Tissue-specific thresholds of mutation burden associated with anti-PD-1/L1 therapy benefit and prognosis in microsatellite-stable cancers.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 or its ligand (PD-1/L1) have expanded the treatment landscape against cancers but are effective in only a subset of patients. Tumor mutation burden (TMB) is postulated to be a generic determinant of ICI-dependent tumor rejection. Here we describe the association between TMB and survival outcomes among microsatellite-stable cancers in a real-world clinicogenomic cohort consisting of 70,698 patients distributed across 27 histologies. TMB was associated with survival benefit or detriment depending on tissue and treatment context, with eight cancer types demonstrating a specific association between TMB and improved outcomes upon treatment with anti-PD-1/L1 therapies. Survival benefits were noted over a broad range of TMB cutoffs across cancer types, and a dose-dependent relationship between TMB and outcomes was observed in a subset of cancers. These results have implications for the use of cancer-agnostic and universal TMB cutoffs to guide the use of anti-PD-1/L1 therapies, and they underline the importance of tissue context in the development of ICI biomarkers.

Natural Killer Cell Infiltration in Prostate Cancers Predict Improved Patient Outcomes.

BACKGROUND: Natural killer (NK) cells are non-antigen specific innate immune cells that can be redirected to targets of interest using multiple strategies, although none are currently FDA-approved. We sought to evaluate NK cell infiltration into tumors to develop an improved understanding of which histologies may be most amenable to NK cell-based therapies currently in the developmental pipeline. METHODS: DNA (targeted/whole-exome) and RNA (whole-transcriptome) sequencing was performed from tumors from 45 cancer types (N = 90,916 for all cancers and N = 3365 for prostate cancer) submitted to Caris Life Sciences. NK cell fractions and immune deconvolution were inferred from RNA-seq data using quanTIseq. Real-world overall survival (OS) and treatment status was determined and Kaplan-Meier estimates were calculated. Statistical significance was determined using X2 and Mann-Whitney U tests, with corrections for multiple comparisons where appropriate. RESULTS: In both a pan-tumor and prostate cancer (PCa) -specific setting, we demonstrated that NK cells represent a substantial proportion of the total cellular infiltrate (median range 2-9% for all tumors). Higher NK cell infiltration was associated with improved OS in 28 of 45 cancer types, including (PCa). NK cell infiltration was negatively correlated with common driver mutations and androgen receptor variants (AR-V7) in primary prostate biopsies, while positively correlated with negative immune regulators. Higher levels of NK cell infiltration were associated with patterns consistent with a compensatory anti-inflammatory response. CONCLUSIONS: Using the largest available dataset to date, we demonstrated that NK cells infiltrate a broad range of tumors, including both primary and metastatic PCa. NK cell infiltration is associated with improved PCa patient outcomes. This study demonstrates that NK cells are capable of trafficking to both primary and metastatic PCa and are a viable option for immunotherapy approaches moving forward. Future development of strategies to enhance tumor-infiltrating NK cell-mediated cytolytic activity and activation while limiting inhibitory pathways will be key.

Smoking Status and Survival in Patients With Early-Stage Primary Cutaneous Melanoma.

Importance: While smoking is associated with a decreased incidence of cutaneous melanoma, the association of smoking with melanoma progression and death is not well defined. Objective: To determine the association of smoking with survival in patients with early-stage primary cutaneous melanoma. Design, Setting, and Participants: This cohort study performed a post hoc analysis of data derived from the randomized, multinational first and second Multicenter Selective Lymphadenectomy Trials (MSLT-I and MSLT-II). Participants were accrued for MSLT-I from January 20, 1994, to March 29, 2002; MSLT-II, from December 21, 2004, to March 31, 2014. Median follow-up was 110.0 (IQR, 53.4-120.0) months for MSLT-I and 67.6 (IQR, 25.8-110.2) months for MSLT-II. Patients aged 18 to 75 years with clinical stages I or II melanoma with a Breslow thickness of 1.00 mm or greater or Clark level IV to V and available standard prognostic and smoking data were included. Analyses were performed from October 4, 2022, to March 31, 2023. Exposure: Current, former, and never smoking. Main Outcomes and Measures: Melanoma-specific survival of patients with current, former, and never smoking status was assessed for the entire cohort and for nodal observation and among subgroups with sentinel lymph node biopsy (SLNB)-negative and SLNB-positive findings. Results: Of 6279 included patients, 3635 (57.9%) were men, and mean (SD) age was 52.7 (13.4) years. The most common tumor location was an extremity (2743 [43.7%]), and mean (SD) Breslow thickness was 2.44 (2.06) mm. Smoking status included 1077 (17.2%) current, 1694 (27.0%) former, and 3508 (55.9%) never. Median follow-up was 78.4 (IQR, 30.5-119.6) months. Current smoking was associated with male sex, younger age, trunk site, thicker tumors, tumor ulceration, and SLNB positivity. Current smoking was associated with a greater risk of melanoma-associated death by multivariable analysis for the entire study (hazard ratio [HR], 1.48 [95% CI, 1.26-1.75]; P < .001). Former smoking was not. The increased risk of melanoma-specific mortality associated with current smoking was greatest for patients with SLNB-negative melanoma (HR, 1.85 [95% CI, 1.35-2.52]; P < .001), but also present for patients with SLNB-positive melanoma (HR, 1.29 [95% CI, 1.04-1.59]; P = .02) and nodal observation (HR, 1.68 [95% CI, 1.09-2.61]; P = .02). Smoking at least 20 cigarettes/d doubled the risk of death due to melanoma for patients with SLNB-negative disease (HR, 2.06 [95% CI, 1.36-3.13]; P < .001). Conclusions and Relevance: The findings of this cohort study suggest that patients with clinical stage I and II melanoma who smoked had a significantly increased risk of death due to melanoma. Smoking status should be assessed at time of melanoma diagnosis and may be considered a risk factor for disease progression.

MRI-derived radiomics assessing tumor-infiltrating macrophages enable prediction of immune-phenotype, immunotherapy response and survival in glioma.

BACKGROUND: The tumor immune microenvironment can influence the prognosis and treatment response to immunotherapy. We aimed to develop a non-invasive radiomic signature in high-grade glioma (HGG) to predict the absolute density of tumor-associated macrophages (TAMs), the preponderant immune cells in the microenvironment of HGG. We also aimed to evaluate the association between the signature, and tumor immune phenotype as well as response to immunotherapy. METHODS: In this retrospective setting, total of 379 patients with HGG from three independent cohorts were included to construct a radiomic model named Radiomics Immunological Biomarker (RIB) for predicting the absolute density of M2-like TAM using the mRMR feature ranking method and LASSO classifier. Among them, 145 patients from the TCGA microarray cohort were randomly allocated into a training set (N=101) and an internal validation set (N=44), while the immune-phenotype cohort (N=203) and the immunotherapy-treated cohort (N=31, patients from a prospective clinical trial treated with DC vaccine) recruited from Huashan Hospital were used as two external validation sets. The immunotherapy-treated cohort was also used to evaluate the relationship between RIB and immunotherapy response. Radiogenomic analysis was performed to find functional annotations using RNA sequencing data from TAM cells. RESULTS: An 11-feature radiomic model for M2-like TAM was developed and validated in four datasets of HGG patients (area under the curve = 0.849, 0.719, 0.674, and 0.671) using MRI images of post contrast enhanced T1-weighted (T1CE). Patients with high RIB scores had a strong inflammatory response. Four hub-genes (SLC7A7, RNASE6, HLA-DRB1 and CD300A) expressed by TAM were identified to be closely related to the RIB, providing important evidence for biological interpretation. Only individuals with a high RIB score were shown to have survival benefits from DC vaccine [DC vaccine vs. Placebo: median progression-free survival (mPFS), 10.0 mos vs. 4.5 mos, HR=0.17, P=0.0056, 95%CI=0.041-0.68; median overall survival (mOS), 15.0 mos vs. 7.0 mos, HR=0.17, P =0.0076, 95%CI=0.04-0.68]. Multivariate analyses also confirmed that treatment by DC vaccine was an independent factor for improved survival in the high RIB score group. However, in the low RIB score group, DC vaccine was not associated with improved survival. Furthermore, a radiomic nomogram based on the RIB score and clinical factors could efficiently predict the 1-, 2-, and 3-year survival rates, as confirmed by ROC curve analysis (AUC for 1-, 2- and 3-year survival: 0.705, 0.729 and 0.684, respectively). CONCLUSIONS: The radiomic model could allow for non-invasive assessment of the absolute density of TAM from MRI images in HGG patients. Of note, our RIB model is the first immunological radiomic model confirmed to have the ability to predict survival benefits from DC vaccine in gliomas, thereby providing a novel tool to inform treatment decisions and monitor patient treatment course by radiomics.

Pilot Study of High-Dose Pemetrexed in Patients with Progressive Chordoma.

PURPOSE: Chordomas are ultrarare tumors of the axial spine and skull-base without approved systemic therapy. Most chordomas have negative expression of thymidylate synthase (TS), suggesting a potential for responding to the antifolate agent pemetrexed, which inhibits TS and other enzymes involved in nucleotide biosynthesis. We evaluated the therapeutic activity and safety of high-dose pemetrexed in progressive chordoma. PATIENTS AND METHODS: Adult patients with previously treated, progressive chordoma participated in an open-label, single-institution, single-arm, pilot clinical trial of intravenous pemetrexed 900 mg/m2 every 3 weeks and supportive medications of folic acid, vitamin B12, and dexamethasone. The primary endpoint was objective response rate according to RECIST v1.1. Secondary endpoints included adverse events, progression-free survival (PFS), tumor molecular profiles, and alterations in tissue and blood-based biomarkers. RESULTS: Fifteen patients were enrolled and the median number of doses administered was 15 (range, 4-31). One patient discontinued treatment due to psychosocial issues after four cycles and one contracted COVID-19 after 13 cycles. Of the 14 response-evaluable patients, 2 (14%) achieved a partial response and 10 (71%) demonstrated stable disease. Median PFS was 10.5 months (95% confidence interval: 9 months-undetermined) and 6-month PFS was 67%. Adverse events were expected and relatively mild, with one grade 3 creatinine increased, and one each of grade 3 and 4 lymphopenia. No grade 5 adverse events, unexpected toxicities, or dose-limiting toxicities were observed. Several patients reported clinical improvement in disease-related symptoms. CONCLUSIONS: High-dose pemetrexed appears tolerable and shows objective antitumor activity in patients with chordoma. Phase II studies of high-dose pemetrexed are warranted.

Diagnostic miRNA Signatures in Paired Tumor, Plasma, and Urine Specimens From Renal Cell Carcinoma Patients.

BACKGROUND: The incidence of patients diagnosed with renal cell carcinoma (RCC) is increasing. There are no approved biofluid biomarkers for routine diagnosis of RCC patients. This retrospective study aims to identify cell-free microRNA (cfmiR) signatures in urine samples that can be utilized as biomarkers for early diagnosis of sporadic RCC patients. METHODS: Tissue, plasma, and urine samples (n = 221) from 56 sporadic RCC patients and respective normal healthy donors were profiled for 2083 microRNAs (miRs) using the next-generation sequencing-based HTG EdgeSeq miR Whole Transcriptome Assay. DESeq2 (FC |1.2|, false discovery rate <0.05) was performed to identify differentially expressed miRs. Data from RCC tissue samples of The Cancer Genome Atlas database were used for miR validation. RESULTS: We found a 10-miR signature that distinguished RCC tissues from remote normal kidney tissue or benign kidney lesion samples. Additionally, we identified subtype-specific miRs (miR-122-5p, miR-210-3p, and miR-21-3p) and miRs specific for all RCC subtypes (miR-106b-3p, miR-629-5p, and miR-885-5p). We observed that miR-155-5p was associated with tumor size. Using The Cancer Genome Atlas data sets, we validated the miRs found in RCC tissue samples. In plasma or urine analysis, we found cfmiRs that were consistently and significantly upregulated in RCC tissue samples. A 15-cfmiR signature was proposed in urine samples of RCC patients, of which miR-1275 was consistently upregulated in tissue, plasma, and urine samples. CONCLUSIONS: This integrative study found diagnostic miRs/cfmiRs for RCC patients, which were validated using The Cancer Genome Atlas data sets. Distinctive cfmiR signatures found in urine may have clinical utility for the diagnosis of RCC.

MiR-181a targets STING to drive PARP inhibitor resistance in BRCA- mutated triple-negative breast cancer and ovarian cancer.

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors (PARPi) are approved for the treatment of BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC) and ovarian cancer (OvCa). A key challenge is to identify the factors associated with PARPi resistance; although, previous studies suggest that platinum-based agents and PARPi share similar resistance mechanisms. METHODS: Olaparib-resistant (OlaR) cell lines were analyzed using HTG EdgeSeq miRNA Whole Transcriptomic Analysis (WTA). Functional assays were performed in three BRCA-mutated TNBC cell lines. In-silico analysis were performed using multiple databases including The Cancer Genome Atlas, the Genotype-Tissue Expression, The Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Gene Omnibus Expression. RESULTS: High miR-181a levels were identified in OlaR TNBC cell lines (p = 0.001) as well as in tumor tissues from TNBC patients (p = 0.001). We hypothesized that miR-181a downregulates the stimulator of interferon genes (STING) and the downstream proinflammatory cytokines to mediate PARPi resistance. BRCA1 mutated TNBC cell lines with miR-181a-overexpression were more resistant to olaparib and showed downregulation in STING and the downstream genes controlled by STING. Extracellular vesicles derived from PARPi-resistant TNBC cell lines horizontally transferred miR-181a to parental cells which conferred PARPi-resistance and targeted STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p = 0.01). In addition, low IFNG response scores were associated with worse response to neoadjuvant treatment including PARPi for high-risk HER2 negative BC patients (p = 0.001). OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to platinum showed resistance to olaparib. Knockout of miR-181a significantly improved olaparib sensitivity in OvCa cell lines (p = 0.001). CONCLUSION: miR-181a is a key factor controlling the STING pathway and driving PARPi and platinum-based drug resistance in TNBC and OvCa. The miR-181a-STING axis can be used as a potential marker for predicting PARPi responses in TNBC and OvCa tumors.

Multi-omic profiling reveals discrepant immunogenic properties and a unique tumor microenvironment among melanoma brain metastases.

Melanoma brain metastases (MBM) are clinically challenging to treat and exhibit variable responses to immune checkpoint therapies. Prior research suggests that MBM exhibit poor tumor immune responses and are enriched in oxidative phosphorylation. Here, we report results from a multi-omic analysis of a large, real-world melanoma cohort. MBM exhibited lower interferon-gamma (IFNγ) scores and T cell-inflamed scores compared to primary cutaneous melanoma (PCM) or extracranial metastases (ECM), which was independent of tumor mutational burden. Among MBM, there were fewer computationally inferred immune cell infiltrates, which correlated with lower TNF and IL12B mRNA levels. Ingenuity pathway analysis (IPA) revealed suppression of inflammatory responses and dendritic cell maturation pathways. MBM also demonstrated a higher frequency of pathogenic PTEN mutations and angiogenic signaling. Oxidative phosphorylation (OXPHOS) was enriched in MBM and negatively correlated with NK cell and B cell-associated transcriptomic signatures. Modulating metabolic or angiogenic pathways in MBM may improve responses to immunotherapy in this difficult-to-treat patient subset.

Spatial profiling of cancer-associated fibroblasts of sporadic early onset colon cancer microenvironment.

The incidence of sporadic early-onset colon cancer (EOCC) has increased worldwide. The molecular mechanisms in the tumor and the tumor microenvironment (TME) in EOCC are not fully understood. The aim of this study is to unravel unique spatial transcriptomic and proteomic profiles in tumor epithelial cells and cancer-associated fibroblasts (CAFs). Here, we divide the sporadic colon cancer tissue samples with transcriptomic data into patients diagnosed with EOCC (<50 yrs) and late-onset colon cancer (LOCC, ≥50 yrs) and then, analyze the data using CIBERSORTx deconvolution software. EOCC tumors are more enriched in CAFs with fibroblast associated protein positive expression (FAP(+)) than LOCC tumors. EOCC patients with higher FAP mRNA levels in CAFs have shorter OS (Log-rank test, p < 0.029). Spatial transcriptomic analysis of 112 areas of interest, using NanoString GeoMx digital spatial profiling, demonstrate that FAP(+) CAFs at the EOCC tumor invasive margin show a significant upregulation of WNT signaling and higher mRNA/protein levels of fibroblast growth factor 20 (FGF20). Tumor epithelial cells at tumor invasive margin of EOCC tumors neighboring FAP(+) CAFs show significantly higher mRNA/protein levels of fibroblast growth factor receptor (FGFR2) and PI3K/Akt signaling activation. NichNET analysis show a potential interaction between FGF20 and FGFFR2. The role of FGF20 in activating FGFR2/pFGFR2 and AKT/pAKT was validated in-vitro. In conclusion, we identify a unique FAP(+) CAF population that showed WNT signaling upregulation and increased FGF20 levels; while neighbor tumor cells show the upregulation/activation of FGFR2-PI3K/Akt signaling at the tumor invasive margin of EOCC tumors.

Heterogeneity in the Metastatic Microenvironment: JunB-Expressing Microglia Cells as Potential Drivers of Melanoma Brain Metastasis Progression.

Reciprocal signaling between melanoma brain metastatic (MBM) cells and microglia reprograms the phenotype of both interaction partners, including upregulation of the transcription factor JunB in microglia. Here, we aimed to elucidate the impact of microglial JunB upregulation on MBM progression. For molecular profiling, we employed RNA-seq and reverse-phase protein array (RPPA). To test microglial JunB functions, we generated microglia variants stably overexpressing JunB (JunBhi) or with downregulated levels of JunB (JunBlo). Melanoma-derived factors, namely leukemia inhibitory factor (LIF), controlled JunB upregulation through Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling. The expression levels of JunB in melanoma-associated microglia were heterogeneous. Flow cytometry analysis revealed the existence of basal-level JunB-expressing microglia alongside microglia highly expressing JunB. Proteomic profiling revealed a differential protein expression in JunBhi and JunBlo cells, namely the expression of microglia activation markers Iba-1 and CD150, and the immunosuppressive molecules SOCS3 and PD-L1. Functionally, JunBhi microglia displayed decreased migratory capacity and phagocytic activity. JunBlo microglia reduced melanoma proliferation and migration, while JunBhi microglia preserved the ability of melanoma cells to proliferate in three-dimensional co-cultures, that was abrogated by targeting leukemia inhibitory factor receptor (LIFR) in control microglia-melanoma spheroids. Altogether, these data highlight a melanoma-mediated heterogenous effect on microglial JunB expression, dictating the nature of their functional involvement in MBM progression. Targeting microglia highly expressing JunB may potentially be utilized for MBM theranostics.

Inhibition of Microsomal Prostaglandin E2 Synthase Reduces Collagen Deposition in Melanoma Tumors and May Improve Immunotherapy Efficacy by Reducing T-cell Exhaustion.

The arachidonic acid pathway participates in immunosuppression in various types of cancer. Our previous observation detailed that microsomal prostaglandin E2 synthase 1 (mPGES-1), an enzyme downstream of cyclooxygenase 2 (COX-2), limited antitumor immunity in melanoma; in addition, genetic depletion of mPGES-1 specifically enhanced immune checkpoint blockade therapy. The current study set out to distinguish the roles of mPGES-1 from those of COX-2 in tumor immunity and determine the potential of mPGES-1 inhibitors for reinforcing immunotherapy in melanoma. Genetic deletion of mPGES-1 showed different profiles of prostaglandin metabolites from that of COX-2 deletion. In our syngeneic mouse model, mPGES-1-deficient cells exhibited similar tumorigenicity to that of COX-2-deficient cells, despite a lower ability to suppress PGE2 synthesis by mPGES-1 depletion, indicating the presence of factors other than PGE2 that are likely to regulate tumor immunity. RNA-sequencing analysis revealed that mPGES-1 depletion reduced the expressions of collagen-related genes, which have been found to be associated with immunosuppressive signatures. In our mouse model, collagen was reduced in mPGES-1-deficient tumors, and phenotypic analysis of tumor-infiltrating lymphocytes indicated that mPGES-1-deficient tumors had fewer TIM3+ exhausted CD8+ T cells compared with COX-2-deficient tumors. CAY10678, an mPGES-1 inhibitor, was equivalent to celecoxib, a selective COX-2 inhibitor, in reinforcing anti-PD-1 treatment. Our study indicates that mPGES-1 inhibitors represent a promising adjuvant for immunotherapies in melanoma by reducing collagen deposition and T-cell exhaustion. Significance: Collagen is a predominant component of the extracellular matrix that may influence the tumor immune microenvironment for cancer progression. We present here that mPGES-1 has specific roles in regulating tumor immunity, associated with several collagen-related genes and propose that pharmacologic inhibition of mPGES-1 may hold therapeutic promise for improving immune checkpoint-based therapies.

Characterization of Lymph Node Tumor Burden in Node-Positive Prostate Cancer Patients after Robotic-Assisted Radical Prostatectomy with Extended Pelvic Lymph Node Dissection.

BACKGROUND: Prostate cancer (PCa) nodal staging does not account for lymph node (LN) tumor burden. The LN anatomical compartment involved with the tumor or the quantified extent of extranodal extension (ENE) have not yet been studied in relation to biochemical recurrence-free survival (BRFS). METHODS: Histopathological slides of 66 pN1 PCa patients who underwent extended pelvic lymph node dissection were reviewed. We recorded metrics to quantify LN tumor burden. We also characterized the LN anatomical compartments involved and quantified the extent of ENE. RESULTS: The median follow-up time was 38 months. The median number of total LNs obtained per patient was 30 (IQR 23-37). In the risk-adjusted cox regression model, the following variables were associated with BRFS: mean size of the largest LN deposit per patient (log2: adjusted hazard ratio (aHR) = 1.91, p < 0.001), the mean total span of all LN deposits per patient (2.07, p < 0.001), and the mean percent surface area of the LN involved with the tumor (1.58, p < 0.001). There was no significant BRFS association for the LN anatomical compartment or the quantified extent of ENE. CONCLUSION: LN tumor burden is associated with BRFS. The LN anatomical compartments and the quantified extent of ENE did not show significant association with BRFS.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

Multiplatform Analysis of Intratumoral PTEN Heterogeneity in Melanoma.

Loss of protein expression of the tumor suppressor PTEN is associated with increased cancer aggressiveness, decreased tumor immune infiltration, and resistance to immune and targeted therapies in melanoma. We assessed a unique cohort of eight melanoma samples with focal loss of PTEN protein expression to understand the features and mechanisms of PTEN loss in this disease. We compared the PTEN-negative (PTEN[-]) areas to their adjacent PTEN-positive (PTEN[+]) areas using DNA sequencing, DNA methylation, RNA expression, digital spatial profiling, and immunohistochemical platforms. Variations or homozygous deletions of PTEN were identified in PTEN(-) areas that were not detected in the adjacent PTEN(+) areas in three cases (37.5%), but no clear genomic or DNA methylation basis for loss was identified in the remaining PTEN(-) samples. RNA expression data from two independent platforms identified a consistent increase in chromosome segregation gene expression in PTEN(-) versus adjacent PTEN(+) areas. Proteomic analysis showed a relative paucity of tumor-infiltrating lymphocytes in PTEN(-) versus adjacent PTEN(+) areas. The findings add to our understanding of potential molecular intratumoral heterogeneity in melanoma and the features associated with the loss of PTEN protein in this disease.

Genomic Amplification of UBQLN4 Is a Prognostic and Treatment Resistance Factor.

Ubiquilin-4 (UBQLN4) is a proteasomal shuttle factor that directly binds to ubiquitylated proteins and delivers its cargo to the 26S proteasome for degradation. We previously showed that upregulated UBQLN4 determines the DNA damage response (DDR) through the degradation of MRE11A. However, the regulatory mechanism at DNA level, transcriptionally and post-transcriptional levels that control UBQLN4 mRNA levels remains unknown. In this study, we screened 32 solid tumor types and validated our findings by immunohistochemistry analysis. UBQLN4 is upregulated at both mRNA and protein levels and the most significant values were observed in liver, breast, ovarian, lung, and esophageal cancers. Patients with high UBQLN4 mRNA levels had significantly poor prognoses in 20 of 32 cancer types. DNA amplification was identified as the main mechanism promoting UBQLN4 upregulation in multiple cancers, even in the early phases of tumor development. Using CRISPR screen datasets, UBQLN4 was identified as a common essential gene for tumor cell viability in 81.1% (860/1,060) of the solid tumor derived cell lines. Ovarian cancer cell lines with high UBQLN4 mRNA levels were platinum-based chemotherapy resistant, while they were more sensitive to poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Our findings highlight the utilities of UBQLN4 as a significant pan-cancer theranostic factor and a precision oncology biomarker for DDR-related drug resistance.

Verification of a Novel Minimally Invasive Device for the Isolation of Rare Circulating Tumor Cells (CTC) in Cancer Patients' Blood.

Circulating tumor cells (CTCs) exist in low quantities in the bloodstream in the early stages of cancers. It, therefore, remains a technical challenge to isolate them in large enough quantities for a precise diagnosis and downstream analysis. We introduce the BMProbe™, a minimally invasive device that isolates CTCs during a 30-minute incubation in the median cubital vein. The optimized geometry of the device creates flow conditions for improved cell deposition. The CTCs are isolated using antibodies that are bound to the surface of the BMProbe™. In this study, flow experiments using cell culture cells were conducted. They indicate a 31 times greater cell binding efficiency of the BMProbe™ compared to a flat geometry. Further, the functionality of isolating CTCs from patient blood was verified in a small ex vivo study that compared the cell count from seven non-small-cell lung carcinoma (NSCLC) patients compared to nine healthy controls with 10 mL blood samples. The median cell count was 1 in NSCLC patients and 0 in healthy controls. In conclusion, the BMProbe™ is a promising method to isolate CTCs in large quantities directly from the venous bloodstream without removing blood from a patient. The future step is to verify the functionality in vivo.

DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy.

The hypoxic tumor microenvironment has been implicated in immune escape, but the underlying mechanism remains elusive. Using an in vitro culture system modeling human T cell dysfunction and exhaustion in triple-negative breast cancer (TNBC), we find that hypoxia suppresses immune effector gene expression, including in T and NK cells, resulting in immune effector cell dysfunction and resistance to immunotherapy. We demonstrate that hypoxia-induced factor 1α (HIF1α) interaction with HDAC1 and concurrent PRC2 dependency causes chromatin remolding resulting in epigenetic suppression of effector genes and subsequent immune dysfunction. Targeting HIF1α and the associated epigenetic machinery can reverse the immune effector dysfunction and overcome resistance to PD-1 blockade, as demonstrated both in vitro and in vivo using syngeneic and humanized mice models. These findings identify a HIF1α-mediated epigenetic mechanism in immune dysfunction and provide a potential strategy to overcome immune resistance in TNBC.