Squamous Cell Carcinoma in Never Smokers: An Insight into SMARCB1 Loss.

Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) constituting 85% of cases. Among NSCLCs, squamous cell carcinoma (SqCC) is strongly associated with smoking. However, lung cancer in never smokers (LCINS) represents approximately 25% of lung cancer cases globally and shows increasing incidence, particularly in East Asia. LCINS-SqCC is less well-characterized, especially regarding its genomic alterations and their impact on clinical outcomes. We conducted a retrospective analysis over a 20-year period (July 2003-July 2023) at two major tertiary centers in the UK. The cohort included 59 patients with LCINS-SqCC who underwent radical surgical resection. Data collected included demographic information, comorbidities, histopathological details, and outcome metrics such as disease-free and overall survival. Molecular sequencing of tumor specimens was performed to identify genomic aberrations. The cohort had a median age of 71 years (IQR 62-77) and a median BMI of 25.4 (IQR 22.8-27.8), with a slight male predominance (53%). The majority of patients (93%) had a preoperative MRC of 1-2. Recurrent disease was observed in 23 patients (39%), and 32 patients (54%) had died at a median follow-up of 3 years. Median disease-free survival was 545 days (IQR 132-1496), and overall survival was 888 days (IQR 443-2071). Preoperative creatinine levels were higher in patients who experienced recurrence (p = 0.037). Molecular analysis identified biallelic SMARCB1 loss in two younger patients, associated with rapid disease progression despite R0 resection. These patients' tumors were PDL1-negative, TTF-1-negative, and positive for cytokeratin, CD56, and p40. SMARCB1-deficient SqCC in never smokers represents a highly aggressive variant with poor disease-free survival, highlighting the importance of integrating advanced molecular diagnostics in clinical practice. This study underscores the necessity for personalized treatment strategies, including targeted therapies such as EZH2 inhibitors and immune checkpoint blockade, to address the unique molecular pathways in SMARCB1-deficient cancers. Further clinical trials are essential to optimize therapeutic approaches for this challenging subgroup of lung cancer.

Predicting programmed death-ligand 1 (PD-L1) expression with fluorine-18 fluorodeoxyglucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT) metabolic parameters in resectable non-small cell lung cancer.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression is a predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). PD-L1 and glucose transporter 1 expression are closely associated, and studies demonstrate correlation of PD-L1 with glucose metabolism. AIM: The aim of this study was to investigate the association of fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) metabolic parameters with PD-L1 expression in primary lung tumour and lymph node metastases in resected NSCLC. METHODS: We conducted a retrospective analysis of 210 patients with node-positive resectable stage IIB-IIIB NSCLC. PD-L1 tumour proportion score (TPS) was determined using the DAKO 22C3 immunohistochemical assay. Semi-automated techniques were used to analyse pre-operative [18F]FDG-PET/CT images to determine primary and nodal metabolic parameter scores (including max, mean, peak and peak adjusted for lean body mass standardised uptake values (SUV), metabolic tumour volume (MTV), total lesional glycolysis (TLG) and SUV heterogeneity index (HISUV)). RESULTS: Patients were predominantly male (57%), median age 70 years with non-squamous NSCLC (68%). A majority had negative primary tumour PD-L1 (TPS < 1%; 53%). Mean SUVmax, SUVmean, SUVpeak and SULpeak values were significantly higher (p < 0.05) in those with TPS ≥ 1% in primary tumour (n = 210) or lymph nodes (n = 91). However, ROC analysis demonstrated only moderate separability at the 1% PD-L1 TPS threshold (AUCs 0.58-0.73). There was no association of MTV, TLG and HISUV with PD-L1 TPS. CONCLUSION: This study demonstrated the association of SUV-based [18F]FDG-PET/CT metabolic parameters with PD-L1 expression in primary tumour or lymph node metastasis in resectable NSCLC, but with poor sensitivity and specificity for predicting PD-L1 positivity ≥ 1%. CLINICAL RELEVANCE STATEMENT: Whilst SUV-based fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography metabolic parameters may not predict programmed death-ligand 1 positivity ≥ 1% in the primary tumour and lymph nodes of resectable non-small cell lung cancer independently, there is a clear association which warrants further investigation in prospective studies. TRIAL REGISTRATION: Non-applicable KEY POINTS: • Programmed death-ligand 1 immunohistochemistry has a predictive role in non-small cell lung cancer immunotherapy; however, it is both heterogenous and dynamic. • SUV-based fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) metabolic parameters were significantly higher in primary tumour or lymph node metastases with positive programmed death-ligand 1 expression. • These SUV-based parameters could potentially play an additive role along with other multi-modal biomarkers in selecting patients within a predictive nomogram.

Global Ring Study to Investigate the Comparability of Total Assay Performance of Commercial Claudin 18 Antibodies for Evaluation in Gastric Cancer.

Claudin 18.2 (CLDN18.2), the dominant isoform of CLDN18 in gastric tissues, is a highly specific tight junction protein of the gastric mucosa with variably retained expressions in gastric and gastroesophageal junction cancers. Additionally, CLDN18.2-targeted treatment with zolbetuximab, in combination with chemotherapy, has recently been assessed in 2 phase-III studies of patients with HER2-negative, locally advanced, unresectable, or metastatic gastric or gastroesophageal junction adenocarcinoma. These trials used the investigational VENTANA CLDN18 (43-14A) RxDx immunohistochemistry (IHC) assay on the Ventana BenchMark platform to identify patients eligible for CLDN18.2-targeted treatment. We report the findings of a global ring study evaluating the analytical comparability of concordance of the results of 3 CLDN18 antibodies (Ventana, LSBio, and Novus) stained on 3 IHC-staining platforms (Ventana, Dako, and Leica). A tissue microarray (TMA), comprising 15 gastric cancer cases, was stained by 27 laboratories across 11 countries. Each laboratory stained the TMAs using at least 2 of the 3 evaluated CLDN18 antibodies. Stained TMAs were assessed and scored using an agreed IHC-scoring algorithm, and the results were collated for statistical analysis. The data confirmed a high level of concordance for the VENTANA CLDN18 (43-14A; Ventana platform only) and LSBio antibodies on both the Dako and Leica platforms, with accuracy, precision, sensitivity, and specificity rates all reaching a minimum acceptable ≥85% threshold and good-to-excellent levels of concordance as measured by Cohen's kappa coefficient. The Novus antibody showed the highest level of variability against the reference central laboratory results for the same antibody/platform combinations. It also failed to meet the threshold for accuracy and sensitivity when used on either the Dako or Leica platform. These results demonstrated the reliability of IHC testing for CLDN18 expression in gastric tumor samples when using commercially available platforms with an appropriate methodology and primary antibody selection.

Adenoid cystic carcinoma of the thymus gland.

BACKGROUND: Thymic carcinomas are rare and aggressive tumours. They constitute a heterogeneous group of tumours with various histological patterns and subtypes resembling epithelial tumours arising from other organs. CASE PRESENTATION: We hereby represent a case of primary thymic carcinoma with adenoid cystic carcinoma-like features (TCACC) which is an extremely rare variant of thymic adenocarcinoma. To date and to the best of our knowledge, there are nine reported cases in literature and ours is the tenth. Our case was treated surgically but the implementation of adjuvant chemoradiotherapy has been reported in few of the published cases. CONCLUSIONS: TCACC constitutes a rare entity of thymic adenocarcinoma with limited available literature. The current data is derived from few case reports and case series. The histological overlap of these tumours and primary ACC of salivary glands poses a diagnostic challenge. Radiological investigations, immunohistochemical phenotyping and genetic analysis are crucial in establishing the diagnosis.

MHC Class II is Induced by IFNγ and Follows Three Distinct Patterns of Expression in Colorectal Cancer Organoids.

Tumor-specific MHC class II (tsMHC-II) expression impacts tumor microenvironmental immunity. tsMHC-II positive cancer cells may act as surrogate antigen-presenting cells and targets for CD4+ T cell-mediated lysis. In colorectal cancer, tsMHC-II negativity is common, in cell lines due to CIITA promoter methylation. To clarify mechanisms of tsMHC-II repression in colorectal cancer, we analyzed colorectal cancer organoids which are epigenetically faithful to tissue of origin. 15 primary colorectal cancer organoids were treated with IFNγ ± epigenetic modifiers: flow cytometry was used for tsMHC-II expression. qRT-PCR, total RNA sequencing, nanopore sequencing, bisulfite conversion/pyrosequencing, and Western blotting was used to quantitate CIITA, STAT1, IRF1, and JAK1 expression, mutations and promoter methylation and chromatin immunoprecipitation to quantitate H3K9ac, H3K9Me2, and EZH2 occupancy at CIITA. We define three types of response to IFNγ in colorectal cancer: strong, weak, and noninducibility. Delayed and restricted expression even with prolonged IFNγ exposure was due to IFNγ-mediated EZH2 occupancy at CIITA. tsMHC-II expression was enhanced by EZH2 and histone deacetylase inhibition in the weakly inducible organoids. Noninducibility is seen in three consensus molecular subtype 1 (CMS1) organoids due to JAK1 mutation. No organoid demonstrates CIITA promoter methylation. Providing IFNγ signaling is intact, most colorectal cancer organoids are class II inducible. Upregulation of tsMHC-II through targeted epigenetic therapy is seen in one of fifteen organoids. Our approach can serve as a blueprint for investigating the heterogeneity of specific epigenetic mechanisms of immune suppression across individual patients in other cancers and how these might be targeted to inform the conduct of future trials of epigenetic therapies as immune adjuvants more strategically in cancer. Significance: Cancer cell expression of MHC class II significantly impacts tumor microenvironmental immunity. Previous studies investigating mechanisms of repression of IFNγ-inducible class II expression using cell lines demonstrate epigenetic silencing of IFN pathway genes as a frequent immune evasion strategy. Unlike cell lines, patient-derived organoids maintain epigenetic fidelity to tissue of origin. In the first such study, we analyze patterns, dynamics, and epigenetic control of IFNγ-induced class II expression in a series of colorectal cancer organoids.

PD-L1 Testing in Urothelial Carcinoma: Analysis of a Series of 1401 Cases Using Both the 22C3 and SP142 Assays.

Immune checkpoint blockade (ICB) drugs are a novel, effective treatment for advanced urothelial carcinoma. Worldwide, several different ICB drugs are approved, each developed and clinically validated with a specific PD-L1 compound diagnostic assay. As a result, PD-L1 testing workflows in routine practice are complex: requiring multiple assays across two platforms, with each assay having a different method of interpretation. Our service tested 1,401 urothelial carcinoma cases for PD-L1 expression, using both the 22C3 PharmDx assay (required prior to Pembrolizumab therapy) and SP142 assay (required prior to Atezolizumab therapy). Of the 1,401 cases tested, 621 cases (44%) were tested with both the 22C3 PharmDx and SP142 assays, 492 cases (35%) with 22C3 PharmDx only, and 288 cases (21%) with SP142 only. Each assay was used and interpreted according to the manufacturer's guidelines. The rate of positivity we observed was 26% with the 22C3 assay and 31% with the SP142 assay, similar to the pre-licensing studies for both drugs. The discrepancy observed between the assays was 11%, which reinforces the requirement for utilisation of the correct assay for each agent, and limits potential cross-utility of assays. This aspect must be considered when setting up a PD-L1 testing strategy in laboratories where both Pembrolizumab and Atezolizumab are available for the treatment of urothelial carcinoma but also has broader implications for testing of other cancers where multiple ICB drugs and their respective assays are approved.

TMP3-NTRK1 rearranged uterine sarcoma: A case report.

INTRODUCTION: Uterine sarcomas are a group of rare tumours with heterogeneous morphological and genetic features. Recent advances in the molecular characterisation of these tumours have identified a novel clinicopathological category underpinned by NTRK gene fusions. CASE REPORT: We present the case of a 42-year-old woman with a polypoid cervical lesion formed of densely cellular, short, haphazard fascicles of monomorphic spindle cells that lacked coagulative necrosis and which showed high mitotic activity. On immunohistochemistry, the tumour was diffusely positive for pan-Trk and weakly positive for CD34 but was negative for a range of other markers, including cytokeratins, smooth muscle markers, hormone receptors and S100. FISH analysis using a NTRK1 break-apart probe was above the threshold for translocation positivity and subsequent next-generation sequencing (NGS) identified a TPM3-NTRK1 fusion. DISCUSSION: NTRK-rearranged uterine sarcomas are a novel subset of gynaecological mesenchymal neoplasms characterised by cytological isomorphism and fibrosarcoma-like morphology. Although distinction from more common mesenchymal neoplasms is possible on the basis of morphology and immunohistochemistry, exclusion of rare differential diagnoses, such as malignant peripheral nerve sheath tumour or the recently described COL1A1-PDGFB fusion sarcoma, requires molecular work-up with FISH or NGS. Identification of these rare tumours is clinically relevant because of their cervical location and the possible role for tropomyosin receptor kinase inhibitors in their treatment.

A review of retroperitoneal liposarcoma genomics.

Retroperitoneal liposarcomas are rare tumours that carry a poorer prognosis than their extremity counterparts. Within their subtypes - well differentiated (WDL), dedifferentiated (DDL), myxoid (MLS) and pleomorphic (PLS) - they exhibit a diverse genomic landscape. With recent advances in next generation sequencing, the number of studies exploring this have greatly increased. The recent literature has deepened our understanding of the hallmark MDM2/CDK4 amplification in WDL/DDL and addressed concerns about toxicity and resistance when targeting this. The FUS-DDIT3 fusion gene remains the primary focus of interest in MLS with additional potential targets described. Whole genome sequencing has driven identification of novel genes and pathways implicated in WDL/DDL outside of the classic 12q13-15 amplicon. Due to their rarity; anatomical location and histologic subtype are infrequently mentioned when reporting the results of these studies. Reports can include non-adipogenic or extremity tumours, making it difficult to draw specific retroperitoneal conclusions. This narrative review aims to provide a summary of retroperitoneal liposarcoma genomics and the implications for therapeutic targeting.

A novel next generation sequencing approach to improve sarcoma diagnosis.

Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 pathologic entities, differential diagnosis can be difficult. Complementing immune-histological diagnosis with current ancillary diagnostic techniques, including FISH and RT-PCR, can lead to inconclusive results in a significant number of cases. We describe here the design and validation of a novel sequencing tool to improve sarcoma diagnosis. A NGS DNA capture panel containing probes for 87 fusion genes and 7 genes with frequent copy number changes was designed and optimized. A cohort of 113 DNA samples extracted from soft-tissue and bone sarcoma FFPE material with clinical FISH and/or RT-PCR results positive for either a translocation or gene amplification was used for validation of the NGS method. Sarcoma-specific translocations or gene amplifications were confirmed in 110 out of 113 cases using FISH and/or RT-PCR as gold-standard. MDM2/CDK4 amplification and a total of 25 distinct fusion genes were identified in this cohort of patients using the NGS approach. Overall, the sensitivity of the NGS panel is 97% with a specificity of 100 and 0% failure rate. Targeted NGS appears to be a feasible and cost-effective approach to improve sarcoma subtype diagnosis with the ability to screen for a wide range of genetic aberrations in one test.

The Clinicopathological and Molecular Associations of PD-L1 Expression in Non-small Cell Lung Cancer: Analysis of a Series of 10,005 Cases Tested with the 22C3 Assay.

PD-L1 expression testing is mandatory prior to pembrolizumab prescription in non-small cell lung cancer. Our service offers PD-L1 testing using the PD-L1 IHC 22C3 pharmDx assay, in parallel with EGFR, ALK, ROS1 and (in some cases) KRAS testing. We correlate PD-L1 expression in 10,005 tumours with patient age and sex, with tumour histological subtypes, with the sampling modality and type of tissue, and with the presence of other molecular alterations. PD-L1 expression testing was performed using the aforementioned assay; tumour proportion scores (TPS) of 1 and 50% were taken as cut-offs for low and high positivity, respectively. EGFR testing was performed using the cobas® EGFR Mutation Test v2. ALK testing was performed using the VENTANA ALK (D5F3) CDx Assay. KRAS testing was performed using pyrosequencing. TPS <1% was seen in 44.4% of tumours, 1-49% in 25.0% and ≥ 50% in 30.6%. We identified no significant relationship with age. Female patients were slightly more likely to express PD-L1. Poorly-differentiated tumour histology and ALK translocation were significantly associated with PD-L1 expression. Rare EGFR mutations tended to be associated with PD-L1 expression. Pleural and nodal metastases were more likely to express PD-L1 than primary tumours, but biopsy and cytological specimens did not show different PD-L1 expression rates. Our data show that the means of acquiring a tumour sample (biopsy versus cytology) does not have a significant impact on PD-L1 expression. However, we found that certain metastatic sites were associated with significantly higher expression rates, which has substantial implications for selection of tissue for testing.