Gopal P. Pathak

Background:Coexisting mutations in non-small cell lung cancer (NSCLC) can influence tumor biology, therapeutic response, and patient outcomes, yet these implications remain underexplored.Methods:5,420 NSCLC from QEII Health Sciences Centre were analyzed. Major driver variants were assessed, and clinical and survival data were analyzed.Results:KRAS mutations were the most common variants, followed by EGFR, with higher variant frequency found in females. Coexisting driver mutations were found in 3.9% of tumours analyzed, with higher proportion in females. KRAS was the most frequent co-mutated driver (67%), and the most common combination of coexisting mutations was KRAS and PIK3CA (35%). 55% of tumours with coexisting alterations had at least one actionable variant. Clonality analysis demonstrated KRAS and EGFR as major initiating drivers, while PIK3CA, AKT1 and IDH1 were secondary events. EGFR+KRAS mutations were associated with worse overall survival compared to patients with KRAS mutations only, in both early (HR: 2.59, 95% CI [1.47-13.33], p= 0.0085) as well as advanced-stage cohorts (HR: 3.20, 95% CI [2.24-18.33], p= 0.0008). Similar outcomes were observed compared to EGFR mutations in early (HR: 4.14, 95% CI [1.99-23.27], p= 0.0025) and advanced-stage cohorts (HR: 3.48, 95% CI [2.46-20.62], p= 0.0007). However, presence of KRAS+PIK3CA or EGFR+PIK3CA co-mutations did not impact prognosis in early-stage but was associated with worse outcomes in advanced-stage.Conclusion:Driver mutations coexist in NSCLC at various stages and can have a significant impact on prognosis. Comprehensive genomic profiling, investigating all oncogenic alterations including coexisting variants, is important for effective therapeutic management.

Background: Liquid biopsy using plasma cfDNA has been established as a tool for informing the management of advanced-stage NSCLC. However, its effectiveness in early lung cancer detection, including the identification of high-risk cases, remains to be determined. Methods: We analyzed plasma cfDNA and matched tumors from 117 stage I–IV lung adenocarcinoma cases and compared the variants identified across all stages using the Oncomine Precision Assay on the Genexus^TM next-generation sequencing platform. Results: Cancer-specific mutations were detected in plasma from approximately 72% (84/117) of cases (all stages), with detection rates increasing by stage. Concordance between cfDNA and tumor tissue also increased with stage 0% (stage I), 19% (stage II), 45% (stage III), and 75% (stage IV). KRAS mutations were concordant in approximately 22% (6/27) of stage II and 46% (11/24) of stage III cases. Clinically important EGFR variants showed concordance in 11% (1/9) of stage II and 80% (8/10) in stage III/IV cases. Actionable mutations, targetable with FDA-approved drugs, were detected in 11% (4/37) of stage II, 27% (12/45) of stage III, and 55% (4/9) of stage IV cases, underscoring the potential of liquid biopsy for early detection of therapeutic targets. Moreover, co-occurring mutations with varying actionability were identified more frequently in plasma than in tumor tissues. Plasma detection of clinically important KRAS and EGFR variants was mostly associated with advanced-stage disease, suggesting the presence of these variants in plasma as a potential indication of disease progression. Conclusions: Liquid biopsy holds promise for identifying high-risk lung adenocarcinoma cases and serves as a complementary diagnostic tool in advanced stages, enhancing disease management strategies.

Objectives: Several studies rely on archived tissue blocks to assess the PD‐L1 scores; however, a detailed analysis of potential variations of scores between fresh and archived tissue blocks still lacks. In addition, the prognostic implications of PD‐L1 in lung cancers have not yet been completely understood. Here, we aimed to investigate the temporal variation in PD‐L1 scores from clinical samples and the clinical implications of PD‐L1 in non–small cell lung cancer (NSCLC).Methods: NSCLC cases from January 2005 to June 2023 were considered for this study, and PD‐L1 scores in archived and fresh tissue blocks were analyzed. Association of PD‐L1 with various driver mutations was explored, and implications of PD‐L1 in progression‐free survival (PFS) and overall survival (OS) were analyzed.Results: Our study revealed a significant disparity in PD‐L1 scores between archived and fresh tissue blocks, and a temporal variation in scores within 6 months of tissue acquisition. Advanced‐stage primary tumors, metastatic lymph nodes, and visceral pleural invasion revealed higher PD‐L1 expression as presented by tumor proportion score (TPS). Notably, in fully resected stage I/II NSCLC cases, OS was better in the high PD‐L1 (≥ 50% TPS) cohort with driver mutations compared to cases without driver mutations (hazard ratio—0.5129, 95% confidence interval 0.2058–1.084, p = 0.0779). In contrast, high PD‐L1 was associated with worse OS compared to no PD‐L1 (< 1% TPS) (hazard ratio—2.431, 95% confidence interval 1.144–6.656, p = 0.0242) in the cohort without driver mutations. Furthermore, the presence of a KRAS mutation favored the outcome of anti‐PD‐L1/PD1 immunotherapy in advanced NSCLC.Conclusion: PD‐L1 detection from tissue blocks was found to vary temporally, urging for a prioritized consideration for patients with marginal scores when archived blocks are employed for its detection. Prognostic roles of PD‐L1 were associated with driver mutations, and KRAS mutations favored the outcome of anti‐PD‐L1/PD1 therapy in advanced NSCLC.

Oncolytic viruses (OV) such as reovirus preferentially infect and kill cancer cells. Thus, the mechanisms that dictate the susceptibility of cancer cells to OV-induced cytotoxicity hold the key to their success in clinics. Here, we investigated whether cancer cell metabolism defines its susceptibility to OV and if OV-induced metabolic perturbations can be therapeutically targeted. Using mass spectrometry–based metabolomics and extracellular flux analysis on a panel of cancer cell lines with varying degrees of susceptibility to reovirus, we found that OV-induced changes in central energy metabolism, pyruvate metabolism, and oxidative stress correlate with their susceptibility to reovirus. In particular, reovirus infection accentuated Warburg-like metabolic perturbations in cell lines relatively resistant to oncolysis. These metabolic changes were facilitated by oxidative stress–induced inhibitory phosphorylation of pyruvate dehydrogenase (PDH) that impaired the routing of pyruvate into the tricarboxylic acid cycle and established a metabolic state unsupportive of OV replication. From the therapeutic perspective, reactivation of PDH in cancer cells that were weakly sensitive for reovirus, either through PDH kinase (PDK) inhibitors dichloroacetate and AZD7545 or short hairpin RNA–specific depletion of PDK1, enhanced the efficacy of reovirus-induced oncolysis in vitro and in vivo. These findings identify targeted metabolic reprogramming as a possible combination strategy to enhance the antitumor effects of OV in clinics. Significance: This study proposes targeted metabolic reprogramming as a valid combinatorial strategy to enhance the translational efficacy of oncolytic virus–based cancer therapies.

Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators. We used this knowledge to develop two different approaches to regulate cellular protein levels with light: a system using CRY2 and CIB1 to induce protein expression with light through stimulation of transcription, and a system using CRY2 and a LOV-fused degron to simultaneously block transcription and deplete protein levels with light. These tools will allow precise, bi-directional control of gene expression in a variety of cells and model systems.

The plant pathogen Pseudomonas syringae (Ps) is a well‐established model organism for bacterial infection of plants. The genome sequences of two pathovars, pv. syringae and pv. tomato, revealed one gene encoding a blue and two genes encoding red/far red light‐sensing photoreceptors. Continuing former molecular characterization of the photoreceptor proteins, we here report selective photoreceptor gene disruption for pv. tomato aiming at identification of potentially regulatory functions of these photoreceptors. Transformation of Ps cells with linear DNA constructs yielded interposon mutations of the corresponding genes. Cell growth studies of the generated photoreceptor knock‐out mutants revealed their role in light‐dependent regulation of cell growth and motility. Disruption of the blue‐light (BL) receptor gene caused a growth deregulation, in line with an observed increased virulence of this mutant (Moriconi et al., Plant J., 2013, 76, 322). Bacterial phytochrome‐1 (BphP1) deletion mutant caused unaltered cell growth, but a stronger swarming capacity. Inactivation of its ortholog, BphP2, however, caused reduced growth and remarkably altered dendritic swarming behavior. Combined knock‐out of both bacteriophytochromes reproduced the swarming pattern observed for the BphP2 mutant alone. A triple knock‐out mutant showed a growth rate between that of the BL (deregulation) and the phytochrome‐2 mutant (growth reduction).