Validation of a microRNA liquid biopsy assay for diagnosis and risk stratification of invasive cutaneous melanoma.

BACKGROUND: Noninvasive molecular biomarkers are needed to improve the early, accurate and precise diagnosis of invasive cutaneous melanoma. OBJECTIVES: To independently validate a previously identified circulating microRNA signature of melanoma (MEL38), and, secondly, to develop a complementary microRNA signature, optimized for prognostication. PATIENTS AND METHODS: MicroRNA expression profiling was performed on plasma samples from a multicentre observational case-control study, involving patients with primary or metastatic melanoma, melanoma in situ, nonmelanoma skin cancer, or benign naevi. MicroRNA profiles from patients with length of survival, treatment and sentinel lymph node biopsy (SLNB) data were used to develop the prognostic signature. The primary outcome of interest for MEL38 was its association with melanoma status, including area under the curve, binary diagnostic sensitivity and specificity, and incidence-adjusted positive and negative predictive values. The prognostic signature was assessed using rates of survival per risk group and relationship to conventional predictors of outcome. RESULTS: Circulating microRNA profiles of 372 patients with invasive melanoma and 210 control individuals were generated. The average age of all participants was 59 years; 49% were male. A MEL38 score > 5.5 indicated the presence of invasive melanoma. Overall, 551/582 (95%) of patients were correctly diagnosed, with 93% sensitivity and 98% specificity. MEL38 score ranged from 0 to 10 with an area under the curve of 0.98 (95% confidence interval 0.97-0.99, P < 0.001). A novel prognostic 12-microRNA signature (MEL12) developed from 232 patients identified low-, standard- or high-risk groups, with 94%, 78% and 58% rates of 10-year melanoma-specific survival, respectively (log-rank P < 0.001). MEL12 prognostic risk groups were significantly associated with clinical staging (χ2, P < 0.001) and SLNB status (P = 0.027). Patients who were classified as high risk by MEL12 were approximately three times more likely to have melanoma detected in their sentinel lymph nodes compared to low-risk patients. CONCLUSIONS: The circulating MEL38 signature may assist in diagnosing patients with invasive melanoma vs. other conditions associated with a lower - or negligible - risk of mortality. A complementary and prognostic MEL12 signature is predictive of SLNB status, clinical stage and probability of survival. Plasma microRNA profiling may help to optimize existing diagnostic pathways as well as enable personalized, risk-informed melanoma treatment decisions.

The contribution of muscle spindles to position sense measured with three different methods.

The sense of limb position is important, because it is believed to contribute to our sense of self-awareness. Muscle spindles, including both primary and secondary endings of spindles, are thought to be the principal position sensors. Passive spindles possess a property called thixotropy which allows their sensitivity to be manipulated. Here, thixotropic patterns of position errors have been studied with three commonly used methods of measurement of position sense. The patterns of errors have been used as indicators of the influence exerted by muscle spindles on a measured value of position sense. In two-arm matching, the blindfolded participant indicates the location of one arm by placement of the other. In one-arm pointing, the participant points to the perceived position of their other, hidden arm. In repositioning, one of the blindfolded participant's arms is placed at a chosen angle and they are asked to remember its position and then, after a delay, reproduce the position. The three methods were studied over the full range of elbow angles between 5° (elbow extension) and 125° (elbow flexion). Different outcomes were achieved with each method; in two-arm matching, position errors were symmetrical about zero and thixotropic influences were large, while in one-arm pointing, errors were biased towards extension. In repositioning, thixotropic effects were small. We conclude that each of the methods of measuring position sense comprises different mixes of peripheral and central influences. This will have to be taken into consideration by the clinician diagnosing disturbances in position sense.

Observation of Collagen-Containing Lesions After Hematoma Resolution in Intracerebral Hemorrhage.

Background and Purpose: The classic presentation of chronic (stage III) hemorrhagic stroke lesions is a fluid-filled cavity. In one of the most commonly used animal models of intracerebral hemorrhage (ICH), we noticed additional solid material within the chronic lesion. We examined the composition of those chronic ICH lesions and compared them with human autopsy cases. Methods: ICH was induced in rats by the injection of collagenase in the striatum. Tissue sections after hematoma resolution corresponding to 3 different chronic time points­28, 42, and 73 to 85 days post-ICH­were selected. Human autopsy reports at the University Hospital of Zurich were searched between 1990 and 2019 for ICH, and 3 chronic cases were found. The rat and human sections were stained with a variety of histopathologic markers. Results: Extensive collagenous material was observed in the chronic lesion after hematoma resolution in both the rat model and human autopsy cases. Additional immunostaining revealed that the material consisted primarily of a loose network of collagen 3 intermingled with occasional GFAP (glial fibrillary acidic protein)-positive processes and collagen 4. Conclusions: A key feature of the chronic ICH lesion is a loose network of collagen 3. The collagenase rat model reproduces the morphology and composition of the chronic human ICH lesion. While identifying new features of ICH lesion pathology, these results are important for treatment and recovery strategies.

Colorectal Cancer Cell Line Proteomes Are Representative of Primary Tumors and Predict Drug Sensitivity.

BACKGROUND AND AIMS: Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. METHODS: Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. RESULTS: Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The 5 proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling, and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared with genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. CONCLUSIONS: Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared with genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine.

Quality of Life, Psychological Functioning, and Treatment Satisfaction of Men Who Have Undergone Penile Prosthesis Surgery Following Robot-Assisted Radical Prostatectomy.

BACKGROUND: Penile prosthesis surgery is last-line treatment to regaining erectile function after radical prostatectomy (RP) for localized prostate cancer. AIMS: To assess quality of life, psychological functioning, and treatment satisfaction of men who underwent penile implantation after RP; the psychosocial correlates of treatment satisfaction and sexual function after surgery; and the relation between patients' and partners' ratings of treatment satisfaction. METHODS: 98 consecutive patients who underwent penile implantation after RP from 2010 and 2015 and their partners were invited to complete a series of measures at a single time point. Of these, 71 patients and 43 partners completed measures assessing sexual function, psychological functioning, and treatment satisfaction. Proportions of patients who demonstrated good sexual function and satisfaction with treatment and clinical levels of anxiety and depression were calculated. Hierarchical regression analyses were conducted to determine psychosocial factors associated with patient treatment satisfaction and sexual function and patient-partner differences in treatment satisfaction. OUTCOMES: Patients completed the Expanded Prostate Cancer Index Composite Short Form (EPIC-26), Erectile Dysfunction Inventory of Treatment Satisfaction (EDITS), Prostate Cancer-Related Quality of Life Scale, Self-Esteem and Relationship Questionnaire (SEAR), Generalized Anxiety Disorder-7 (GAD-7), and Patient Health Questionnaire-9 (PHQ-9). Partners completed the GAD-7, PHQ-9, EDITS (partner version), and SEAR. RESULTS: 94% of men reported satisfaction with treatment (EDITS score > 50). 77% of men reported good sexual function (EPIC-26 score > 60). Lower depression scores were associated with higher sexual confidence and sexual intimacy, and these were correlated with better treatment satisfaction and sexual function. Patients experienced higher sexual relationship satisfaction (median score = 90.6) than their partners (median score = 81.2), but there was no difference in treatment satisfaction between groups. Higher patient treatment satisfaction was more likely to be reported for couples whose depression scores were more similar. CLINICAL IMPLICATIONS: It is important to provide preoperative penile implant counseling and encourage patients to seek postoperative counseling if needed. STRENGTHS AND LIMITATIONS: This is one of the first Australian-based studies comprehensively assessing treatment satisfaction and psychosocial health of men after penile prosthesis surgery after RP. This was a retrospective cross-sectional study, so there is a possibility of recall bias, and causal associations could not be determined. CONCLUSION: Men in this Australian series who underwent penile prosthesis surgery after RP generally reported good sexual function and treatment satisfaction. Nevertheless, patient and partner mental health influenced their reported experience of the treatment. Pillay B, Moon D, Love C, et al. Quality of Life, Psychological Functioning, and Treatment Satisfaction of Men Who Have Undergone Penile Prosthesis Surgery Following Robot-Assisted Radical Prostatectomy. J Sex Med 2017;14:1612-1620.

Much more than prescribing a pill - Assessment and treatment of erectile dysfunction by the general practitioner.

BACKGROUND: Erectile dysfunction is a common but often neglected condition. Prevalence increases with age, but is not insignificant in younger men. OBJECTIVE: This article will broadly describe the epidemiology, classification and risk factors of erectile dysfunction. It will also discuss assessment and current treatment modalities, with a particular focus on the unique role of the general practitioner (GP). DISCUSSION: Erectile dysfunction may be classified as vasculogenic, neurogenic, endocrinological, drug-related, psychogenic or mixed. Commonly, erectile dysfunction is a cause of anxiety and even depression. Risk factors, such as smoking and hypertension, and reversible causes, such as hypogonadism or offending medications, should be addressed. At present, oral pharmacotherapy represents the first-line option for most patients with erectile dysfunction. It is of utmost importance to evaluate and treat comorbidities, such as depression, metabolic syndrome and cardiovascular disease, that often accompany erectile dysfunction. Patients will undoubtedly benefit from comprehensive management by a dedicated GP. Occasionally, referral to a urologist, psychologist or sexual health physician may be required.

Adult male stress and urge urinary incontinence - A review of pathophysiology and treatment strategies for voiding dysfunction in men.

BACKGROUND: Male urinary incontinence adversely affects health-related quality of life and is associated with significant psychosexual and financial burden. The two most common forms of male incontinence are stress urinary incontinence (SUI) and overactive bladder (OAB) with concomitant urge urinary incontinence (UUI). OBJECTIVE: The objectives of this article are to briefly review the current understandings of the pathophysiological mechanisms in SUI and OAB/UUI, and offer a set of practical, action-based recommendations and treatment strategies. DISCUSSION: The initial evaluation of male urinary incontinence usually occurs in general practice, and the basic work-up aims to identify reversible causes. First-line treatment is conservative management, such as lifestyle interventions, pelvic floor muscle training with or without biofeedback, and bladder retraining. Treatment options include male slings and artificial urinary sphincter surgery for men with persistent SUI, and medical therapy, intravesical botulinum toxin, sacral neuromodulation or surgery in refractory cases for those with predominant OAB/UUI.

Urolithiasis - Ten things every general practitioner should know.

BACKGROUND: Upper urinary tract stones are a common problem in Australia, with an incidence of 0.13% per year, and a lifetime prevalence of up to 15% in males and 8% in females. Many of these patients first present to general practitioners (GPs), so a thorough understanding of the diagnosis, treatment and prevention of stone disease is an important part of any GP's arsenal. OBJECTIVE: In this article, we present evidence-based guidelines regarding urolithiasis, from diagnosis, through to conservative and operative management, and prevention, as a reference for GPs and other primary care physicians. DISCUSSION: The majority of urolithiasis cases can be conservatively managed. However, prior to conservative management, adequate imaging must be obtained and emergent conditions must be excluded. Conservative management should not be initiated without a plan in the event the management fails, and adequate analgesia and medical expulsive therapy should be prescribed. Should surgery be necessary, the majority of operations can be performed as minimally invasive day procedures.

Peyronie's disease - Watch out for the bend.

BACKGROUND: Peyronie's disease is a relatively common condition in urological practice, but is still poorly identified and understood in the wider medical community and by most of the public. Identifying the condition and appropriate referral for expert opinion can significantly lessen the physical and psychological effect on patients. OBJECTIVE: The objective of this article is to provide general practitioners with a concise and updated review of Peyronie's disease, with the aim of helping them to provide appropriate advice to their patients. DISCUSSION: Peyronie's disease is an aberrant wound healing process culminating in excess scar formation in the penis, which may cause penile pain, shortening and curvature. It is often accompanied by erectile dysfunction, and can result in progressive and severe impairment of penetrative intercourse. The course of the disorder is divided into active inflammatory and chronic stable phases. Oral therapy is usually of limited efficacy, while penile traction may only be beneficial in motivated patients. Intralesional injections of collagenase were recently introduced as a non-surgical measure to decrease penile curvature. Surgery remains the most effective treatment for Peyronie's disease and is considered the gold standard.

The discovery and preclinical characterization of 6-chloro-N-(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists.

The synthesis, SAR and preclinical characterization of a series of 6-chloro-N-(2-(4,4-difluoropiperidin-1-yl)-2-(2-(trifluoromethyl)pyrimidin-5-yl)ethyl)quinoline-5-carboxamide based P2X7 antagonists is described herein. The lead compounds are potent inhibitors in Ca(2+) flux and whole blood IL-1ß P2X7 release assays at both human and mouse isoforms. Compound 1e showed a robust reduction of IL-1ß release in a mouse ex vivo model with a 50mg/kg oral dose. Evaluation of compound 1e in the mouse SNI tactile allodynia, carrageenan-induced paw edema or CIA models resulted in no analgesic or anti-inflammatory effects.

Wild-type APC predicts poor prognosis in microsatellite-stable proximal colon cancer.

BACKGROUND: APC mutations (APC-mt) occur in ∼70% of colorectal cancers (CRCs), but their relationship to prognosis is unclear. METHODS: APC prognostic value was evaluated in 746 stage I-IV CRC patients, stratifying for tumour location and microsatellite instability (MSI). Microarrays were used to identify a gene signature that could classify APC mutation status, and classifier ability to predict prognosis was examined in an independent cohort. RESULTS: Wild-type APC microsatellite stable (APC-wt/MSS) tumours from the proximal colon showed poorer overall and recurrence-free survival (OS, RFS) than APC-mt/MSS proximal, APC-wt/MSS distal and APC-mt/MSS distal tumours (OS HR⩾1.79, P⩽0.015; RFS HR⩾1.88, P⩽0.026). APC was a stronger prognostic indicator than BRAF, KRAS, PIK3CA, TP53, CpG island methylator phenotype or chromosomal instability status (P⩽0.036). Microarray analysis similarly revealed poorer survival in MSS proximal cancers with an APC-wt-like signature (P=0.019). APC status did not affect outcomes in MSI tumours. In a validation on 206 patients with proximal colon cancer, APC-wt-like signature MSS cases showed poorer survival than APC-mt-like signature MSS or MSI cases (OS HR⩾2.50, P⩽0.010; RFS HR⩾2.14, P⩽0.025). Poor prognosis APC-wt/MSS proximal tumours exhibited features of the sessile serrated neoplasia pathway (P⩽0.016). CONCLUSIONS: APC-wt status is a marker of poor prognosis in MSS proximal colon cancer.

Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome.

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3;1,4)-ß-d-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Survival From Revision Surgery for New Rigicon Infla10 Three-piece Inflatable Penile Prosthesis Is Comparable to Preceding Devices.

OBJECTIVE: To report the incidence of the reoperation surgeries of nearly all the Rigicon Infla10 implants performed since device introduction in 1/2019. Inflatable penile prosthesis has some of the highest survival from revision surgery of any medical device implanted in humans [1]. We expand on previous Rigicon Infla10 research, adding more patients and increasing follow-up duration [2]. MATERIALS AND METHODS: 535 patients had Rigicon Infla10 devices implanted from 1/2019 to 8/2022. 103 surgeons from 26 centers in 15 countries participated in the study. Patient information forms were analyzed from virtually all implantations. Explantation or revision surgery for mechanical failure, infection, other medical reasons, and patient dissatisfaction were cataloged. SPSS 25.0 (IBM) was used for the statistical analysis of Kaplan Meier survival statistics. RESULTS: Mean follow-up was 24.2months (7-43months). Mean patient age was 56years. Reoperation was necessary for 3.5% of subjects. Revision for mechanical failure occurred in 2.24% (12/535). The rate of explant for patient dissatisfaction was 0.56% (3/535). Revision for component out of place was 0.37% (2/535) with an infection rate and unsuccessful Peyronie's correction being 0.19% (1/535). Survival from requiring another corrective surgery at 1, 2, and 3years was 96.4%, 95.0%, and 94.0%, respectively. These initial survival rates compare favorably to devices currently available, which have been repeatedly enhanced to improve reliability. CONCLUSION: In its first 2-3years of availability, The Rigicon Infla10 inflatable penile prosthesis shows freedom from revision comparable to existing enhanced devices that have been on the market for decades.

Regulatory hotspots are associated with plant gene expression under varying soil phosphorus supply in Brassica rapa.

Gene expression is a quantitative trait that can be mapped genetically in structured populations to identify expression quantitative trait loci (eQTL). Genes and regulatory networks underlying complex traits can subsequently be inferred. Using a recently released genome sequence, we have defined cis- and trans-eQTL and their environmental response to low phosphorus (P) availability within a complex plant genome and found hotspots of trans-eQTL within the genome. Interval mapping, using P supply as a covariate, revealed 18,876 eQTL. trans-eQTL hotspots occurred on chromosomes A06 and A01 within Brassica rapa; these were enriched with P metabolism-related Gene Ontology terms (A06) as well as chloroplast- and photosynthesis-related terms (A01). We have also attributed heritability components to measures of gene expression across environments, allowing the identification of novel gene expression markers and gene expression changes associated with low P availability. Informative gene expression markers were used to map eQTL and P use efficiency-related QTL. Genes responsive to P supply had large environmental and heritable variance components. Regulatory loci and genes associated with P use efficiency identified through eQTL analysis are potential targets for further characterization and may have potential for crop improvement.

InterStoreDB: a generic integration resource for genetic and genomic data.

Associating phenotypic traits and quantitative trait loci (QTL) to causative regions of the underlying genome is a key goal in agricultural research. InterStoreDB is a suite of integrated databases designed to assist in this process. The individual databases are species independent and generic in design, providing access to curated datasets relating to plant populations, phenotypic traits, genetic maps, marker loci and QTL, with links to functional gene annotation and genomic sequence data. Each component database provides access to associated metadata, including data provenance and parameters used in analyses, thus providing users with information to evaluate the relative worth of any associations identified. The databases include CropStoreDB, for management of population, genetic map, QTL and trait measurement data, SeqStoreDB for sequence-related data and AlignStoreDB, which stores sequence alignment information, and allows navigation between genetic and genomic datasets. Genetic maps are visualized and compared using the CMAP tool, and functional annotation from sequenced genomes is provided via an EnsEMBL-based genome browser. This framework facilitates navigation of the multiple biological domains involved in genetics and genomics research in a transparent manner within a single portal. We demonstrate the value of InterStoreDB as a tool for Brassica research. InterStoreDB is available from: http://www.interstoredb.org.

Epidemiology and Outcomes of Patients With Healthcare Facility-Onset Clostridioides difficile Infection.

BACKGROUND: Clostridioides difficile infection (CDI) has become a rising public health threat. Our study aims to characterize the epidemiology and measure the attributable cost, length of stay, and in-hospital mortality of healthcare facility-onset Clostridioides difficile infection (HO-CDI) among patients in the U.S. Military Health System (MHS). METHODS: We performed a case-control and cross-sectional inpatient study of HO-CDI using MHS database billing records. Cases included those who were at least 18 years of age admitted to a military treatment facility with a stool sample positive for C. difficile obtained >3 days after admission. Risk factors in the preceding year were identified. Patient case-mix adjusted outcomes including in-hospital mortality, length of stay, and hospitalization cost were evaluated by high-dimensional propensity score adjusted logistic regression. RESULTS: Among 474,518 admissions within the MHS from 2008 to 2015, we identified 591 (0.12%) patients with HO-CDI and found a significant increase in the trend of HO-CDI over the 7-year study period (P < .001). Patients with HO-CDI had significantly higher hospitalization cost (attributable difference $66,044, P < .001), prolonged hospital stay (attributable difference 12.4 days, P < 0.001), and increased odds of in-hospital mortality (case-mix adjusted odds ratio 1.98; 95% CI, 1.43-2.74). CONCLUSIONS: Healthcare facility-onset Clostridioides difficile infection is rising in patients within the MHS and is associated with increased length of stay, hospital costs, and in-hospital mortality. We identified a significantly increased burden of hospitalization among patients admitted with HO-CDI, highlighting the importance of infection control and antimicrobial stewardship initiatives aimed at decreasing the spread of this pathogen.

Findings from precision oncology in the clinic: rare, novel variants are a significant contributor to scaling molecular diagnostics.

BACKGROUND: Next generation sequencing for oncology patient management is now routine in clinical pathology laboratories. Although wet lab, sequencing and pipeline tasks are largely automated, the analysis of variants for clinical reporting remains largely a manual task. The increasing volume of sequencing data and the limited availability of genetic experts to analyse and report on variants in the data is a key scalability limit for molecular diagnostics. METHOD: To determine the impact and size of the issue, we examined the longitudinally compiled genetic variants from 48,036 cancer patients over a six year period in a large cancer hospital from ten targeted cancer panel tests in germline, solid tumour and haematology contexts using hybridization capture and amplicon assays. This testing generated 24,168,398 sequenced variants of which 23,255 (8214 unique) were clinically reported. RESULTS: Of the reported variants, 17,240 (74.1%) were identified in more than one assay which allowed curated variant data to be reused in later reports. The remainder, 6015 (25.9%) were not subsequently seen in later assays and did not provide any reuse benefit. The number of new variants requiring curation has significantly increased over time from 1.72 to 3.73 variants per sample (292 curated variants per month). Analysis of the 23,255 variants reported, showed 28.6% (n = 2356) were not present in common public variant resources and therefore required de novo curation. These in-house only variants were enriched for indels, tumour suppressor genes and from solid tumour assays. CONCLUSION: This analysis highlights the significant percentage of variants not present within common public variant resources and the level of non-recurrent variants that consequently require greater curation effort. Many of these variants are unique to a single patient and unlikely to appear in other patients reflecting the personalised nature of cancer genomics. This study depicts the real-world situation for pathology laboratories faced with curating increasing numbers of low-recurrence variants while needing to expedite the process of manual variant curation. In the absence of suitably accurate automated methods, new approaches are needed to scale oncology diagnostics for future genetic testing volumes.

Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy.

Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8+ T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.