RECENT ADVANCES IN LUNG TUMORS AND

TUMOR LIKE LESIONS

Ekta Jajodia

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

Require evidence of either keratinization or intercellular bridging

Well , moderately or poorly differentiated carcinoma

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

SCC WITH PAPILLARY GROWTH PATTERN

BASALOID VARIANT OF SCC

SMALL CELL CARCINOMAStrongly a/w smoking

Variants-1. Small cell (pure)

2. Combined small cell/ non small cell

IHC- Chromogranin A ( specific ) synaptophysin ( sensitive)NSEN CAM (CD 56)bombesin

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

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

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

MUCINOUS NON MUCINOUS

Neoplastic cells grow exclusively along lining of alveolar walls without any evidence of infiltration into interstitium – LEPIDIC pattern

BRONCHOALVEOLAR CARCINOMA

CLEAR CELL ADENOCARCINOMA

Mucinous (“colloid”) carcinoma

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

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

LYMPHOEPITHELIOMA-LIKE CARCINOMA

Tumor cell islands surrounded by prominent lymphoplasmacytic host response

May be associated with Epstein-Barr virus

CARCINOID

Typical and atypical carcinoids

Uniform polygonal cells

Inconspicous nucleoli

Scant to moderate cytoplasm

Necrosis and mitosis 2-10/10 HPF

TUMOR LIKE LESIONS

Minute meningothelial nodulesNodular lymphoid hyperplasiaLocalized organizing pneumoniaNodular amyloidHyalinizing granulomaMicronodular pneumocyte hyperplasiaEndometriosisRounded atelectasisInflammatory pseudotumorBronchial inflammatory polypCongenital lesions

Minute Meningothelial Nodules

Epithelial membrane antigen and vimentin - positive

Cytokeratin, actin, and neuroendocrine markers - negative

Solid with irregular borders caused by widening of adjacent alveolar walls.

Whorls of cells are typical

Numerous secondary lymphoid follicles and fibrosis

Nodular Lymphoid Hyperplasia

Secondary lymphoid follicles surrounded by lymphocytes and plasma cells

Numerous plasma cells associated with russel bodies (but absence of dutcher bodies)

FOCAL ORGANIZING PNEUMONIA

Nonencapsulated nodule

Fibroblastic intraalveolar proliferations that fill alveolar spaces, alveolar ducts, and respiratory bronchioles

This airway contains an accumulation of fibroblastic cells and inflammatory cells that extends into the adjacent respiratory bronchiole

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

At interface with normal lung the alveoli shows emphysematous changesApical cap showing abundant elastic tissue and collagen and occasional fibroblast

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

Nodular amyloid is densely eosinophilic, with scant cellularity and without the fibrillar appearance of collagen

Amorphous eosinophilic material with sparse lymphoplasmacytic infiltrate

Multinucleated giant cells can be present

Ossification is often present in pulmonary amyloid

PULMONARY HYALINIZING GRANULOMA

Idiopathic condition

SFTs can also have areas of dense collagen but PHG is less cellular

Also, cells of SFT are CD34 positive

A circumscribed nodule, centrally acellular with a rim of inflammatory cells, is typical of pulmonary hyalinizing granuloma

The central eosinophilic zone is composed of dense ropy collagen with scant spindled cells and no histocyticinflammation or necrosis

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

markedly thickened and fibrotic pleura, with parietal pleural adhesion and encircled lung tissue

Underlying lung tissue is collapsed, adjacent to pleural fibrosis

INTERNATIONAL ASSOCIATION FOR THE STUDY OF LUNGCANCER/AMERICAN THORACIC SOCIETY/EUROPEAN

RESPIRATORY SOCIETY INTERNATIONAL MULTIDISCIPLINARY CLASSIFICATION OF LUNG

ADENOCARCINOMA

IASLC/ATS/ERS Classification of LungAdenocarcinoma in Resection Specimens

Preinvasive lesions

Atypical adenomatous hyperplasiaAdenocarcinoma in situ (3 cm formerly BAC) Nonmucinous Mucinous Mixed mucinous/nonmucinous

Minimally invasive adenocarcinoma (3 cm lepidic predominant tumor with 0.5cm invasion)

NonmucinousMucinousMixed mucinous/nonmucinous

Invasive adenocarcinoma

Lepidic predominant (formerly nonmucinous BAC pattern, with 5 mm invasion)Acinar predominantPapillary predominantMicropapillary predominantSolid predominant with mucin production

Variants of invasive adenocarcinoma

Invasive mucinous adenocarcinoma (formerly mucinous BAC)ColloidFetal (low and high grade)Enteric

RECOMMENDATION 1

Discontinuing use of the term BRONCHOALVEOLAR

CARCINOMA(BAC)

RECOMMENDATION 2

ADENOCARCINOMA IN-SITU – for small ( ≤3cm) , solitary adenocarcinoma with pure lepidic growth

Has 100% disease specific survival

Mucinous adenocarcinoma in situ

Nonmucinous adenocarcinoma in situ

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 pleura2. Contains tumour necrosis

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

RECOMMENDATION 4

INVASIVE ADENOCARCINOMA – most tumors consist of mixture of subtypes

Individual tumors are classified according to predominant pattern and percentage of subtypes

RECOMMENDATION 5

In patients with multiple lung adenocarcinoma - determine whether tumors are metastasis or separate synchronous or metachronous primaries

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

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

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 carcinoma2. Potential life threatening hemorrhage can occur if patients with squamous cell carcinoma receive bevacizumab3. EGFR and ALK mutation in adenocarcinoma respond to TKIs

Other recommendations are-

Small cell variant of squamous cell carcinoma is removed in the new classification

Other variants are papillary, clear cell and basaloid

Classification of Lung Cancer into Molecular Subtypes

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

1. CHARACTERIZED BY MUTATIONS IN THE EGFR GENE

Includes three classes of mutations –

a. Class I - exon 19 in-frame deletions- most commonb. Class II - single amino acid changesc. Class III - exon 20 in-frame duplication/insertions

Treatment – 1st generation EGFR inhibitors – erlotinib and gefitinib

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

SUBTYPE 2 - mutations in the K-ras gene

Ras has many isoforms-1. H-Ras mutation – in bladder cancer2. N-Ras mutation – in melanomas3. 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

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.

SUBTYPE 3 - ALK REARRANGEMENTSIt 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 anaplasticlymphomas and in about 50% of inflammatory myofibroblastic tumors (IMTs)Treatment – ALK inh - crizotinib

SUBTYPE 4 - ABERRATIONS IN C-MET

Characterized by either dysregulation or mutation of mesenchymal-epithelial transition factor receptor tyrosine kinase (c-MET)

SUBTYPE 5 – ABBERATION IN AKT/PI3K PATHWAY

SUBTYPE 6 – ABBERATION IN VASCULAR ENDOTHELIAL GROWTH FACTOR PATHWAY

SUBTYPE 7 – ROS-1 MUTATION

Molecular Testing Guideline for Selection of Lung Cancer

Patients for EGFR and ALK Tyrosine Kinase Inhibitors

Guideline from the College of American Pathologists, InternationalAssociation for the Study of Lung Cancer, and Association forMolecular Pathology

Almost all EGFR and ALK abberations are seen inadenocarcinoma 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

INDICATIONS –

1. Adenocarcinoma or adenocarcinoma component2. In tissues with incomplete sampling (biopsies,cytology) in which adenocarcinoma cant be

excluded3. 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

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

HOW RAPIDLY TEST RESULT BE AVAILABLE

Within 1 week(5 working days) to a maximum of 2 week (10 working days)

PROCESSING OF SPECIMEN FOR EGFR MUTATION TESTING

Formalin-fixed, paraffin-embedded (FFPE) specimensor 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.

SPECIMEN REQUIREMENTS FOR EGFR TESTING

EGFR mutation testing can be performed on specimensprocured by almost any procedure: surgical resection, open biopsy, endoscopy, transthoracic needle biopsy, fine-needle aspiration, or thoracentesis

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

There are 3 main types of EGFR IHC: IHCfor total EGFR, IHC for phosphorylated EGFR, and IHC formutated 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

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 neededSo IHC is too insensitive

For patients with low cellularity specimen which is inadequate for DNA analysis, IHC may be the best option available

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

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

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 tyrosinekinase domain have been described in AR specimens, including L747S, D761Y, and T854A

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

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 commercialassay as a “companion diagnostic” to select patients toreceive an FDA-approved ALK TKI

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

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

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.

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

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)

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

Stepwise-testing algorithms, if used, shouldnonetheless 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

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

IHC SCORING FOR ALK FUSION

3 criteria: 3-intense, granular cytoplasmic staining2- moderate, smooth cytoplasmic staining1-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 G1serum substitution for the primary antibody (ALK).

Score 3 showing intense,granular cytoplasmic staining

score 2 showing moderate, smooth(without the apparent granularityseen in score 3) cytoplasmic staining

score 1 showing faint, barelydiscernable cytoplasmic staining

score 0 showing no staining

14 DAYS AND 2 MONTHS AFTER ERLOTINIB THERAPY

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 CancerPatients for EGFR and ALK Tyrosine Kinase InhibitorsGuideline from the College of American Pathologists, InternationalAssociation for the Study of Lung Cancer, and Association forMolecular Pathology

4. Benign Tumors and Tumorlike Conditions of the LungAlain C. Borczuk, MD

REFERENCES

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