- Solitary lung mass on radiology in a chronic smoker patient; no evidence of any other mass elsewhere in the body.
- Few small irregular and angulated glands seen in an invasive pattern with desmoplastic/ myofibroblastic stroma.
- Occasional glands melt within fibrous stroma.
- Nuclei show enlargement and hyperchromasia, along with anisonucleosis
Practical Points of Pathoclinics:
- Purpose of biopsy and/or cytology is diagnostic as well as evaluation of several molecular markers, for targeted therapy. Surgical pathologists and histo-technician must be aware of the importance of molecular testing, along with careful handling of the tissue biopsy and cytology specimen.
- Repeat lung biopsies for detection of predictive molecular markers are necessary in cases with inadequate tissue. Complications arising from repeat biopsies are low and most are of minor degree like pneumothoraces, which doesn’t required active intervention. Slightly higher complication rates seen with wide bore needle (18-gauge needles). Multiple passes with 20‐gauge biopsy needles increase the tissue yield. Tissue cores should be obtained as much as possible, at least 5 cores are good. Submission of one or two cores in one paraffin block is ideal for better cutting and preservation of tissue by preventing unnecessary trimming of blocks. These tissue cores must be handled carefully. It is good practice to obtained initial multiple sections of the paraffin blocks, for diagnostic as well as predictive immunohistochemistry (IHC) staining, Fluorescence In Situ Hybridization (FISH) analyses, and sequencing studies. This approach prevents the loss of tissue during second time cutting of paraffin block. Diagnostic purpose should be fulfilled with first and last sections, and IHC (some around 3 or 4 IHC markers only) should be done on middle sections. In cases of very low tumor cellularity, more sections needs to be stained. After diagnostic process, even stained slides can be reused for molecular work-up. However, digital images should be taken and archived before sacrificing the diagnostic slides for molecular work-up.
- Needle biopsy is preferable over cytology for molecular evaluation. However, any sample with adequate tumor cellularity should be the choice. Rapid on‐site evaluation of cellularity also ensures the specimen adequacy. Quality and ease of DNA yield is better with alcohol fixed cytology samples than formalin fixed tissue. Cell block preparations are more preferred than direct cytology smears. However, cell block preparations can be cell poor despite adequately cellular aspirates. FNA needle rinses samples or liquid-based preparations can be used for molecular studies.
- Metastatic deposits in bone biopsies are not preferred for molecular testing, as decalcification procedure decrease the yield of DNA for molecular profiling. Before decalcification, soft tissue should be separated and processed to avoid significant denaturation of DNA.
- Grading: G1: Well-differentiated adenocarcinoma: lepidic pattern (consider only single row of cells) with 0 to 1 mitotic figure/10 HPF and mild degree of pleomorphism only (round, regular nuclei with evenly dispersed chromatin and without inconspicuous nucleoli, up to 2× to 3× the size of a lymphocyte) and with no or less than 20% of high-grade patterns (solid, micro papillary, and/or complex glandular patterns). G2: Moderately differentiated adenocarcinomas: acinar and papillary pattern (more than single layer of cells) with mitotic counts of 2 to 4/10 HPF and mild to moderate nuclear pleomorphism (round, mildly irregular, pleomorphic nuclei without inconspicuous nucleoli, up to 2× to 3× the size of a lymphocyte) with no or less than 20% of high-grade patterns. G3: Poorly differentiated adenocarcinoma: 20% or more of high-grade patterns – solid, cribriform and micropapillary and with 5 or more mitotic figures/10 HPF and moderate to marked nuclear pleomorphism (pleomorphic nuclei with prominent nucleoli, greater than 5× the size of a lymphocyte). Due to heterogeneity of lung adenocarcinomas, grading should be recorded as per the highest-grade area including presence or absence of tumor necrosis. Grading is an independent predictor of prognosis.
- Morphological heterogeneity is common in lung adenocarcinoma. Most common pattern is mixed type, with variable combination of acinar and papillary morphology. Other uncommon patterns of growth are solid with mucin, micropapillary, cribriform, mucinous (colloid), signet-ring cell, clear cell, fetal type, endometrioid-like, and hepatoid type. In small biopsies, it is important to mention the type of pattern, view of prognostic implication.
- Desmoplastic stromal response is helpful in the diagnosis of invasion. However, sometimes the stroma may be very scanty, so it is difficult to diagnose the invasion. Immunohistochemical stains like collagen IV and fibronectin can help in the evaluation of invasion. Occasionally, lymphatic or blood vessel invasion can be seen. Prominent desmoplastic reaction may create distortion of the glands or sometimes completely obliterate the glandular lumen, creating a cord like pattern, make diagnosis difficult.
- Mucinous component has no impact on prognosis in invasive adenocarcinoma. KRAS mutation and fusion of NRG1 are the most frequent abnormalities in invasive mucinous adenocarcinoma.
- Clear cell changes can occur in any non-small cell carcinoma type, and has no role in the diagnosis or prognosis.
- In majority of cases of NSCLC, use of two or three IHC markers (TTF-1, Napsin A and p40 or p63) sort out the issues of adeno vs squamous origin. Combined positivity for TTF-1 (nuclear) and for napsin A (cytoplasmic) are highly specific for primary lung adenocarcinoma. Clinical findings of no tumour elsewhere in the body also help in selecting the few IHC markers and preservation of tissue for molecular predictive markers.
- Mucinous lung adenocarcinoma is more often positive for CK7 and variable positive for TTF-1 with weak and patchy positivity. Some cases may show positivity for CK20 and CDX2, along with staining for TTF1 in other cell populations.
- Adenocarcinomas arising from more proximal locations, like bronchioles and bronchi are more likely to be negative or only weak positive for TTF-1 and focal/patchy positive for p63. More proximal adenocarcinomas are focal positive for CK7 and positive for CK20, but negative for CDX2.
- Next step after the diagnosis is assessment of tissue adequacy for the molecular testing – DNA sequencing techniques, immunohistochemistry, and FISH analyses. Immunohistochemistry and fluorescence in situ hybridization evaluate the single gene or type of mutation or a limited panel of gene alterations. In contrast, next generation sequencing (NGS) technology yields a very large number of mutations, fusions and copy number variations, even with low amounts of extracted DNA and RNA. Targeted NGS, whole exome NGS and whole genome NGS are available techniques. Targeted NGS (amplicon-based) is widely used. This process must be monitored in each step by well-trained technicians, molecular biologists, and pathologists to avoid the production of unreliable data and subsequent misinterpretation of the results.
- Standardization and validation of predictive markers and internal and external quality control procedures should be strictly followed. Pathology laboratory must be accredited either at the national or at the international level. It is important to take note about pre-analytical variables like tissue characteristics, fixation and processing, section oven-baking, deparaffination, and protease digestion.
- Testing for both epidermal growth factor (EGFR) mutations and an anaplastic lymphoma kinase (ALK) translocation should be preferred on initial work-up in all cases with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors. Recommended testing methods are polymerase chain reaction (PCR)–based assays for EGFR mutations and fluorescence in situ hybridization (FISH) assays for ALK testing.
- For ALK IHC internal and external controls are important. IHC for ALK with 3+ intensity can be considered for treatment without further molecular work-up, as it is almost always positive by FISH. Any cases with ALK score of 2 plus must be checked with FISH. Newer antibody for ALK clone 1A4/1H7 (OriGene Tech.) give good results with high concordance with FISH testing. FISH for ALK is done on cytology, as cytology smears may not reliable. FISH is the gold standard for evaluation of number and type of rearrangement (translocation vs 5’ deletion). Fusion partner with ALK should be reported for treatment purpose.
- For ROS1 translocation, immunohistochemistry should be used to sort out the negative cases. FISH is usually performed after borderline or positive results with immunohistochemistry. Break-apart, dual-color FISH test is the gold standard method for for ROS1 assessment. Normal pneumocytes and macrophages are occasionally expressed ROS1.
- PD-L1 evaluation should be done in cases with EGFR, ALK and ROS1 negative metastatic non-squamous carcinomas and for all metastatic squamous cell carcinomas. Percentage of tumor cell positivity is different with different clone. Only 22C3 clone on Agilent platform and SP263 on Ventana platform are FDA approved systems. Agilent clone 22C3 has cut-off for treatment is at least 1% positive tumor cells. For SP263 clone on the Ventana platform has a cut-off of 25% of PD-L1 positive tumor cells. Heterogeneity is seen in expression of PD-L1, also related to previous chemotherapy and pre-analytic factors. PD-L1 epitopes are also potentially unstable with prolonged tissue fixation or inadequate tissue handling before fixation, or prolonged paraffin block storage.
- Patients with lung adenocarcinomas with RET fusion gene are younger, usually never-smokers and show poorly differentiated morphology.
- A new activating mutation of MET has been found: exon 14 skipping mutation for which a targeted therapy is available.
- KRAS mutations and NRG1 fusion is much more common in mucinous adenocarcinomas. Targeted therapy for both available so both should be included in the tests.
- Neurotrophic receptor tyrosine kinases 1–3 (NTRK) fusions are seen in large cell neuroendocrine carcinomas, squamous cell, and in adenocarcinomas, including many non-pulmonary tumors. NTRK should be included in molecular profiling as newer drugs are available.
- Amplifications and mutations in ERBB2 (HER2Neu) and BRAF are rare in lung adenocarcinoma. Also Herceptin do not show the good efficacy in pulmonary adenocarcinomas as compared to breast carcinoma.
- ERCC1 is a member of the DNA repair enzyme machinery. High expression of ERCC1 has very less or no response with platinum-based chemotherapy.
- The liquid biopsy may be used for detection of activating and resistant EGFR mutations, in cases with an inadequate or unavailable tissue sample, through circulating tumor DNA (ctDNA), which is helpful to select patients with NSCLC for treatment with tyrosine kinase inhibitors (TKIs). Also, liquid biopsy is a good alternative to tissue samples, for the detection of resistance to TKIs treatment, in cases with difficult re-biopsy and the heterogeneity of mutations (e.g. EGFR T790M), especially in patients with widespread diseases. ctDNA levels may reflect the tumor burden. Liquid biopsies have several limitations. Common limitation of liquid biopsy is the content of the circulating tumor DNA, which is usually below the limit of detection of currently available assays.
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