Ashish Mehta

Background: Tumour mutation burden (TMB) is an emerging pan‐cancer biomarker with predictive value for immune checkpoint inhibitor (ICI) outcomes, yet evidence is inconsistent due to methodological variability and cut‐off thresholds. This systematic review and meta‐analysis evaluated the impact of TMB on overall survival (OS) and progression‐free survival (PFS) across solid tumours in ICI‐treated cohorts and its predictive relevance in non‐ICI‐treated cohorts. Methods: Following PRISMA 2020 guidelines, we searched PubMed, Scopus, ScienceDirect and Cochrane for studies published between 2010 and 2024 reporting hazard ratios (HRs) and 95% confidence intervals (CIs) for OS and PFS in high‐ versus low‐TMB cohorts. High and low TMB were defined by study‐specific cut‐offs, and ultra‐high TMB was defined as the top 20% of cohort‐specific values. Study quality was assessed with the Newcastle‐Ottawa Scale; heterogeneity with I ² ; publication bias with funnel plots/Egger's test; and robustness by leave‐one‐out analysis. Results: 5278 patients across 28 studies were analysed. High TMB, defined by cohort‐specific cut‐offs, was significantly associated with improved OS and PFS, particularly in non‐small cell lung cancer (OS: HR = 0.56), selected gastrointestinal cancers (OS: HR = 0.36), and advanced/recurrent tumours (OS: HR = 0.52). Benefits were greatest in ICI‐treated patients, especially with combined anti‐PD‐L1/PD‐1 and anti‐CTLA‐4 therapy (OS: HR = 0.47; PFS: HR = 0.50). Chemotherapy‐treated cohorts also showed better outcomes, but less consistently (OS: HR = 0.60; PFS: HR = 0.55). Ultra‐high TMB had better OS than the universal 10 mut/Mb cut‐off (HR = 0.44 vs. 0.58). Non‐beneficial associations were observed in glioma and penile squamous cell carcinoma, highlighting disease‐specific variability. Sequencing platforms and cut‐off definitions remained sources of heterogeneity. Conclusion: TMB demonstrates prognostic relevance and predictive utility in a histology‐ and treatment‐context‐dependent manner, with the most consistent associations in selected ICI‐treated tumours. Associations in non‐ICI‐treated cohorts were weaker and inconsistent, indicating putative predictive value. Standardising TMB assessment and refining relevant thresholds are essential for optimising its role in precision oncology. Trial Registration: PROSPERO Registration Number: CRD42024608809

Background Tumour mutation burden (TMB) is an emerging pan-cancer biomarker with potential predictive value for immune checkpoint inhibitor (ICI) therapy outcomes. However, its prognostic significance remains inconsistent due to methodological variability and differing cut-off thresholds. This systematic review and meta-analysis evaluated the impact of TMB on overall survival (OS) and progression-free survival (PFS) across solid tumours. Methods Following PRISMA 2020 guidelines, we systematically searched PubMed, Scopus, ScienceDirect, and Cochrane databases for studies published between 2010 and 2024. Eligible studies reported hazard ratios (HRs) and 95% confidence intervals (CIs) comparing OS and PFS in high versus low TMB cohorts. Heterogeneity was assessed using the I² statistic, and publication bias via funnel plots and Egger’s test. Results A total of 5,278 patients across 28 studies were analysed. High TMB was significantly associated with improved OS and PFS, particularly in non-small cell lung cancer (OS: HR = 0.56), gastrointestinal cancers (OS: HR = 0.36), and advanced/recurrent tumours (OS: HR = 0.52). Survival benefits were most pronounced in ICI-treated patients, especially those receiving combination anti-PD-L1/PD-1 and anti-CTLA4 therapy (OS: HR = 0.47; PFS: HR = 0.50). Ultra-high TMB cases had superior outcomes (OS: HR = 0.44) compared to a universal 10 mut/Mb cut-off (OS: HR = 0.58). Variability in TMB measurement across sequencing platforms highlights the need for standardisation. Conclusion High TMB is a strong prognostic and predictive biomarker in ICI-treated cancers, yet methodological inconsistencies hinder clinical implementation. Standardising TMB assessment and refining clinically relevant thresholds are essential for optimising its role in precision oncology. PROSPERO registration number The protocol of this systematic review is registered on PROSPERO (CRD42024608809).

Cell delivery is a major determinant of success in mesenchymal stem cell (MSC) therapies. Cellular modifications and biomaterial-assisted approaches can enhance the engraftment, retention, and survival of implanted cells for improved tissue repair.

Replacing cardiac tissues lost to myocardial infarction remains a therapeutic goal for regenerative therapy in recovering cardiac function. We assessed the feasibility of constructing a macrosized human cardiac tissue construct using pluripotent stem cell‐derived cardiomyocytes or control fibroblasts infused fibrin/collagen hydrogel and performed ectopic implantation in peripheral vascular system of a porcine model for 3 weeks. Finally, an optimized vascularized cardiac construct was explanted and grafted onto porcine myocardium for 2 weeks. Myocardial‐grafted human cardiac constructs showed a nascent tissue‐like organization with aligned cardiomyocytes within the remodelled collagen matrix. Nevertheless, no significant changes in intraconstruct density of cardiomyocytes were observed in the myocardial‐grafted constructs (human embryonic stem cell [hESC]‐derived cardiomyocyte [n = 4]: 70.5 ± 22.8 troponin I⁺ cardiomyocytes/high power field [HPF]) as compared to peripherally implanted constructs (hESC‐derived cardiomyocyte [n = 4]: 59.0 ± 19.6 troponin I⁺ cardiomyocytes/HPF; human induced pluripotent stem cell‐derived cardiomyocyte [n = 3]: 50.9 ± 8.5 troponin I⁺ cardiomyocytes/HPF, p = ns). However, the myocardial‐grafted constructs showed an increased in neovascularization (194.4 ± 24.7 microvessels/mm² tissue, p < .05), microvascular maturation (82.8 ± 24.7 mature microvessels/mm², p < .05), and tissue‐like formation whereas the peripherally implanted constructs of hESC‐derived cardiomyocyte (168.3 ± 98.2 microvessels/mm² tissue and 68.1 ± 33.4 mature microvessels/mm²) and human induced pluripotent stem cell‐derived cardiomyocyte (86.8 ± 57.4 microvessels/mm² tissue and 22.0 ± 32.7 mature microvessels/mm²) were not significantly different in vascularized response when compared to the control human fibroblasts (n = 3) constructs (65.6 ± 34.1 microvessels/mm² tissue and 30.7 ± 20.7 mature microvessels/mm²). We presented results on technical feasibility and challenges of grafting vascularized centimetre‐sized human cardiac construct that may spur novel approaches in cardiac tissue replacement strategy.

Activation of signal transducer and activator of transcription 3 (STAT3) is imperative for mammalian development, specifically cardiogenesis. STAT3 phosphorylation and acetylation are key post-translational modifications that regulate its transcriptional activity. Significance of such modifications during human cardiogenesis remains elusive. Using human pluripotent stem cells to recapitulate cardiogenesis, two independently modified STAT3α (92 kDa) isoforms (phosphorylated and acetylated), which perform divergent functions were identified during cardiomyocyte (CM) formation. Phosphorylated STAT3α functioned as the canonical transcriptional activator, while acetylated STAT3α underwent caspase-3-mediated cleavage to generate a novel STAT3ζ fragment (∼45 kDa), which acted as a molecular adaptor integral to the ErbB4-p38γ signaling cascade in driving CM formation. While STAT3α knockdown perturbed cardiogenesis by eliminating both post-translationally modified STAT3α isoforms, caspase-3 knockdown specifically abrogates the function of acetylated STAT3α, resulting in limited STAT3ζ formation thereby preventing nuclear translocation of key cardiac transcription factor Nkx2-5 that disrupted CM formation. Our findings show the coexistence of two post-translationally modified STAT3α isoforms with distinct functions and define a new role for STAT3 as a molecular adaptor that functions independently of its canonical transcriptional activity during human cardiogenesis.

Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and Epidemal growth factor receptor (EGFR or ErbB1) in determining cardiac differentiation in vitro as these receptor tyrosine kinases are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation, cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment toward neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (>twofold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/noncanonical Wnt signaling determines cardiogenic fate in hPSCs.

Activation of ErbB4 receptor signaling is instrumental in heart development, lack of which results in embryonic lethality. However, mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells, we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38γ MAPK isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38γ/NKx2.5 downregulation is followed by p38α/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38γ MAPK similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38γ signaling hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38γ to the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development.

Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by “foot-print free” reprogramming of somatic cells to induced pluripotent stem cells (iPSC). In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA) based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA) recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.

Long QT syndrome 2 (LQTS2) caused by missense mutations in hERG channel is clinically associated with abnormally prolonged ventricular repolarization and sudden cardiac deaths. Modelling monogenic arrhythmogenic diseases using human-induced pluripotent stem cells (hiPSCs) offers unprecedented mechanistic insights into disease pathogenesis. We utilized LQTS2-hiPSC-derived cardiomyocytes (CMs) to elucidate pathological changes and to demonstrate reversal of LQTS2 phenotype in a therapeutic intervention using a pharmacological agent, (N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine) (ALLN). We generated LQTS2-specific CMs (A561V missense mutation in KCNH2) from iPSCs using the virus-free reprogramming method. These CMs recapitulate dysfunction of hERG potassium channel with diminished I_Kr currents, prolonged repolarization durations, and elevated arrhythmogenesis due to reduced membrane localization of glycosylated/mature hERG. Dysregulated expression of folding chaperones and processing proteasomes coupled with sequestered hERG in the endoplasmic reticulum confirmed trafficking-induced disease manifestation. Treatment with ALLN, not only increased membrane localization of mature hERG but also reduced repolarization, increased I_Kr currents and reduced arrhythmogenic events. Diverged from biophysical interference of hERG channel, our results show that modulation of chaperone proteins could be therapeutic in LQTS2 treatment. Our in vitro study shows an alternative approach to rescue diseased LQTS2 phenotype via corrective re-trafficking therapy using a small chemical molecule, such as ALLN. This potentially novel approach may have ramifications in other clinically relevant trafficking disorders.