclusions
• Current practice of pathology in lung
cancer requires more precise classification
of tumor type
• For poorly differentiated NSCLC, a minimal
set of immunohistochemistry markers
(TTF1, mucin, p63 and CK5) should reduce
the number of NOS to <5%.
• Cell block preparation should be part of
the routine practice of cytology (especially
for FNA and fluid)
• For both small biopsy and cytology, every
ACKNOWLEDGEMENTS
• Dr. Diana Ionescu (British Columbia
Cancer Agency)
• Dr. Tony Magliocco (Tom Baker Cancer
Centre and Calgary Laboratory Services)
• Dr. Christian Couture (Université Laval)
• Maritz Canada & Eli Lilly Canada