2. WHO CLASSIFICATION
SQUAMOUS CELL CARCINOMA
The central type and the peripheral type
According to a study the patient population of the
peripheral type was older, improved survival, lower
lymphatic vessel involvement, and lymph node metastasis
IHC : Low and High Molecular weight keratins ( CK 5/6)
P63, p40
Involucrin
3. Require evidence of either keratinization or intercellular
bridging
Well , moderately or poorly differentiated carcinoma
4. 1) Small cell variant - vs small cell carcinoma
( lack the nuclear characteristics of small cell ca
and may show focal keratinization )
2) Clear cell variant
3) Papillary variant
4) Basaloid – Very aggressive clinical course
, peripheral palisading of cells
7. SMALL CELL CARCINOMA
Strongly a/w smoking
Variants-
1. Small cell (pure)
2. Combined small cell/ non small cell
IHC-
Chromogranin A ( specific )
synaptophysin ( sensitive)
NSE
N CAM (CD 56)
bombesin
8. Microscopy
Diffuse growth of small tumor cells
Hyperchromatic finely granular nuclei
Inconspicuous nucleoli
Thin nuclear membrane
Scant , faintly stained finely granular
cytoplasm
Ill defined cell borders
Nuclear moulding
Stroma is delicate , finely vascular
Frequent mitotic figures and individual
cell necrosis
9. Necrosis leading to diffusion of chromatin to wall of the
blood vessels which appear hematoxyphillic
+ve for feulgen reaction
Also seen in lymphomas, seminomas
AZZOPARDI EFFECT
10. Adenocarcinoma
Adenocarcinoma, mixed subtype
Acinar adenocarcinoma
Papillary adenocarcinoma
Bronchioloalveolar carcinoma
Nonmucinous
Mucinous
Mixed nonmucinous and mucinous or indeterminate
Solid adenocarcinoma with mucin production
Fetal adenocarcinoma
Mucinous (“colloid”) carcinoma
Mucinous cystadenocarcinoma
Signet ring adenocarcinoma
Clear cell adenocarcinoma
11. MUCINOUS NON MUCINOUS
Neoplastic cells grow exclusively along lining of alveolar
walls without any evidence of infiltration into interstitium –
LEPIDIC pattern
BRONCHOALVEOLAR CARCINOMA
13. Large cell neuroendocrine carcinoma
Combined large cell neuroendocrine carcinoma
Basaloid carcinoma
Lymphoepithelioma-like carcinoma
Clear cell carcinoma
Large cell carcinoma with rhabdoid phenotype
LARGE CELL CARCINOMA
14. LARGE CELL
NEUROENDOCRINE
CARCINOMA Organoid , nesting, trabecular
growth, rosettes and perilobular
palisading patterns
Cells are generally large
Moderate to abundant cytoplasm
Nucleoli prominent
Mitosis >11/10 HPF
Positive NE markers –
synaptophysin, chromogranin,CD56
25. This airway contains an accumulation of fibroblastic
cells and inflammatory cells that extends into the
adjacent respiratory bronchiole
26. Apical cap
Irregular apical nodule (upper lobe > lower lobe)
Subpleural, triangular contours with broad pleural base
Irregular borders – radiologically- spiculated
Overlying pleura can be thickened, resembling
hyalinized pleural plaque
27. At interface with normal lung the alveoli shows
emphysematous changes
Apical cap showing abundant elastic tissue and collagen and
occasional fibroblast
28. NODULAR AMYLOIDOSIS
Nodular amyloidosis can be 1. Organ isolated ( idiopathic or
due to chronic inflammation)
2. Part of systemic disease
(Sjogren syndrome, multiple myeloma, lymphoma, or light
chain disease)
Detected as a solitary lung nodule
Primary amyloidosis type
Serum and urine testing in these patients does not show a
monoclonal protein in many cases
29. Nodular amyloid is densely eosinophilic, with scant
cellularity and without the fibrillar appearance of
collagen
34. A circumscribed nodule, centrally acellular with a rim of
inflammatory cells, is typical of pulmonary hyalinizing
granuloma
35. The central eosinophilic zone is composed of dense ropy
collagen with scant spindled cells and no histocytic
inflammation or necrosis
36. Peripheral subpleural area of lung tissue that becomes
folded into an area of visceral pleural fibrosis
Asbestos exposure causing parietal pleural plaques
followed by visceral pleural adhesion is the typical
scenario and then adjacent lung gets entrapped in
pleural adhesions
At the time of surgery the lysis of adhesions and
reexpansion of the folded lung causes the nodule to
disappear
ROUNDED ATELECTASIS
37. markedly thickened and fibrotic pleura, with parietal
pleural adhesion and encircled lung tissue
39. INTERNATIONAL ASSOCIATION FOR THE STUDY OF LUNG
CANCER/AMERICAN THORACIC SOCIETY/EUROPEAN
RESPIRATORY SOCIETY INTERNATIONAL
MULTIDISCIPLINARY CLASSIFICATION OF LUNG
ADENOCARCINOMA
40. IASLC/ATS/ERS Classification of Lung
Adenocarcinoma in Resection Specimens
Preinvasive lesions
Atypical adenomatous hyperplasia
Adenocarcinoma in situ (3 cm formerly BAC)
Nonmucinous
Mucinous
Mixed mucinous/nonmucinous
Minimally invasive adenocarcinoma (3 cm lepidic
predominant tumor with 0.5cm invasion)
Nonmucinous
Mucinous
Mixed mucinous/nonmucinous
41. Invasive adenocarcinoma
Lepidic predominant (formerly nonmucinous BAC pattern, with 5 mm invasion)
Acinar predominant
Papillary predominant
Micropapillary predominant
Solid predominant with mucin production
Variants of invasive adenocarcinoma
Invasive mucinous adenocarcinoma (formerly mucinous BAC)
Colloid
Fetal (low and high grade)
Enteric
46. RECOMMENDATION 3
MINIMALLY INVASIVE ADENOCARCINOMA (MIA)- for small
(≤3cm) , solitary adenocarcinoma with predominant lepidic
growth and small foci of invasion measuring (≤0.5cm)
Near 100% disease specific survival
The invasive component is defined as – 1. histological
subtype other than a lepidic pattern
(acinar, papillary, micropappilary, solid)
2. Tumor cells infiltrating myofibroblastic stroma
MIA is excluded if – 1. invades blood vessels, lymphatics or
pleura
2. Contains tumour necrosis
47. Size of the largest invasive area should be measured in
the largest dimension
Size of invasion is not summation of all such foci, if more
than one occurs
48. RECOMMENDATION 4
INVASIVE ADENOCARCINOMA – most tumors consist of
mixture of subtypes
Individual tumors are classified according to predominant
pattern and percentage of subtypes
49. RECOMMENDATION 5
In patients with multiple lung adenocarcinoma -
determine whether tumors are metastasis or separate
synchronous or metachronous primaries
50. RECOMMENDATION 6
LEPIDIC PREDOMINANT ADENOCARCINOMA (LPA) -
replaces use of term “mixed subtype”
Invasive adenocarcinoma is present in atleast one focus
measuring more than 0.5cm in greatest dimension
Invasion is defined as- 1. histological subtype other than
lepidic pattern (acinar , papillary , micropapillary, solid)
2. Tumor cells invading myofibroblastic stroma
Diagnosis of LPA is made if – 1. tumor invades
lymphatics, blood vessels and pleura
2. contains tumor necrosis
51. RECOMMENDATION 7
Addition of one more subtype of invasive
adenocarcinoma – micropapillary type
Others are – solid, papillary, acinar, lepidic
Micropapillary type- tumor cells grow in papillary tufts
lacking fibrovascular core
Associated with poor prognosis
52. RECOMMENDATION 8
NSCLC- NOS to be used as little as possible
Recommended to further classify into more specific type
such as adenocarcinoma or squamous cell carcinoma
because- 1. adenocarcinoma respond to pemetrexed
therapy better than squamous cell carcinoma
2. Potential life threatening hemorrhage can occur if
patients with squamous cell carcinoma receive
bevacizumab
3. EGFR and ALK mutation in adenocarcinoma respond
to TKIs
53. Other recommendations are-
Small cell variant of squamous cell carcinoma
is removed in the new classification
Other variants are papillary, clear cell and
basaloid
55. EGFR gene encodes a transmembrane receptor
binds to epidermal growth factor becomes
activated EGFR tyrosine kinase activity stimulates
activation of downstream pathways leading to DNA
synthesis and cell proliferation
Found in approx 15% of NSCLC
SUBTYPE 1 - aberrations in the EGFR
gene/pathway
56. 1. CHARACTERIZED BY MUTATIONS IN
THE EGFR GENE
Includes three classes of mutations –
a. Class I - exon 19 in-frame deletions- most common
b. Class II - single amino acid changes
c. Class III - exon 20 in-frame duplication/insertions
Treatment – 1st generation EGFR inhibitors – erlotinib
and gefitinib
57. 2. Harbours a T790M mutation in exon
20 of the EGFR gene
Mutations emerge in response to treatment with EGFR
TKIs (erlotinib or gefitinib )
EGFR TKI compete with ATP for ATP binding site on the
EGFR prevents autophosphorylation and
activation of EGFR’s kinase domain T790M
mutation increases EGFR’s affinity for ATP
reduces potency of competitive tyrosine kinase
inhibitors confers drug resistance
2nd gen EGFR inh - afatinib
58. SUBTYPE 2 - mutations in the K-ras gene
Ras has many isoforms-
1. H-Ras mutation – in bladder cancer
2. N-Ras mutation – in melanomas
3. K-Ras mutation – in adenocarcinoma of lung, colon,
pancreas
Point mutations at codons 12, 13, or 60 in the K-ras
oncogene lead to activation of K-ras protein
Reported in 15% to 20% of all patients with NSCLC
59. Mutations at codon 12 most commonly detected
K-ras mutations are seen almost exclusively in smokers
K-ras mutations in NSCLC are associated with decreased
response to EGFR TKIs.
60. SUBTYPE 3 - ALK REARRANGEMENTS
It harbors the EML4-ALK fusion oncogene, a fusion between
echinoderm microtubule-associated protein-like 4 (EML4)
and anaplastic lymphoma kinase (ALK)
The fusion generates a transforming tyrosine kinase, with as
many as nine different variants identified
Patients with EML4-ALK mutant tumors are characteristically
younger, female, and never to light smokers
ALK fusions have also been described in anaplastic
lymphomas and in about 50% of inflammatory
myofibroblastic tumors (IMTs)
Treatment – ALK inh - crizotinib
61. SUBTYPE 4 - ABERRATIONS IN C-MET
Characterized by either dysregulation or mutation of
mesenchymal-epithelial transition factor receptor tyrosine
kinase (c-MET)
63. Molecular Testing Guideline for
Selection of Lung Cancer
Patients for EGFR and ALK
Tyrosine Kinase Inhibitors
Guideline from the College of American Pathologists, International
Association for the Study of Lung Cancer, and Association for
Molecular Pathology
64. Almost all EGFR and ALK abberations are seen in
adenocarcinoma or NSCLC with adenocarcinoma
component
EGFR mutation seen in 15% of NSCLCs
ALK rearrangements seen in 4% of adenocarcinomas
Seen in younger age, females, non-smokers
Rare EGFR mutation seen in pulmonary salivary gland-type
tumors, large cell carcinoma, sarcomatoid carcionoma
65. INDICATIONS –
1. Adenocarcinoma or adenocarcinoma component
2. In tissues with incomplete sampling
(biopsies,cytology) in which adenocarcinoma cant
be excluded
3. In undifferentiated or large cell carcinoma – testing
done if IHC positive for adenocarcinoma lineage(TTF-
1) or IHC negative for squamous cell carcionma
lineage(P63 or P40)
Not recommended in pure squamous cell carcinoma or
pure small cell carcinoma
66. WHEN TO TEST
EGFR mutation and ALK rearrangement testing ordered at
time of diagnosis for patients presenting with advanced-
stage disease (stage IV) or at time of recurrence or
progression in patients who originally presented with
lower-stage disease but were not previously tested
67. HOW RAPIDLY TEST RESULT BE
AVAILABLE
Within 1 week(5 working days) to a maximum of 2 week
(10 working days)
68. PROCESSING OF SPECIMEN FOR
EGFR MUTATION TESTING
Formalin-fixed, paraffin-embedded (FFPE) specimens
or fresh, frozen, or alcohol-fixed specimens for polymerase
chain reaction (PCR)–based EGFR mutation tests
Other tissue treatments (eg, acidic or heavy metal
fixatives, or decalcifying solutions) should be avoided in
specimens destined for EGFR testing
Cytologic samples are also suitable for EGFR and ALK
testing, with cell blocks being preferred over smear
preparations.
69. SPECIMEN REQUIREMENTS FOR
EGFR TESTING
EGFR mutation testing can be performed on specimens
procured by almost any procedure: surgical resection,
open biopsy, endoscopy, transthoracic needle biopsy, fine-
needle aspiration, or thoracentesis
70. HOW EGFR TESTING BE
PERFORMED
Should use EGFR test methods that are able to detect
mutations in specimens with at least 50% cancer cell
content
Sanger sequencing with and without mutated allele
enrichment is the recommended method
EGFR copy number testing, by FISH or CISH, is less
predictive than mutation testing and should not be used
as a method for EGFR TKI treatment selection
71. There are 3 main types of EGFR IHC: IHC
for total EGFR, IHC for phosphorylated EGFR, and IHC for
mutated forms of EGFR
IHC for total EGFR is not an acceptable test for EGFR TKI
treatment selection because it correlates poorly or not at
all with presence of EGFR mutations
IHC FOR EGFR
72. IHC FOR MUTATED FORMS- antibodies directed against
the most common mutated forms of EGFR: the 15-bp/5-
amino-acid deletion in exon 19 and the L858R point
mutation in exon 21
Even if IHC negative - molecular testing is still needed
So IHC is too insensitive
For patients with low cellularity specimen which is
inadequate for DNA analysis, IHC may be the best option
available
73. Role of KRAS analysis in
selecting patients for EGFR TKI
The most common (~30%) oncogene mutated in lung
adenocarcinomas is KRAS.
EGFR and KRAS mutations are mutually exclusive
Testing for KRAS mutations as a negative predictor of
response to EGFR TKI has become part of molecular
diagnostic algorithms for lung adenocarcinoma in many
centers if sample is sufficient
74. ADDITIONAL TEST IN SECONDARY
OR ACQUIRED RESISTANCE TO
EGFR TKI
Such tests should be able to detect the secondary EGFR
T790M mutation in as few as 5% of cells
Most common mechanism of AR involves the emergence
of an additional EGFR tyrosine kinase domain
mutation, T790M
This mutation is seen in approximately 50% of tumors at
the time of treatment failure
75. T790M is often not present in every tumor cell, so
conventional Sanger sequencing, even with
microdissection, is considered insufficient for this
testing
Sanger sequencing with a mutation enriching strategy
such as peptide nucleic acid/locked nucleic acid
clamps, or a more sensitive assay (eg, allele-specific
PCR) that targets the T790M mutation to be used
Other rare second-site mutations in the EGFR tyrosine
kinase domain have been described in AR
specimens, including L747S, D761Y, and T854A
76. METHODS USED FOR ALK TESTING
ALK FISH assay is done using dual-labeled break-apart
probes
Most common ALK rearrangements involves a pericentric
inversion on the short arm of chromosome 2, which
creates a fusion gene encoding the aminoterminal portion
of EML4 and the intracellular region of ALK
77. The NPM-ALK translocation seen in anaplastic large cell
lymphoma - not reported in lung cancer
A commercial assay is available that contains an orange
and green labeled probes
With this probe set, wild-type configuration appears as
fused yellow signal
ALK rearrangement is seen as distinct and separated
orange and green signals
In the USA, FDA has approved this commercial
assay as a “companion diagnostic” to select patients to
receive an FDA-approved ALK TKI
78. ALK IHC may be considered as a screening methodology
to select specimens for ALK FISH testing
If IHC is negative – not tested for ALK rearrangement by
FISH
Tumors positive for ALK IHC, either weakly or
strongly, referred to FISH for confirmation of a
rearrangement
79. Specimen requirements for ALK FISH are generally similar
to those for EGFR mutation testing:
Formalin fixation is acceptable, specimens should have
enough cancer cells to analyze clearly, and DNA-
damaging fixatives or acidic decalcifying agents should be
avoided, as should specimens with abundant necrosis
FISH – Not performed on alcohol fixed samples
Ideally performed on recently cut sections
80. Proper interpretation of the FDA-
approved commercial break-apart
assay
The most common positive result of a break-apart dual-
labeled FISH assay in lung cancer will result in nuclei with 1
separate orange and 1 separate green signal that are
separated by a gap larger than 2 signal diameters
The native unaltered ALK region will remain as a yellow
fusion signal but also commonly appears as 2 narrowly split
orange and green signals.
81. A case was considered positive if ≥15% of 50 nuclei
assessed in a tumor-rich portion of the section showed
the classic split-signal pattern
Secondary mutations in ALK is seen that confer acquired
resistance to crizotinib
Number of such cases are too small to recommend
testing for these mutations
Anticipated for such testing in near future as effective
second line therapies become available
82. MUST ALL ADENOCARCINOMAS BE TESTED
FOR BOTH EGFR AND ALK
First algorithm - test for EGFR mutations first and proceed to
ALK FISH if the EGFR results are wild type
Second algorithm - initial sensitive and rapid EGFR mutation
screening test by a method such as denaturing high-
performance liquid chromatography, high-resolution melting
analysis, or single-stranded conformational polymorphism.
could detect a mutation but fail to characterize it
completely (ie, fail to define the size of an exon 19 deletion
or distinguish between L858R and L861Q point mutations)
83. SCREENING FOR EGFR MUTATION
MUTATION DETECTED MUTATION NOT DETECTED
SPECIFIC METHOD LIKE
SEQUENCING PERFORMED
FOR DEFINITIVE DIAGNOSIS
SCREENING BY ALK IHC
POSITIVE
ALK FISH
NEGATIVE
SOME OTHER
MUTATION PRESENT
84. Stepwise-testing algorithms, if used, should
nonetheless be completed within 10 working days
THIRD ALGORITHM- Test for KRAS mutation
KRAS mutation occurs in 25-30% of lung adenocarcinoma
and such tumors do not have EGFR or ALK abberations
If positive- no other test to be done
If negative – can go for either 1st or 2nd algorithm
Should not be undertaken if KRAS testing will exhaust the
sample and thereby preclude EGFR and ALK testing
85. HOW SHOULD EGFR AND ALK
RESULTS BE REPORTED?
The preclinical section of reports should include
specimen’s morphologic characteristics:
diagnosis and tumor content (percentage of total nuclei
that are malignant)
The results section of reports should include, prominently,
the names of any clinically significant mutations identified
In case of ALK testing - The results section should also
include the number of cells analyzed, and the number and
percentage of cells with each finding
86. IHC SCORING FOR ALK FUSION
3 criteria:
3-intense, granular cytoplasmic staining
2- moderate, smooth cytoplasmic staining
1-faint cytoplasmic staining in 10% of tumor cells
0- no Staining
Positive control was from a known CD30-positive ALCL
Negative control was a mouse immunoglobulin G1
serum substitution for the primary antibody (ALK).
93. 1. International Association for the Study of Lung Cancer/American
Thoracic Society/European Respiratory Society International
Multidisciplinary Classification of Lung Adenocarcinoma
2. WHO classification of lung tumors
3. Molecular Testing Guideline for Selection of Lung Cancer
Patients for EGFR and ALK Tyrosine Kinase Inhibitors
Guideline from the College of American Pathologists, International
Association for the Study of Lung Cancer, and Association for
Molecular Pathology
4. Benign Tumors and Tumorlike Conditions of the Lung
Alain C. Borczuk, MD
REFERENCES