Non-small Cell Lung Carcinoma Subtypes Research Articles

SAMPLER: unsupervised representations for rapid analysis of whole slide tissue images

Deep learning has revolutionized digital pathology, allowing automatic analysis of hematoxylin and eosin (H&E) stained whole slide images (WSIs) for diverse tasks. WSIs are broken into smaller images called tiles, and a neural network encodes each tile. Many recent works use supervised attention-based models to aggregate tile-level features into a slide-level representation, which is then used for downstream analysis. Training supervised attention-based models is computationally intensive, architecture optimization of the attention module is non-trivial, and labeled data are not always available. Therefore, we developed an unsupervised and fast approach called SAMPLER to generate slide-level representations. Slide-level representations of SAMPLER are generated by encoding the cumulative distribution functions of multiscale tile-level features. To assess effectiveness of SAMPLER, slide-level representations of breast carcinoma (BRCA), non-small cell lung carcinoma (NSCLC), and renal cell carcinoma (RCC) WSIs of The Cancer Genome Atlas (TCGA) were used to train separate classifiers distinguishing tumor subtypes in FFPE and frozen WSIs. In addition, BRCA and NSCLC classifiers were externally validated on frozen WSIs. Moreover, SAMPLER's attention maps identify regions of interest, which were evaluated by a pathologist. To determine time efficiency of SAMPLER, we compared runtime of SAMPLER with two attention-based models. SAMPLER concepts were used to improve the design of a context-aware multi-head attention model (context-MHA). SAMPLER-based classifiers were comparable to state-of-the-art attention deep learning models to distinguish subtypes of BRCA (AUC=0.911±0.029), NSCLC (AUC=0.940±0.018), and RCC (AUC=0.987±0.006) on FFPE WSIs (internal test sets). However, training SAMLER-based classifiers was >100 times faster. SAMPLER models successfully distinguished tumor subtypes on both internal and external test sets of frozen WSIs. Histopathological review confirmed that SAMPLER-identified high attention tiles contained subtype-specific morphological features. The improved context-MHA distinguished subtypes of BRCA and RCC (BRCA-AUC=0.921±0.027, RCC-AUC=0.988±0.010) with increased accuracy on internal test FFPE WSIs. Our unsupervised statistical approach is fast and effective for analyzing WSIs, with greatly improved scalability over attention-based deep learning methods. The high accuracy of SAMPLER-based classifiers and interpretable attention maps suggest that SAMPLER successfully encodes the distinct morphologies within WSIs and will be applicable to general histology image analysis problems. This study was supported by the National Cancer Institute (Grant No. R01CA230031 and P30CA034196).

Concentration of Secreted Frizzled-Related Proteins (SFRPs) in Non-Small Cell Lung Carcinoma Subtypes-A Preliminary Study.

Non-small cell lung carcinoma (NSCLC) is the most common lung cancer worldwide. Secreted frizzled-related proteins (SFRPs) are important tumour suppressors and antagonists of the Wnt signalling pathway, which is linked with cancer development. The aim of this study was to evaluate the concentrations of SFRP1, SFRP2, and SFRP5 proteins in tumour and non-tumour (NT) samples obtained from 65 patients with primary NSCLC. An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of SFRPs in the tissue homogenates. A significantly lower SFRP2 protein concentration was found in the total NSCLC tumour samples and the following NSCLC subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (AC) (p > 0.05, p = 0.028 and p = 0.001, respectively). AC tumour samples had a higher SFRP1 level than NT samples (p = 0.022), while the highest SFRP1 concentration was found in NSCLC samples from patients with clinical stage T4 cancer. Increased concentrations of SFRP1 and SFRP5 were present in stage III NSCLC samples, while the tumour samples with high pleural invasion (PL2) had an increased level of SFRP2. The results from this study suggest that the tumour suppressor or oncogenic roles of SFRPs could be connected with the NSCLC subtype. The levels of SFRPs varied according to the clinicopathological parameters of NSCLC.

Programmed death ligand-1 expression and its association with the degree of differentiation and the presence of necrosis in non-small cell lung carcinoma

The mechanisms underlying the expression of programmed death ligand-1 (PD-L1) in non-small cell lung carcinoma (NSCLC) are not yet fully clarified. In this study, surgical resections of 730 lung cancer patients with diagnosed NSCLC were analyzed. Results of PD-L1 immunohistochemistry (using clone 22C3) were correlated with clinicopathological variables including the degree of tumor differentiation and the presence of confluent areas of coagulative necrosis. PD-L1 immunohistochemistry was analyzed in tumor cells, whereas PD-L1 positivity was defined as membranous staining in ≥1 of tumor cells. A significantly higher proportion of PD-L1 positive cases was noted in poorly differentiated (grade 3) adenocarcinomas compared to better differentiated (grade 1 and grade 2) subtypes (63.8% vs. 28.7%; p<0.001). Contrary to this, better differentiated (keratinizing) and less differentiated (non-keratinizing) squamous cell carcinoma subtypes were found to have a similar proportion of PD-L1 positive cases (51.4% vs. 55.8%; p=0.570). High levels of PD-L1 expression significantly correlated with the presence of necrosis in NSCLC: seventy-nine of 109 NSCLC cases with the presence of necrosis were PD-L1 positive compared to 256 out of 621 NSCLC without necrosis (72.5% vs. 41.2%; p<0.001). High PD-L1 expression was not positively correlated with age, gender, and advanced T stage but a significant association between PD-L1 positivity and higher N stage was observed (p<0.001) in NSCLC patients. In conclusion, the proportion of PD-L1 positive cases is higher only in poorly differentiated NSCLC of the adenocarcinoma type. A significantly higher overall rate of PD-L1 positive cases was noted in NSCLC with the presence of necrosis. Further investigation is suggested to elucidate the intricated interconnections between the plethora of hypoxic biomarkers and immunological factors in different types and subtypes of NSCLC.

Subtyping of Non-Small Cell Lung Carcinoma into Adenocarcinoma and Squamous Cell Carcinoma by Cytological Structural Features

Introduction: This study aimed to clarify the diagnostic structural features in cytology specimens that are useful in subtyping non-small cell lung carcinoma (NSCLC) into adenocarcinoma (ADC) and squamous cell carcinoma (SQCC). Methods: Cytology specimens (n = 233) of NSCLCs, which included ADCs (n = 149) and SQCCs (n = 84), were analyzed. The following cytological features were evaluated: isolated cell, flat sheet, three-dimensional cluster with irregular arrangement, papillary-like structure, micropapillary-like structure, acinar-like structure, palisading pattern, protrusion of nuclei at the periphery of the cluster, honeycomb pattern, streaming arrangement, three-dimensional sheets with regular arrangement, flattening at the periphery of the cluster, fuzzy pattern at the periphery of the cluster, and mutual inclusion. Results: ADCs exhibited significantly higher frequencies of flat sheet (p < 0.001), papillary-like structure (p < 0.001), micropapillary-like structure (p = 0.028), acinar-like structure (p < 0.001), and protrusion of nuclei at the periphery of the cluster (p < 0.001) than SQCCs. The latter exhibited significantly higher frequencies of streaming arrangement (p < 0.001), three-dimensional sheets with regular arrangement (p < 0.001), flattening at the periphery of the cluster (p < 0.001), fuzzy pattern at the periphery of the cluster (p < 0.001), and mutual inclusion (p < 0.001) than ADCs. Discussion: Cytological structural features, such as flat sheet, papillary-like structure, micropapillary-like structure, acinar-like structure, and protrusion of nuclei at the periphery of the cluster, indicated ADC, whereas streaming arrangement, three-dimensional sheets with regular arrangement, flattening at the periphery of the cluster, fuzzy pattern at the periphery of the cluster, and mutual inclusion indicated SQCC. Paying attention to these cytological structural features can enable the accurate subtyping of NSCLC into ADC and SQCC.

A Beclin 1-targeting stapled peptide synergizes with erlotinib to potently inhibit proliferation of non-small-cell lung cancer cells

Epidermal Growth Factor Receptor (EGFR) is a major drug target for non-small-cell lung carcinoma (NSCLC). Tyrosine Kinase Inhibitors (TKIs) like erlotinib are potent inhibitors of EGFR and have achieved impressive clinical success against NSCLC. However, NSCLC cells readily develop resistance to TKIs by acquiring mutations in EGFR or other oncogenes. Novel strategies to inhibit EGFR are needed to overcome this urgent problem of TKI resistance. Beclin 1 is an essential autophagy protein and is intimately involved in tumorigenesis and EGFR signaling. Here we present data to show that a Beclin 1-targeting stapled peptide Tat-SP4 can inhibit the EGFR signaling pathway by enhancing the Beclin 1-mediated endolysosomal degradation of EGFR. This inhibition mechanism is orthogonal to that employed by TKIs and is effective against either wild-type or mutant EGFR. Tat-SP4 alone showed moderate anti-proliferative efficacy in NSCLC cells but synergized with erlotinib to potently inhibit NSCLC proliferation. These results suggest that Beclin 1-targeting stapled peptides may be used in combination with TKIs to enhance their efficacy, particularly for NSCLC subtypes refractory to current regiments.

Evaluation of Microscopic Tumour Extension in Localized Stage Non-Small-Cell Lung Cancer for Stereotactic Radiotherapy Planning.

Simple SummaryStereotactic radiotherapy for localised stage non-small-cell lung carcinoma is an alternative indication for patients who are inoperable or refuse surgery. A study showed that the microscopic tumour extension of non-small-cell lung carcinoma varied according to the histological type, which allowed us to deduce adapted margins for the clinical target volume. The objectives of our retrospective study are. The objective of our study is to measure the microscopic tumour extension of T1N0 or T2aN0 primary lung tumors who underwent surgery. The margin required to cover the microscopic tumour extension with a 95% probability is 4.4 mm and 2.9 mm for squamous cell carcinoma and adenocarcinoma, respectively. Multivariate analysis showed a statistically significant relationship between the maximum microextension distance and size with the shrinkage coefficient.Background: Stereotactic radiotherapy for localised stage non-small-cell lung carcinoma (NSCLC) is an alternative indication for patients who are inoperable or refuse surgery. A study showed that the microscopic tumour extension (ME) of NSCLC varied according to the histological type, which allowed us to deduce adapted margins for the clinical target volume (CTV). However, to date, no study has been able to define the most relevant margins for patients with stage 1 tumours. Methods: We performed a retrospective analysis including patients with adenocarcinoma (ADC) or squamous cell carcinoma (SCC) of localised stage T1N0 or T2aN0 who underwent surgery. The ME was measured from this boundary. The profile of the type of tumour spread was also evaluated. Results: The margin required to cover the ME of a localised NSCLC with a 95% probability is 4.4 mm and 2.9 mm for SCC and ADC, respectively. A significant difference in the maximum distance of the ME between the tumour-infiltrating lymphocytes (TILs), 0–10% and 50–90% (p < 0.05), was noted for SCC. There was a significant difference in the maximum ME distance based on whether the patient had chronic obstructive pulmonary disease (COPD) (p = 0.011) for ADC. Multivariate analysis showed a statistically significant relationship between the maximum microextension distance and size with the shrinkage coefficient. Conclusion: This study definitively demonstrated that the ME depends on the pathology subtype of NSCLC. According to International Commission on Radiation Units and Measurements (ICRU) reports, 50, 62 and 83 CTV margins, proposed by these results, should be added to the GTV (Gross tumour volume). When stereotactic body radiation therapy is used, this approach should be considered in conjunction with the dataset and other margins to be applied.

Frequency of Epidermal Growth Factor Receptor and T790M Mutations Among Patients With Non-Small Cell Lung Carcinoma: A Hospital-Based Study in the King Khalid University Hospital (KKUH) Since 2009-2017.

ObjectivesTo estimate the proportion of positive epidermal growth factor receptor (EGFR) mutations among patients diagnosed with non-small cell lung carcinoma (NSCLC) and T790M at the King Khalid University Hospital (KKUH).MethodsA retrospective cohort study that included all patients that were diagnosed with NSCLC from 2009 to 2017 at KKUH. Data obtained from both electronic and paper medical records and the following information were studied: age, gender, smoking, region, subtype of NSCLC, EGFR mutation test result, treatment, T790M mutation test (if required), comorbidities, metastasis. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, version 21.0; SPSS Inc., Chicago, IL, USA).ResultsAmong 71 patients with NSCLC 18 cases were identified for EGFR positive mutation and only one case for T790M. Deletion mutation in exon 19 represented 50% of total cases. Moreover, it showed that it is more frequent in males and non-smokers with 61.1% (11) and 66.7% (12), respectively. Majority of the cases were above the age of 60 years by 61.1% (11). The mutations reported highest in those living in Najd with a 44.4% (8) and all the mutated cases were adenocarcinoma. There was no statistical significance in the association between EGFR mutation and disease variables.ConclusionUltimately, we found that the frequency of EGFR and T790M mutations among NSCLC patients at KKUH from 2009 to 2017 was 25.4% and 1.4%, respectively. Moreover, this result was conspicuous among non-smokers.

Untargeted Lipidomics of Non-Small Cell Lung Carcinoma Demonstrates Differentially Abundant Lipid Classes in Cancer vs. Non-Cancer Tissue.

Lung cancer remains the leading cause of cancer death worldwide and non-small cell lung carcinoma (NSCLC) represents 85% of newly diagnosed lung cancers. In this study, we utilized our untargeted assignment tool Small Molecule Isotope Resolved Formula Enumerator (SMIRFE) and ultra-high-resolution Fourier transform mass spectrometry to examine lipid profile differences between paired cancerous and non-cancerous lung tissue samples from 86 patients with suspected stage I or IIA primary NSCLC. Correlation and co-occurrence analysis revealed significant lipid profile differences between cancer and non-cancer samples. Further analysis of machine-learned lipid categories for the differentially abundant molecular formulas identified a high abundance sterol, high abundance and high m/z sphingolipid, and low abundance glycerophospholipid metabolic phenotype across the NSCLC samples. At the class level, higher abundances of sterol esters and lower abundances of cardiolipins were observed suggesting altered stearoyl-CoA desaturase 1 (SCD1) or acetyl-CoA acetyltransferase (ACAT1) activity and altered human cardiolipin synthase 1 or lysocardiolipin acyltransferase activity respectively, the latter of which is known to confer apoptotic resistance. The presence of a shared metabolic phenotype across a variety of genetically distinct NSCLC subtypes suggests that this phenotype is necessary for NSCLC development and may result from multiple distinct genetic lesions. Thus, targeting the shared affected pathways may be beneficial for a variety of genetically distinct NSCLC subtypes.

Can cytology reliably subtype non-small cell lung carcinomas?

Cytology specimens play an important role in the diagnosis and predictive testing of lung cancer. While morphological characterisation of small cell and non-small cell lung carcinomas (NSCLC) on cytology is possible, further subtyping of NSCLC into adenocarcinoma and squamous cell carcinoma morphology is also mandatory in the current era of personalised medicine. Notably, cytology specimens in different forms (fine needle aspiration, exfoliative, and cell block) with or without immunocytochemistry are reliable sources for accurate diagnosis of adenocarcinoma and squamous cell carcinoma as evidenced by numerous studies present in the literature. However, there are instances where subtyping of NSCLC based on morphology alone is challenging on cytology samples, especially non-cell block preparations. In this paper, we will discuss current concepts, advances, and challenges of subtyping NSCLC in cytology specimens.

New Insights on Old Biomarkers Involved in Tumor Microenvironment Changes and Their Diagnostic Relevance in Non-Small Cell Lung Carcinoma.

Background: Lung cancer is a multifactorial disease with a heterogeneous tumor group that hampers diagnostic and therapeutic approaches, as well as understanding of the processes that underlie its pathogenesis. Current research efforts are focused on examining alterations in the tumor microenvironment, which may affect the pathogenesis and further malignant progression in lung cancer. The aim of this study was to investigate changes in the levels of biomarkers involved in the lung tumor microenvironment and their diagnostic utility in differentiating lung cancer subtypes and stages. Methods: This study comprised 112 lung cancer patients, 50 with adenocarcinoma, 35 with squamous cell carcinoma, 13 with other non-small cell lung carcinoma subtypes, and 14 with other lung neoplasms than non-small cell lung carcinoma. Tumor markers (CEA, CYFRA 21-1, and NSE) were measured in the patients’ sera and plasmas, along with IL-6, TNF-α, SAA1, CRP, MMP-2, MMP-9, glucose, lactate, and LDH, utilizing enzyme-linked immunosorbent assays, enzyme immunoassays, and automated clinical chemistry and turbidimetry systems. The results were statistically analyzed across patient groups based on the subtype and stage of lung cancer. Results: Glucose concentrations showed statistically significant (p < 0.05) differences both between lung cancer subtypes and stages, with the highest levels in patients with other lung neoplasms (me = 130.5 mg/dL) and in patients with stage IIB lung cancer (me = 132.0 mg/dL). In patients with advanced lung cancer, IL-6 and LDH had considerably higher concentration and activity. There was also a significant positive correlation between IL-6 and MMP-9 in adenocarcinoma and SqCC, with correlation coefficients of 0.53 and 0.49, respectively. The ROC analyses showed that the best single biomarkers for distinguishing adenocarcinoma from squamous cell carcinoma are glucose, CRP, and CYFRA 21-1; however, their combination did not significantly improve sensitivity, specificity, and the AUC value. The combinations of IL-6, glucose, LDH and CEA, IL-6, SAA1, MMP-9, and lactate can distinguish patients with stage IIB lung cancer from those with stage IIA with 100% sensitivity, 100% specificity, and with an AUC value of 0.8333 and 1.0000, respectively, whereas the combination of CEA, IL-6, and LDH can identify patients with stage IIIA lung cancer from those with stage IIB with 72.73% sensitivity, 94.44% specificity, and an AUC value of 0.8686. Conclusion: There is a link between biomarkers of tumor microenvironment changes and tumor markers, and combinations of these markers may be clinically useful in the differential diagnosis of adenocarcinoma and squamous cell carcinoma, as well as lung cancer stages IIB and IIA, and IIIA and IIB.

Identification and characterization of non-small cell lung cancer associated sialoglycoproteins

Aberrantly sialylated cellular glycoconjugates were found to be involved in different processes during tumorigenesis. Such alteration was also noted in case of lung cancer, an important cause of cancer-related death throughout the world. Thus, study on lung cancer associated sialoglycoproteins is of paramount relevance to have a deeper insight into the mechanism of the disease pathogenesis. In the present study, sialic acid specific lectin (Maackia amurensis agglutinin and Sambcus nigra agglutinin)-based affinity chromatography followed by 2D-PAGE and MALDI-TOF/TOF mass spectrometric analysis were done to explore the disease-associated serum proteins of squamous cell carcinoma and adenocarcinoma [the major two subtypes of NSCLC (non-small cell lung carcinoma)] patients. Among seven identified proteins, α1-antitrypsin and haptoglobin-β were preferred for further studies. These two proteins were characterized as the disease associated serum-sialoglycoproteins of NSCLC-patients by western immunoblotting using each lectin specific inhibitor. The presence of these sialoglycoproteins was found on NSCLC cell lines (NCI-H520 & A549) by confocal microscopy. Both these proteins were also present in tissue samples of NSCLC origin and involved in proliferation, invasion and migration of NSCLC cells. Our findings suggest that α1-antitrypsin and haptoglobin-β may be the disease-associated sialoglycoproteins in NSCLC, which seem to be involved in disease progression. SignificanceOur contribution regarding the identification of the NSCLC associated sialoglycoproteins may provide a new vision towards the development of clinically useful newer strategies for the treatment of this disease.

Analysis of The Relationship between Blood Groups and Lung Cancer Risk Depending on Histology

Aim: Blood group antigens are the first genetic markers discovered. ABO blood groups which are expressed on red blood cell membranes are reported to be associated with malignancy risk. The present study aimed to investigate whether there is an association between blood groups and patients with lung cancer. Material and Methods: In our study, ıt was reviewed 587 patients with lung cancer whose ABO/Rh blood types were serologically confirmed. Patients were classified according to blood groups (O, A, B, AB), Rh factor (+, −) and histological types of lung cancer as Non-small cell lung carcinoma (NSCLC) and Small cell lung carcinoma (SCLC). The adenocarcinoma (AC) and squamous carcinoma (SC) as subtypes of NSCLC were recorded. The relationship of ABO/Rh blood groups with clinical features, such as age, sex and histological subtype were evaluated. Results: There was no significant association between pathological results and blood groups O, A, B and AB. SCLC patients had significantly higher frequency of blood type B Rh (-) when compared to NSCLC patients. Conclusion: We observed an association between risk of lung cancer and ABO blood groups. Although the Rh factor is not effective by itself, patients with the B Rh (-) blood group may have a higher risk than SCLC. Examining the relationship of blood group subtypes with lung cancer cells at the molecular level would be useful to clarify the relationship between lung cancer and blood groups.

Circular RNA hsa_circ_0072309 promotes tumorigenesis and invasion by regulating the miR-607/FTO axis in non-small cell lung carcinoma.

Emerging evidence has demonstrated that circular RNAs (circRNAs) are abnormally expressed in non-small cell lung carcinoma (NSCLC). However, the contributions of circRNAs to the tumorigenesis of lung adenocarcinoma (LUAD), one of the subtypes of NSCLC, remain unclear. Based on a microarray assay, we found that hsa_circ_0072309 was significantly upregulated in NSCLC compared with matched normal samples. Moreover, functional experiments demonstrated that hsa_circ_0072309 promotes the proliferation, migration, and invasion of NSCLC cells. In vitro precipitation of circRNAs, luciferase reporter assays, and biotin-coupled microRNA capture assays were carried out to investigate the mechanisms by which hsa_circ_0072309 regulates NSCLC. Through the above work, we found that hsa_circ_0072309 interacted with miR-607 via its miRNA response element to upregulate the expression of FTO, an m6A demethylase and downstream target of miR-607, thus promoting tumorigenesis of NSCLC. In total, our findings indicated the oncogenic role of hsa_circ_0072309 in NSCLC and provide a potential target for treatment.

Large Cell Neuro-Endocrine Carcinoma of the Lung: Current Treatment Options and Potential Future Opportunities.

Large-cell neuroendocrine carcinomas of the lung (LCNECs) are rare tumors representing 1–3% of all primary lung cancers. Patients with LCNEC are predominantly male, older, and heavy smokers. Histologically, these tumors are characterized by large cells with abundant cytoplasm, high mitotic rate, and neuroendocrine immunohistochemistry-detected markers (chromogranin-A, synaptophysin, and CD56). In 2015 the World Health Organization classified LCNEC as a distinct subtype of pulmonary large-cell carcinoma and, therefore, as a subtype of non-small cell lung carcinoma (NSCLC). Because of the small-sized tissue samples and the likeness to other neuroendocrine tumors, the histological diagnosis of LCNEC remains difficult. Clinically, the prognosis of metastatic LCNECs is poor, with high rates of recurrence after surgery alone and overall survival of approximately 35% at 5 years, even for patients with early stage disease that is dramatically shorter compared with other NSCLC subtypes. First-line treatment options have been largely discussed but with limited data based on phase II studies with small sample sizes, and there are no second-line well defined treatments. To date, no standard treatment regimen has been developed, and how to treat LCNEC is still on debate. In the immunotherapy and targeted therapy era, in which NSCLC treatment strategies have been radically reshaped, a few data are available regarding these opportunities in LCNEC. Due to lack of knowledge in this field, many efforts have been done for a deeper understanding of the biological and molecular characteristics of LCNEC. Next generation sequencing analyses have identified subtypes of LCNEC that may be relevant for prognosis and response to therapy, but further studies are needed to better define the clinical impact of these results. Moreover, scarce data exist about PD-L1 expression in LCNEC and its predictive value in this histotype with regard to immunotherapy efficacy. In the literature some cases are reported concerning LCNEC metastatic patients carrying driver mutations, especially EGFR alterations, showing targeted therapy efficacy in this setting of disease. Due to the rarity and the challenging understanding of LCNEC, in this review we aim to summarize the management options currently available for treatment of LCNEC.

The Diagnostic and Prognostic Roles of Combined Expression of Novel Biomarkers in Lung Adenocarcinoma and Lung Squamous Cell Carcinoma: An Immunohistochemical Study.

Background & Objective:Diagnosis and discrimination of lung adenocarcinoma (LUAD) from lung squamous cell carcinoma (LUSC) is critical to select the appropriate treatment regimen as recently targeted therapies require accurate subtyping of nonsmall-cell lung carcinoma (NSCLCs). There are currently several biomarkers that could be used for differentiation between LUAD and LUSC, but they have less sensitivity, specificity, and clinical applicability. The aim of this study was to assess the diagnostic and prognostic values of CLCA2, SPATS2, ST6GALNAC1, and Adipophilin tissue expression in the tissues retrieved from LUAD and LUSC patients using immunohistochemistry.Methods:The current study was performed on the samples retrieved from sixty primary lung masses that were diagnosed as LUAD and LUSC. Immunohistochemistry was performed by using a panel of CLCA2, SPATS2, and ST6GALNAC1. We assessed the diagnostic roles of the studied markers in the discrimination between LUAD and LUSC and their prognostic values. Results:SPATS2 and CLCA2 were expressed higher in LUSC than LUAD. ST6GALNAC1 and Adipophilin showed higher expression in LUAD than LUSC (P<0.001). The sensitivity and specificity of CLCA2, SPATS2, ST6GALNAC1 and Adipophilin in adequate subtyping and reaching the accurate diagnosis was 100%. We found only significant difference in survival rate between the patients with negative and positive CLCA2 expression (P=0.038 and P=0.019, respectively). Conclusion:The combination of biomarkers of CLCA2, SPATS2, ST6GALNAC1, and Adipophilin may lead to an appropriate subtyping of lung cancer and reaching accurate diagnosis with the highest sensitivity and specificity.

Differential MUC22 expression by epigenetic alterations in human lung squamous cell carcinoma and adenocarcinoma.

Disruption in mucins (MUCs) is involved in cancer development and metastasis and is thus used as a biomarker. Non-small cell lung carcinoma (NSCLC) is characterized by heterogeneous genetic and epigenetic alterations. Lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are the two primary subtypes of NSCLC that require different therapeutic interventions. Here, we report distinct expression and epigenetic alterations in mucin 22 (MUC22), a new MUC family member, in LUSC vs. LUAD. In lung cancer cell lines and tissues, MUC22 was downregulated in LUSC (MUC22Low) but upregulated in LUAD (MUC22High) with co-expression of MUC21. The aberrant expression of MUC22 was inversely correlated with its promoter hypermethylation in LUSC and hypomethylation in LUAD cells and tissues, respectively. Decreased MUC22 expression in NSCLC cell lines was restored upon treatment with epigenetic modifiers 5-aza-2′-deoxycytidine (5-Aza) or trichostatin A (TSA), accompanied by reduction in global protein level of histone deacetylase 1 (HDAC1) but increased enrichment of histone H3 lysine 9 acetylation (H3K9ac) specifically in the MUC22 promoter in the SK-MES-1 cell line. MUC22 knockdown increased the growth and motility of lung cancer cells and an immortalized human bronchial epithelial BEAS-2B cell line via NF-κB activation. Clinically, MUC22Low in LUSC and MUC22High in LUAD were shown to be indicators of unfavorable overall survival for patients with early cancer stages. Our study reveals that changes in MUC22 expression due to epigenetic alterations in NSCLC may have important biological significance and prognostic potential in LUSC when compared to LUAD. Thus, MUC22 expression and epigenetic alterations may be used for molecular subtyping of NSCLC in precision medicine.

Automated TTF-1 Immunohistochemistry Assay for the Differentiation of Lung Adenocarcinoma Versus Lung Squamous Cell Carcinoma.

Due to therapeutic advances, the subclassification of non-small cell lung carcinomas (NSCLC) between the adenocarcinomas and squamous cell carcinomas subtypes is essential for the practice of personalized and targeted medicine. The clinical management for these two NSCLC subtypes is different due to their different molecular properties and histological origins. Immunohistochemistry (IHC) markers such is TTF-1 play a key role in the differentiation of lung adenocarcinomas and squamous cell carcinomas. However, immunohistochemistry is a complex process involving many critical steps and the reliability of results depends on the standardization of the assay as well as the appropriate interpretation. Different laboratories use different reagents and different IHC approaches for the detection of TTF-1 in lung cancer tumors. Here we describe an automated IHC protocol used in our laboratory for the detection of TTF-1 in formalin-fixed, paraffin-embedded (FFPE) tissue sections from lung tumors.

Minimalistic Immunomarker Approach for the Diagnosis of Non Small Cell Lung Carcinoma with Emphasis on Tumours with Ambiguous Morphology

Introduction: Diagnosing Non Small Cell Lung Carcinoma (NSCLC) by morphology on small biopsy specimens are on demand. With major advances in molecular testing of lung cancers and introduction of targeted therapies, accurate subtyping of NSCLC has become essential. Immunophenotyping with various immunomarkers is helpful in identification of different tumour subtypes. As sparing material for molecular testing is mandatory, a minimalistic Immunohistochemistry (IHC) based diagnostic approach is warranted by means of reliable and accessible immunomarkers. In this study, one immunomarker Thyroid Transcription Factor (TTF1) for glandular and protein40 (p40) for squamous differentiation was applied to maximise the proportion of accurately subtyped NSCLC on small biopsy specimens. Aim: To find utility of TTF1/p40 minimalistic immunopanel in subtyping NSCLC especially those with ambiguous morphology. Materials and Methods: This retrospective observational study was conducted from 15 December 2020 to 5 March 2021 in a tertiary care hospital in which 93 (mean age 64.8 years, Male:female ratio 4.8:1) consecutive lung biopsies from suspected lung carcinoma obtained from January 2019 to December 2020 were studied. The morphological diagnosis and diagnosis after application of TTF1 and p40 minimalistic immunopanel was evaluated using chi-square test. The p-value &lt;0.01 was considered significant. Results: Based on morphology, only 21 (22.5%) cases out of 93 cases of NSCLC were characterised into Adenocarcinoma (ADC) and Squamous Cell Carcinoma (SCC), whereas 72 (77.4%) were grouped as NSCLC ambiguous type. With addition of IHC (p40 and TTF1), the latter category reduced to 10 (10.7%) which constituted final NSCLC Not Otherwise Specified (NOS) category and a sum of 79 (84.9%) cases were accurately subtyped into SCC and ADC. Significance of adding TTF1/p40 immunopanel in the diagnosis of non small cell carcinoma was found to be very significant(p-value&lt;0.001) p40 showed 100% sensitivity and 92.6% specificity for SCC where as TTF1 showed sensitivity of 92.59% and 100% specificity for ADC. Conclusion: Study showed that a minimalistic approach using only two antibodies (p40 and TTF1) might help in subtyping NSCLC especially those with ambiguous morphology and reduction of category of NSCLC NOS significantly and contribute in sparing material for molecular testing.

Identification of prognostic biomarkers for major subtypes of non-small-cell lung cancer using genomic and clinical data.

Intra-tumor heterogeneity and high mortality among patients with non-small-cell lung carcinoma (NSCLC) emphasize the need to identify reliable prognostic markers unique to each subtype. In this study, univariate cox regression and prognostic index (PI)-based approaches were used to develop models for predicting NSCLC patients' subtype-specific survival. Prognostic analysis of TCGA dataset identified 1334 and 2129 survival-specific genes for LUSC (488 samples) and LUAD (497 samples), respectively. Individually, 32 and 271 prognostic genes were found and validated in GSE study exclusively for LUSC and LUAD. Nearly, 9-10% of the validated genes in each subtype were already reported in multiple studies thus highlighting their importance as prognostic biomarkers. Strong literature evidence against these prognostic genes like "ELANE" (LUSC) and "AHSG" (LUAD) instigates further investigation for their therapeutic and diagnostic roles in the corresponding cohorts. Prognostic models built on five and four genes were validated for LUSC [HR = 2.10, p value = 1.86 × 10-5] and LUAD [HR = 2.70, p value = 3.31 × 10-7], respectively. The model based on the combination of age and tumor stage performed well in both NSCLC subtypes, suggesting that despite having distinctive histological features and treatment paradigms, some clinical features can be good prognostic predictors in both. This study advocates that investigating the survival-specific biomarkers restricted to respective cohorts can advance subtype-specific prognosis, diagnosis, and treatment for NSCLC patients. Prognostic models and markers described for each subtype may provide insight into the heterogeneity of disease etiology and help in the development of new therapeutic approaches for the treatment of NSCLC patients.

SMARCA4/BRG1-Deficient Non-Small Cell Lung Carcinomas: A Case Series and Review of the Literature.

Somatic mutations in SMARCA4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4) gene and/or BRG1 (Brahma-related gene 1) loss identifies a subset of non-small cell lung carcinomas (NSCLCs) lacking alterations in EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), and ROS1 (ROS proto-oncogene 1) genes. Preliminary observations suggest responsiveness to immunotherapy and targeted therapies. To study BRG1 loss in NSCLCs and elucidate the clinicopathologic profile of such SMARCA4-deficient NSCLCs. Non-small cell lung carcinomas diagnosed during 6 years were subject to immunohistochemistry for BRG1 and BRM (Brahma). Tumors with BRG1 loss were stained with antibodies against thyroid transcription factor 1 (TTF-1), p40, cytokeratins, hepatocyte paraffin 1 (Hep Par 1), Sal-like protein 4 (SALL4), CD34, SRY-box 2 (SOX2), chromogranin, synaptophysin, p53, integrase interactor 1, ALK, and ROS1. EGFR mutation testing was performed by polymerase chain reaction-based method. Among 100 NSCLCs tested, 4 cases (4%) showed BRG1 loss. The histology ranged from solid adenocarcinomas (n = 1) to large cell/poorly differentiated carcinomas (n = 3) with clear cell cytology in 2 cases. All showed loss/reduction of BRM with variable cytokeratin and SALL4 expression, and were negative for TTF-1, p40, Hep Par 1, ALK, ROS1, and EGFR mutations. CD34 and SOX2 were negative in all 4 cases. Isolated BRM loss was common (21%), distributed across all NSCLC subtypes including squamous cell carcinomas and a hepatoid adenocarcinoma. BRG1 loss occurs in a subset of TTF-1/p40-negative poorly differentiated NSCLCs. Identification and follow-up will clarify the prognosis, diagnostic criteria, and potential for therapeutic personalization.