Cancer Research Communications

Blood-based metabolomic profiling has been widely investigated for breast cancer (BC) detection; however, clinical implementation remains limited due to variability in sample handling, analytical reproducibility, and overfitting during statistical analysis. We established a plasma GC/MS metabolomics workflow for discriminating BC from healthy controls (HC) using conventional machine-learning algorithms. Plasma samples (n = 360; BC = 180, HC = 180) were collected prospectively under standardized preanalytical conditions before surgery and the initiation of systematic anticancer therapy and analyzed using a quantitative GC/MS platform with automated derivatization. Feature selection and model development were conducted using three machine-learning (ML) algorithms (Lasso logistic regression (LR), random forest classifier (RFC), and support vector machine (SVM)). A total of 45 metabolite candidate biomarkers were identified, and the optimal number of metabolite features for each algorithm was estimated by a recursive feature elimination (RFE)-based strategy. The best-performing models achieved area under the ROC curve values (AUC) of 0.910 (LR), 0.893 (RFC), and 0.843 (SVM). We selected prioritizing candidate biomarkers consistently expressed across the multi-algorithm pipeline. A bagging ensemble model improved stability (AUC = 0.911) and reduced false-positive predictions in the independent HC dataset. In addition, model stability with respect to false-positive predictions was assessed using an independent HC cohort (n = 15) that was collected at a separate institution. These results indicate that a plasma metabolomics workflow combined with conventional multi-algorithm ML, algorithm-specific feature selection, and independent assessment provides stable discrimination between BC and HC in a moderately sized cohort.

Germ cell tumors (GCTs) are highly sensitized to cell death in response to DNA damaging agents, a property that underlies the success of current chemotherapeutic regimens. To address the molecular basis for this, known as apoptotic priming, we evaluated how different BCL2 family members modulate the heightened sensitivity of GCTs to therapy. Our analysis of human GCTs finds consistently high expression of the pro-survival factors MCL1 and BCL2L1 (BCLX) in a cohort of primary tumors and in their embryonic precursor cells, frequently accompanied by copy number gains of these loci and reciprocal losses of their pro-apoptotic interaction partners and inhibitors, PMAIP1 (NOXA) and BAD. We find that co-inhibition of MCL1 and BCLX using selective BH3 mimetics results in a potent synthetic lethality in multiple GCT embryonal carcinoma cell lines. When these cell lines were cultured with the DNA damaging agents cisplatin or etoposide, inhibition of MCL1 or BCLX potentiated their apoptotic effect in undifferentiated embryonal carcinoma cell lines, but not in retinoic acid-differentiated cells. The inhibition of MCL1 also heightened cisplatin sensitivity in p53-deficient or -mutant cell lines, which is associated with resistance to therapy. Employing an in ovo human xenograft model, we validate that the combination of cisplatin and MCL1 inhibition enhanced the therapeutic response by eliminating tumor cells. Our findings identify MCL1 and BCLX as critical factors to maintain GCT viability and as putative therapeutic targets to further augment GCT responsiveness to DNA damaging agents.

Background/Objectives: In high-grade serous ovarian cancer (HGSOC), the abilities of tumor cells to resist cytotoxic treatment and relapse are largely driven by ovarian cancer stem cells (CSCs). CSCs are a population of cells within a tumor that maintain stem-like qualities and have the capacity to produce metastasis and drive drug resistance. Based on the complexity of CSC signaling and emerging evidence of differential isoform usage in development, we hypothesize that CSCs possess a patient-specific, heterogeneous landscape which exploits isoform diversity to sustain stemness, metastasis and drug resistance. Methods: RNA-seq was generated from 3 patient-derived HGSOC (PD-HGSOC) to investigate stemness and differentiation status comparing standard culture and CSCs. The data was analyzed at both gene- and isoform-levels to identify patient-specific CSC features and define mechanisms of cancer-related stemness and plasticity. Results: CSCs exhibited higher stemness characteristics and identified differentially expressed gene sets across PD-HGSOCs. We noted that each patient had a distinct subset of genes regulating stemness phenotypes which suggested multiple mechanisms of CSC development and maintenance. Additionally, a large proportion of gene-level expression changes were driven by the differential expression of a single predominant isoform. Importantly, our isoform-level analysis enabled the identification of differential isoform usage that would not be detected with gene level expression analysis. Conclusions: CSC phenotypes such as chemoresistance, metastasis and self-renewal are conserved across patients, but arise from largely patient-specific gene and isoform programs. This CSC heterogeneity is reflective of the heterogeneity seen in patients and should be further evaluated in deriving precision-medicine approaches for HGSOC.

PurposeSWI/SNF (BAF) chromatin remodeling complex alterations are common in urothelial carcinoma, yet no biomarker-directed therapeutic strategies have been established for this population. We investigated whether BAF alterations delineate a biologically distinct, therapeutically actionable urothelial carcinoma subtype. Experimental DesignWe performed integrative genomic and transcriptomic analyses of 792 urothelial carcinoma tumors from the Oncology Research Information Exchange Network (ORIEN) and validated findings in the TCGA-BLCA cohort. Mechanistic studies incorporated RNA sequencing and ATAC-seq following histone deacetylase (HDAC) inhibition. Functional dependencies were assessed using patient-derived xenograft organoids and cell line models. Clinical relevance was explored in a biomarker-enriched investigator-initiated trial. ResultsApproximately half of urothelial carcinoma tumors exhibited BAF alterations, defining a previously unrecognized chromatin-altered molecular subtype characterized by activation of proliferative programs, loss of lineage identity, and altered metabolic signaling. This subtype was enriched for transcriptomic programs associated with HDAC inhibitor sensitivity and depleted of HDAC inhibitor resistance signatures. Mechanistically, HDAC inhibition induced widespread chromatin remodeling with reduced accessibility at AP-1 and TEAD-associated regions, and downregulation of E2F- and MYC-driven transcriptional networks. Functional studies confirmed enhanced HDAC inhibition sensitivity in ARID1A-mutated cell lines and a patient-derived organoid model. Early clinical observations demonstrated a durable responder treated with HDAC inhibitors and immunotherapy. ConclusionsBAF alterations define a chromatin-dependent tumor state in urothelial carcinoma that is selectively vulnerable to HDAC inhibition. Integrating genomic, epigenomic, functional, and early clinical evidence, these findings provide a rationale for biomarker-enriched clinical trials and HDAC inhibitor-based combination strategies in urothelial carcinoma.

Endometrial cancer (EC), the most common gynecologic malignancy in the USA, has seen limited improvement in patient outcomes over recent decades, underscoring the need for relevant preclinical models. To address EC heterogeneity, we established an integrated platform of patient-derived xenografts (PDXs) and matched patient-derived primary cancer cells (PDCs) for disease modeling and systematic drug sensitivity testing. Fresh tumor specimens (n=103) were collected from EC patients to generate PDXs in immunodeficient mice and corresponding PDCs. Fifty-three PDX models were successfully established (52% engraftment rate), with higher success observed in high-grade, recurrent, metastatic tumors (70%), compared with their low-grade counterparts (56%). Histopathologic and immunohistochemical analyses confirmed that PDX tumors faithfully preserved morphology, hormone receptor status, and intertumoral heterogeneity across multiple passages. Using 13 PDC models, we performed an unbiased screening of 179 FDA-approved oncology drugs, revealing marked intertumoral variability in drug response. Almost all PDC models exhibited limited sensitivity to NCCN-recommended therapies, highlighting the need for alternative treatment strategies. In contrast, multiple FDA-approved agents including epigenetic modulators, dual PI3-kinase/HDAC inhibitors, topoisomerase II inhibitors, and proteasome inhibitors demonstrated potent antitumor activity. Importantly, a low-dose combination of the DNA methyltransferase inhibitor 5-azacytidine and the histone deacetylase inhibitor romidepsin significantly suppressed tumor growth across six independent PDX models. Together, these findings establish a comprehensive PDX and PDC platform as a robust translational resource. By capturing the histopathologic and molecular diversity of EC and identifying clinically actionable therapeutic advantages, including an epigenetic combination regimen, this study offers a translational resource for preclinical drug evaluation.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian and breast cancers with homologous recombination deficiency (HRD). Despite substantial initial efficacy, the frequent development of acquired resistance poses a critical clinical challenge, necessitating a deeper understanding of the underlying molecular mechanisms. While genomic profiling provides information on baseline HRD status, it is insufficient to capture the dynamic and functional adaptations accompanying resistance during treatment. In this study, we investigated the role of alternative splicing in PARPi resistance using longitudinal pre- and post-treatment RNA-sequencing data from an ovarian cancer patient cohort (n=40). We demonstrated that profiling alternative splicing dynamics and differentially used transcripts (DUTs) provides transcriptomic insights distinct from conventional differential gene expression analysis. Notably, we observed significant differences between acquired resistance (AR) and innate resistance (IR) groups. Specifically, longitudinal analysis revealed that AR tumors exhibit a distinct transcript usage pattern, characterized by an increase in canonical protein-coding transcripts involved in cell-cycle processes and a decrease in non-canonical non-coding transcripts within the homologous recombination pathway following treatment. Extending this comparison across AR and IR tumors at both pre- and post-treatment stages, these transcriptomic features were most pronounced in the AR post-treatment group. Further analysis of subsequent treatment outcomes showed that, among AR patients, greater resistance-associated transcriptomic remodeling was associated with poorer response and shorter progression-free survival. The resistance-associated transcriptomic signatures identified through longitudinal profiling were further recapitulated in an independent pre-treatment cohort (n=71; 43 AR and 28 IR), with more pronounced differences among BRCA wild-type patients. Taken together, our findings identify differential transcript usage as dynamic transcriptomic features associated with acquired PARPi resistance and establish a framework for utilizing longitudinal transcriptome dynamics to investigate the molecular basis of clinical drug resistance.

Triple negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Conventional treatments include neoadjuvant chemo-immunotherapy followed by surgical resection and may include further adjuvant immunotherapy and/or radiotherapy of the tumor bed and lymph nodes. Nonetheless, TNBC patients with residual disease have rapid metastatic recurrence. While the roles of metabolic and mitochondrial adaptations in chemotherapeutic resistance have been the subject of many studies, their importance in the context of ionizing radiation (IR) therapy remains poorly understood. We established longitudinal in vitro models of post-IR human TNBC, characterized by cellular regression to a residual phenotypic state, then eventual cell repopulation. This was accompanied by plastic adoption of unique metabolic, proteomic, and morphologic features that largely reverted when cells regrew. Following IR, residual cells exhibited extensive mitochondrial rewiring, including elevated mitochondrial content, oxidative phosphorylation (oxphos) rates, cristae structures, and metabolite levels. Concomitantly, levels of the short protein isoform of the mitochondrial inner membrane protein optic atrophy 1 (OPA1) were significantly elevated in residual cells, and OPA1 knockout ablated mitochondrial adaptations induced by IR. OPA1 genetic or pharmacologic perturbations led to improved cellular responses to IR. Metabolomic and proteomic analyses of radio-residual cells uncovered a coordinated program of antioxidant and redox capacity elevation with mitochondrial metabolism, which was corroborated by analyses of external datasets. Together, these findings provide evidence that TNBC cells surviving radiotherapy adopt an OPA1-dependent program of mitochondrial reorganization that supports their survival and regrowth, thereby positioning OPA1 as a therapeutic dependency that could improve radiotherapy efficacy in TNBC.

Patients with majority variant subtype histology including sarcomatoid urothelial carcinoma (SUC) have poor outcomes and limited treatment options, and there are few preclinical resources to investigate rare histologic subtypes. We developed three novel variant subtype histology bladder cancer (BC) cell lines by finding that basal BC tumor cells derived from mice treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) were initially not competent to grow in immune competent mice, but then acquired tumorigenicity following passaging (recycling) in mice lacking mature B and T cells. Three genitourinary oncology pathologists identified BBN966 as SUC, BBN964 as a hybrid between SUC and squamous, and BBN975 as squamous, and each had unique growth characteristics. Using a publicly available dataset and our spatial transcriptomics analysis of a cohort of human tumors with mixed SUC and UC as comparators, we found that consistent with human SUC, BBN966 lack expression of basal and luminal genes. Consistent with human SUC and spatial transcriptomics analysis of canine SUC, BBN966 had the highest epithelial to mesenchymal transition (EMT) upregulation, and all three recycled tumor cells had increased EMT pathway expression in recycled tumor cells. Moreover, increased expression of genes upregulated in all three recycled tumor cells associated with worse overall survival in patients with muscle invasive UC. Similar to human SUC, BBN966 tumors had higher proportion of M2 macrophage gene expression. Finally, we found in the largest cohort of PD-L1 IHC to date that 20 of 38 (52.6%) human SUC had CPS > 50, which was driven mostly by tumor proportion score. Consistent with this, we found anti-PD1 immune checkpoint inhibitor (ICI) significantly inhibited BBN966 tumor growth, while the hybrid BBN964 tumors had mixed response to ICI. Thus, the murine BBN966 tumors exhibited similarities to human SUC, and can be used to examine novel therapies for translation into future SUC clinical studies.

Abstract Background: Experimental models that retain endogenous tumor immune complexity are needed to investigate heterogeneous responses to immune checkpoint inhibitors (ICIs) in melanoma. We established patient-derived melanoma organoids (PDMOs) to examine retention of parental tumor cellular components and investigate patient-specific and context-dependent responses to checkpoint blockade. Methods: Fresh melanoma specimens (n=50), including primary tumors, neoadjuvant-treated tumors and tumor-infiltrating lymphocyte (TIL) associated samples were processed for Matrigel-embedded culture of patient-derived melanoma organoids (PDMOs). Characterization and functional studies were performed in subsets of established cultures. Immunofluorescence assessed tumor, stromal, immune, and checkpoint-marker expression in PDMOs and matched parental tissues. NGFR expression was evaluated in relation to organoid establishment and growth. Responses to anti PD1 alone or combined with anti-LAG3 or anti-CTLA4 therapies were evaluated using viability and morphological analyses, with selected models undergoing immune phenotyping and multiplex cytokine profiling. Available clinical outcomes were used for exploratory comparison. PDMOs and matched two-dimensional cultures were evaluated under 21% and 5% oxygen conditions. Results: PDMOs were established from 30 of 50 specimens (60%) and retained melanoma, stromal, lymphoid, and myeloid components identified in matched parental tissues during early culture, together with immune checkpoint expression. Higher NGFR (CD271) expression was associated with greater organoid-forming capacity in an exploratory subset. Responses to checkpoint blockade varied across patient-derived models and regimens, with combination blockade not uniformly producing lower viability than PD1 monotherapy. Ex vivo responses showed parallels with available clinical outcomes, with discordant cases also observed. In three selected PDMOs, divergent PD-1 responses were accompanied by differences in T-cell and myeloid representation, MHCII and AXL expression, and inflammatory versus immunoregulatory cytokine profiles. Under normoxic conditions, PDMOs and matched two-dimensional cultures exhibited divergent treatment sensitivities, including clinically responding cases in which sensitivity was retained in PDMOs but attenuated in monolayers. Changing oxygen tension further altered checkpoint sensitivity within individual PDMOs, with the direction and magnitude of the observed shifts varying by model and regimen. Conclusions: Early passage PDMOs retain key endogenous tumor, immune, and stromal components and support functional investigation of patient-derived melanoma biology. Their heterogenous responses across checkpoint regimens and oxygen conditions provide a platform for investigating how tumor-immune composition and environmental context influence treatment sensitivity.

Epithelioid sarcoma (EpS) is an ultra-rare, aggressive soft tissue sarcoma (STS) driven by INI1 loss and consequent hyperactivation of the chromatin-modifying enzyme EZH2. Although the EZH2 inhibitor tazemetostat has shown clinical activity, responses remain limited, highlighting the need for improved treatment strategies. Here, using two in-house generated patient-derived xenograft models and matched cell lines derived from INI-1 deficient EpS, we investigated EZH2 inhibition in combination with doxorubicin, the first-line standard for advanced STSs, identifying distinct patterns of response and resistance. Integrated transcriptomic and functional analyses revealed that response to EZH2 inhibition-based therapy was associated with chromatin remodeling, characterized by increased H3K27 acetylation and downregulation of histone deacetylase (HDAC)-related transcriptional programs. Conversely, the intrinsically resistant model failed to undergo this epigenetic transition despite EZH2 inhibition. Pharmacological HDAC inhibition restored H3K27 acetylation, promoted apoptosis, and enhanced the activity of EZH2 inhibition-based therapy. These findings identify failure to accumulate H3K27 acetylation as a hallmark of resistance to EZH2 inhibition-based treatment, and show that pharmacological HDAC inhibition can restore this chromatin transition and re-sensitize resistant tumors, providing a rationale for combined epigenetic targeting strategies in INI1-deficient malignancies. Translational relevanceProspective trials are challenging in rare tumors such as epithelioid sarcoma (EpS), limiting the level of evidence for existing therapies and the development of new agents. This is particularly relevant for EpS, where drug regimens are those used for all soft tissue sarcomas (STSs), and the specific mechanisms of drug response remain poorly understood. This preclinical study of tazemetostat in combination with doxorubicin shows differential outcomes in two INI1-deficient proximal-type EpS models, providing evidence of the heterogeneity that exists even within the same tumor subtype and fostering the need to better understand the molecular mechanisms driving drug sensitivity/resistance in this disease. In addition, we demonstrated the potential to treat EpS models through modulation of epigenetic mechanisms, showing that HDAC inhibition may restore sensitivity to EZH2-targeted therapy. These findings support the rationale for developing combination strategies incorporating epigenetic modulators and provide a preclinical framework for overcoming resistance to current therapies in EpS.

Neuroblastoma (NB) is one of the most common solid malignancies in children, and high-risk patients have poor prognosis, underscoring the need for novel therapeutic agents. The small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various protein functions, and the first-in-class SUMOylation inhibitor TAK-981 (subasumstat) is currently in clinical trials in adult cancer patients. However, the role of SUMOylation in NB pathogenesis remains poorly characterized. We show that SUMOylation-catalyzing enzymes, SAE1, SAE2, and UBE2I, are highly expressed in malignant neuroendocrine cells, and their expression correlate with poor survival in NB patients and associate with advanced stage, MYCN amplifications, and downregulation of late differentiation markers. The peripheral nervous system lineage and NB cell lines are highly sensitive to the CRISPR knockout of SAE1 and SAE2, suggesting SUMOylation as promising therapeutic target in NB. Accordingly, TAK-981-mediated inhibition of SUMOylation reduces growth of NB cells in both in vitro and in vivo models through induction of apoptosis and perturbation of differentiation-associated pathways. Sensitivity to SUMOylation inhibition is greatest in NB cells with low expression of favorable late neuroblast markers and is independent of MYCN amplification status. Moreover, TAK-981 is effective in combination with differentiation-inducing all-trans retinoic acid and the DNA methyltransferase inhibitor decitabine. Mechanistically, combination of TAK-981 and retinoic acid potently downregulates retinoic acid receptor alpha (RAR) expression, whereas inhibition of SUMOylation combined with decitabine induces a strong accumulation of DNA damage. Taken together, these findings establish SUMO pathway components as potential prognostic markers and SUMOylation as a therapeutic vulnerability in high-risk NB.

Traditional animal models are often inbred and genetically uniform. This makes them powerful for controlled experiments, but it limits how well they represent the patient-to-patient variation seen in real-world disease. Comparative oncology seeks to address this gap by studying naturally occurring cancers in outbred companion animals, especially dogs. Canine medicine offers two important advantages: first, prospective trials can often be completed faster than in humans and second, dogs are already part of the translational pipeline through pharmacokinetic and toxicology studies. Here, we assessed the transcriptional fidelity of human and canine invasive urothelial carcinoma in primary tumors and patient-derived organoids. We then used single-cell and spatial data to resolve the underlying cellular organization. Despite strong species and platform differences, human and canine tumors preserved the same major luminal-basal structure and a similar tumor microenvironment. The two species reached this shared biology through different recurrent mutations. These included FGFR3 alterations in humans and BRAF alterations in dogs, which converged on overlapping pathways and a luminal phenotype. Human and canine organoids also underwent a similar shift in culture. Both became more proliferative and metabolic while losing inflammatory programs. Thus, organoids preserved important tumor biology while introducing predictable platform effects. Single-cell and spatial analyses showed that the luminal-basal axis reflects a gradient of cell states organized around the tumor-stroma boundary, rather than two discrete tumor types. This helps explain why bulk RNA-sequencing subtypes are reproducible but coarse. Together, these findings define where canine and human bladder cancer agree, where they differ, and how dogs can support parallel therapeutic and diagnostic development.

Cisplatin resistance remains a major barrier in head and neck squamous cell carcinoma (HNSCC) treatment. ATP-binding cassette (ABC) transporters contribute to chemoresistance by limiting intracellular drug accumulation. Bitter taste receptor 10 (T2R10) has been implicated in ABC transporter regulation, but its role in HNSCC remains undefined. HNSCC cell lines were treated with T2R10-agonist caffeine (100 or 200 M), cisplatin, or a combination, and viability was assessed by crystal violet assay. T2R10 promoter activity and expression following caffeine exposure were evaluated using a promoter-driven mCherry reporter and RT-qPCR. ABC transporter expression was measured after caffeine treatment and T2R10 gene (TAS2R10) knockdown or overexpression. Associations between tumor TAS2R10 expression and survival were assessed using TCGA data through GEPIA2. Caffeine enhanced the cisplatin-associated reduction in viability in a cell line- and concentration-dependent manner, with the strongest effect seen in UM-SCC47. A significant effect was observed in FaDu at 200 M of caffeine, and minimal response in RPMI 2650. RPMI 2650 cells and FaDu cells exhibited lower baseline TAS2R10 expression and RPMI 2650 cells did not demonstrate enhanced cisplatin sensitivity following caffeine treatment. Caffeine treatment increased TAS2R10 promoter activity and expression and was associated with decreased ABCG2 expression. TAS2R10 knockdown increased ABCG2 and ABCF1 expression, whereas TAS2R10 overexpression reduced ABCG2 and ABCC1 expression. High tumor TAS2R10 expression was associated with improved disease-free survival (log-rank p=0.0071; HR=0.61) but not overall survival. Caffeine enhances cisplatin sensitivity in selected HNSCC models. Caffeine exposure is associated with increased TAS2R10 expression and reduced expression of chemoresistance-associated transporters, particularly ABCG2.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are the only targeted treatment available for early triple-negative breast cancer (TNBC), an aggressive subtype with limited treatment options. While PARPi are effective in patients with BRCA mutations, resistance to these inhibitors can arise. We previously developed BRCA-mutant cell lines, HCC1395, with acquired resistance to talazoparib and olaparib, and have now developed PARPi-resistant MX1 cell lines in an analogous manner. Ribosomal pathways were upregulated in 3/4 cell lines with acquired PARPi resistance. The resistant cell lines showed high and low basal nucleolar stress based on nucleolar morphology and nucleophosmin delocalization from nucleolus to nucleoplasm. Treatment of these cell lines with the combination of olaparib and a ribosome biogenesis inhibitor, BMH-21, demonstrated synergy in the PARPi-resistant cell lines, but not in the parental cells. Interestingly, HCC1395 cells, which had lower baseline nucleolar stress, showed greater nucleophosmin delocalization with combination treatment than MX1 cells, which had higher baseline nucleolar stress. All four cell lines demonstrated sustained DNA damage with the combination. We extended our investigation to 3 BRCA wild-type TNBC cell lines, BT549, Hs578T, and MDAMB231 and identified synergy in the context of intrinsic PARPi resistance. In MDAMB231 cells, combining olaparib with BMH-21 induced a senescence-like phenotype, increasing gH2AX-positive cells by up to 7.3-fold, senescence-associated B-galactosidase (SA-B-gal) staining by 5.6-fold, and p21 by 3.6-fold. These results were mirrored with a second ribosome biogenesis inhibitor, CX-5461. Finally, we identified distinct patterns of ribosomal pathway activity in a cohort of patients with treatment-resistant TNBC, and found correlations between ribosomal pathways with MYC, proliferation, DNA damage, and senescence. Together, these data identify ribosome biogenesis as a potential therapeutic vulnerability in PARPi-resistant TNBC and support its targeting in combination with PARPi, while demonstrating a broader for ribosome biogenesis in treatment-resistant TNBC.

The immunomodulatory effects of chemotherapy (chemoimmunomodulation; CIM) are clinically consequential and heterogeneous, yet no systematic framework exists for classifying the immunomodulatory trajectory a tumor follows in response to treatment (CIM trajectory). Here, we present the CIM Induction Classifier (CIMIC), an unsupervised clustering pipeline leveraging delta gene expression across 3,189 CIM-related genes to classify specimens chemoimmunomodulatory trajectory. Applied to two pre- and post-chemotherapy breast cancer (BC) datasets (NKI/SMC, N = 36; NEO, N = 19) and nine epirubicin-perturbed triple-negative BC (TNBC) cell lines, CIMIC identified two divergent CIM trajectories: a functional CIM (Fun-CIM) trajectory, broadly conserved across tumors and cell lines and characterized by induction of inflammatory cell death, antigen presentation, viral mimicry, and adaptive immune activation programs, and a dysfunctional CIM (Dys-CIM) trajectory, characterized by induction of proteostatic and metabolic stress-adaptation programs, reduced immune cell abundances and cytotoxic activity, and enrichment of aggressive BC subtypes. Using survival and longitudinal transcriptomic data in NKI/SMC (N = 20), treatment-induced increases in Fun-CIM-associated genes and ssGSEA scores were associated with reduced recurrence, whereas Dys-CIM-associated genes and scores were associated with increased recurrence. In multivariable analyses within independent chemotherapy-treated BC cohorts (METABRIC, N = 412; SCAN-B, N = 2,462), higher baseline Fun-CIM ssGSEA scores were associated with better outcomes, whereas higher baseline Dys-CIM ssGSEA scores were associated with worse outcomes. These findings establish CIM as a dynamic, trajectory-level process and position CIMIC as a framework for defining CIM trajectories and supporting future efforts to identify predictors, mechanisms, and therapeutic strategies that maximize beneficial CIM.

Approximately 10% of bladder, upper urinary tract, and endometrial cancers harbor deleterious somatic alterations in nucleotide excision repair (NER) pathway genes. Although platinum-based chemotherapy is the standard treatment for NER-deficient tumors, its utility is limited by nephrotoxicity, motivating alternative strategies that avoid direct DNA targeting. Here, we employed a genome-wide CRISPR-Cas9 screen to identify synthetic lethal vulnerabilities in NER-deficient cancer cells. An ERCC4-null RT112/84 bladder cancer cell line was generated and validated by Sanger sequencing and Western blotting, and functional NER deficiency was confirmed using a host cell reactivation assay. Parallel screens in ERCC4-deficient and isogenic wild-type cells. nominated SLC25A28 as the top-ranked synthetic lethal candidate in ERCC4-deficient cells, an interaction further supported by DepMap dependency. Clonogenic assays confirmed that loss of SLC25A28 selectively impaired cell viability in cells deficient for ERCC2, ERCC3, ERCC4, or ERCC5 and inducible knockout selectively suppressed ERCC4-deficient xenograft growth in-vivo. Mechanistically, RNA sequencing followed by gene set enrichment analysis revealed enrichment of oxidative stress-related pathways, including cellular responses to reactive oxygen species (ROS) and DNA damage signatures, upon SLC25A28 loss. These transcriptional changes are consistent with increased oxidative stress and DNA damage, placing heightened demand on NER-mediated repair. Direct measurement confirmed increased intracellular ROS. Collectively, our nominates SLC25A28 as a previously unrecognized synthetic lethal vulnerability in NER-deficient cancers and implicate mitochondrial iron homeostasis as a candidate metabolic dependency that may be targeted independently of platinum-based chemotherapy.

Genomic, transcriptomic and epigenomic characterization has accelerated the discovery of clinically-relevant alterations in cancer, predominantly using fresh frozen (FF) specimens. However, clinical molecular pathology laboratories prefer formalin-fixed paraffin-embedded (FFPE) methods, known to introduce artifacts at the nucleic acid level, over fresh frozen methods. Extending the multi-platform analysis to FFPE specimens for comprehensive clinical molecular diagnosis requires a thorough understanding of the consequence of formalin-fixation. We present a detailed multi-platform characterization of FFPE preservation using paired FF specimens as the 'gold standard'. DNA and RNA were obtained from 38 patients across 6 cancer types using a FFPE optimized co-isolation. The impact of FFPE on exome sequencing was dependent on filtering, where a minimum coverage or supporting read filter can mitigate FFPE-specific false positives. Copy number alterations, MSI assessment, mutational signatures, and DNA methylation were comparable between FFPE and FF. FFPE biases in RNA expression can be overcome when using biology-relevant genes and we describe a novel consequence of FFPE on miRNA species diversity. Collectively, this data provides a broad view of FFPE artifact and offers best practices for overcome these biases.

Background: Small cell lung cancer (SCLC) is an aggressive, high-grade neuroendocrine carcinoma (hgNEC) with poor prognosis. Tarlatamab, a DLL3-targeting T cell engager was approved in 2024 for relapsed SCLC, with almost doubled overall survival benefit compared to chemotherapy. However, over half of patients do not respond to tarlatamab and others develop resistance within months. With multiple DLL3-targeting therapies in clinical trials, there are currently no validated predictive biomarkers to identify patients most likely to benefit from tarlatamab. In this study, we evaluate baseline DLL3 IHC level in patients as a predictive biomarker to tarlatamab clinical outcome. Methods: We assembled a cohort of 138 patients within IRB-approved MD Anderson GEMINI database (PA13-0589) with DLL3 expression by CLIA-validated immunohistochemistry (IHC), and those treated with tarlatamab from 7/1/2024 to 3/30/2026. Only those with DLL3 IHC results and treated with tarlatamab were included in this predictive biomarker study. All DLL3 levels were reported as percentage cells with cytoplasmic or surface DLL3 expression, including 24 with additional intensity score and H-score calculation. demographic, clinical, and outcome data were collected. Systemic and intracranial response data were assessed by RECIST and mRANO BM criteria respectively. Longitudinal liquid biopsies were taken from a subset of patients for circulating tumor DNA (ctDNA) and (Cytometry by Time-of-Flight) CyTOF analyses. Results: In the predictive biomarker cohort of 54 patients with SCLC treated with tarlatamab, Median DLL3% level in tarlatamab treated cohort was 75%. Median follow-up time was 9.9 months, median time on tarlatamab treatment (ToT) was 5.8 months. At the data cut-off, 61% (33 out of 54) patients were alive. Kaplan-Meier curve stratified by DLL3 % of <=75% vs. >75 showed significant longer ToT and a trend toward longer median OS (mOS). In DLL3>75% compared to DLL3 <=75%: mToT was 10.1 months vs. 2.9 months (p=0.036), mOS: not reached vs. 9.5 months (p= 0.066). Among 41 patients who completed one cycle of tarlatamab treatment and were eligible for systemic (extra-cranial) response per RECIST criteria; we observed partial response (PR) in 51% (21) patients, stable disease (SD) in 22% (9) patients; and progressive disease (PD) in 27% (11) patients. The median DLL3 % in those with PR vs. PD were 80% vs. 40% (p=0.006); median DLL3% comparing PR and SD were 80% vs. 60% (p=0.051). Among 35 patients evaluable for intracranial response, best objective response prior to any brain radiation were 5 (14%) CR,11(31%) PR, 8 (23%) SD and 11 (31%) PD. 9 received brain radiation (8 SRS, 1 WBRT) during tarlatamab to achieve better intracranial control while systemic control is maintained. DLL3% does not appear to be correlated with intracranial response. Longitudinal ctDNA and CyTOF analyses showed persistent ctDNA positivity, as well as increasing NEUROD1 subtype cells in circulating tumor cells in post-tarlatamab progression samples. We additionally profiled DLL3 expression by IHC in 138 patients with SCLC and found heterogenous DLL3 expression, with Median DLL3% of 70% across different biopsy sites. Conclusions: Our data suggests DLL3 expression in patients with SCLC is heterogeneous and not ubiquitous, with median DLL3 IHC of 70%. DLL3 IHC predicts objective response and long-term outcome with tarlatamab. In addition, dynamic monitoring of ctDNA level and CTCs by CyTOF are suggestive of development of tarlatamab resistance, however further studies with larger cohort of paired longitudinal samples are needed to validate the novel blood-based biomarkers. To our knowledge, this study is the first to validate DLL3 IHC as a predictive biomarker for tarlatamab in SCLC, which leads the way for optimizing treatment selection and combinatorial therapies for the subset of patients less likely to respond to tarlatamab.

Tumor cellular composition--including malignant cell states, immune populations, and stromal populations--is increasingly recognized as a determinant of therapeutic response and resistance to anti-cancer agents, yet comprehensive cellular profiling remains largely confined to research settings. Here, we present a clinically compatible sample-to-report workflow for tumor composition profiling from routine formalin-fixed paraffin-embedded (FFPE) clinical specimens. By combining low-input single-nucleus RNA sequencing with foundation model- based automated cell annotation, this workflow enables prospective sample-by-sample analysis without dedicated research material or cohort-based processing. Across 116 clinical specimens representing six cancer types, we generated reproducible measurements of cellular composition and cell-type-specific gene expression, demonstrated high technical reproducibility, and showed concordance with pathological assessment of immune infiltration. The workflow was similarly applicable to archival FFPE material and ultra-low-input biopsy specimens. Together, these findings establish a practical framework for routine single-cell profiling from standard pathology specimens and open the perspective of prospective evaluation of cellular composition as a clinical biomarker in precision oncology.

Homologous recombination deficiency (HRD) can force cancer cells to rely on alternative DNA repair pathways, including microhomology-mediated end joining (MMEJ), an error-prone mechanism that can generate deletions with microhomology at repair junctions. Because such patterns may reflect DNA repair defects with clinical relevance, this study aimed to assess the biological and prognostic significance of microhomology-associated deletions and optimize their detection parameters to improve patient stratification and inform targeted therapeutic strategies, including PARP inhibition. We optimized the microhomology length threshold (Mlt parameter) using signal-to-noise ratios and survival models. We evaluated the utility of whole-exome (WES) versus whole-genome sequencing (WGS). We then investigated how Loss of Function (LoF) alterations affect microhomology-associated deletion burden and assessed their prognostic significance across an ovarian cancer (OV) cohort and a TCGA Pan-Cancer dataset. Applying an Mlt = 2 threshold maximized the precision and statistical reliability of microhomology-associated deletion identification. WGS yielded higher reproducibility, whereas WES proved insufficient for accurate estimation. Inactivation of tumor suppressor genes, including RB1 and CCDC122, was associated with increased microhomology-associated deletion burden. Importantly, higher burden correlated with extended overall survival in ovarian cancer. Furthermore, while baseline microhomology-associated deletion burden varies by tumor type, Pan-Cancer analysis identified candidate gene-level alterations, including FDX1 and PDE8B, whose LoF increased microhomology-associated deletion burden across tumors. Precise parameterization combined with WGS resolution highlights the extent to which specific gene losses affect deletion burden consistent with MMEJ-mediated repair. Identifying these genetic vulnerabilities and the resulting deletion burden may provide a candidate prognostic biomarker and foundation for personalized therapies.