Reinterpretation of prostate cancer pathology by Appl1, Sortilin and Syndecan-1 biomarkers.

The diagnosis of prostate cancer using histopathology is reliant on the accurate interpretation of prostate tissue sections. Current standards rely on the assessment of Haematoxylin and Eosin (H&E) staining, which can be difficult to interpret and introduce inter-observer variability. Here, we present a digital pathology atlas and online resource of prostate cancer tissue micrographs for both H&E and the reinterpretation of samples using a novel set of three biomarkers as an interactive tool, where clinicians and scientists can explore high resolution histopathology from various case studies. The digital pathology prostate cancer atlas when used in conjunction with the biomarkers, will assist pathologists to accurately grade prostate cancer tissue samples.

Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression.

BACKGROUND: This study focuses on the role of lysosomal trafficking in prostate cancer, given the essential role of lysosomes in cellular homoeostasis. METHODS: Lysosomal motility was evaluated using confocal laser scanning microscopy of LAMP-1-transfected prostate cells and spot-tracking analysis. Expression of lysosomal trafficking machinery was evaluated in patient cohort databases and through immunohistochemistry on tumour samples. The roles of vesicular trafficking machinery were evaluated through over-expression and siRNA. The effects of R1881 treatment on lysosome vesicular trafficking was evaluated by RNA sequencing, protein quantification and fixed- and live-cell microscopy. RESULTS: Altered regulation of lysosomal trafficking genes/proteins was observed in prostate cancer tissue, with significant correlations for co-expression of vesicular trafficking machinery in Gleason patterns. The expression of trafficking machinery was associated with poorer patient outcomes. R1881 treatment induced changes in lysosomal distribution, number, and expression of lysosomal vesicular trafficking machinery in hormone-sensitive prostate cancer cells. Manipulation of genes involved in lysosomal trafficking events induced changes in lysosome positioning and cell phenotype, as well as differential effects on cell migration, in non-malignant and prostate cancer cells. CONCLUSIONS: These findings provide novel insights into the altered regulation and functional impact of lysosomal vesicular trafficking in prostate cancer pathogenesis.

Identification of temporal shifts of oral bacteria in bone regeneration following mandibular bone defect injury and therapeutic surgery in a porcine model.

BACKGROUND: Considered the second largest and most diverse microbiome after the gut, the human oral ecosystem is complex with diverse and niche-specific microorganisms. Although evidence is growing for the importance of oral microbiome in supporting a healthy immune system and preventing local and systemic infections, the influence of craniomaxillofacial (CMF) trauma and routine reconstructive surgical treatments on community structure and function of oral resident microbes remains unknown. CMF injuries affect a large number of people, needing extensive rehabilitation with lasting morbidity and loss of human productivity. Treatment efficacy can be complicated by the overgrowth of opportunistic commensals or multidrug-resistant pathogens in the oral ecosystem due to weakened host immune function and reduced colonization resistance in a dysbiotic oral microbiome. AIMS: To understand the dynamics of microbiota's community structure during CMF injury and subsequent treatments, we induced supra-alveolar mandibular defect in Hanford miniature swine (n = 3) and compared therapeutic approaches of immediate mandibullar reconstructive (IMR) versus delayed mandibullar reconstructive (DMR) surgeries. METHODS: Using bacterial 16S ribosomal RNA gene marker sequencing, the composition and abundance of the bacterial community of the uninjured maxilla (control) and the injured left mandibula (lingual and buccal) treated by DMR were surveyed up to 70-day post-wounding. For the injured right mandibula receiving IMR treatment, the microbial composition and abundance were surveyed up to 14-day post-wounding. Moreover, we measured sera level of biochemical markers (e.g., osteocalcin) associated with bone regeneration and healing. Computed tomography was used to measure and compare mandibular bone characteristics such as trabecular thickness between sites receiving DMR and IMR therapeutic approaches until day 140, the end of study period. RESULTS: Independent of IMR versus DMR therapy, we observed similar dysbiosis and shifts of the mucosal bacteria residents after CMF injury and/or following treatment. There was an enrichment of Fusobacterium, Porphyromonadaceae, and Bacteroidales accompanied by a decline in Pasteurellaceae, Moraxella, and Neisseria relative abundance in days allotted for healing. We also observed a decline in species richness and abundance driven by reduction in temporal instability and inter-animal heterogeneity on days 0 and 56, with day 0 corresponding to injury in DMR group and day 56 corresponding to delayed treatment for DMR or injury and immediate treatment for the IMR group. Analysis of bone healing features showed comparable bone-healing profiles for IMR vs. DMR therapeutic approach.

Synthesis and cellular uptake of neutral rhenium(I) morpholine complexes.

Morpholine motifs have been used extensively as targeting moieties for lysosomes, primarily in fluorescence imaging agents. Traditionally these imaging agents are based on organic molecules which have several shortcomings including small Stokes shifts, short emission lifetimes, and susceptibility to photobleaching. To explore alternative lysosome targeting imaging agents we have used a rhenium based phosphorescent platform which has been previously demonstrated to have an improved Stokes shift, a long lifetime emission, and is highly photostable. Rhenium complexes containing morpholine substituted ligands were designed to accumulate in acidic compartments. Two of the three complexes prepared exhibited bright emission in cells, when incubated at low concentrations (20 µM) and were non-toxic at concentrations as high as 100 µM, making them suitable for live cell imaging. We show that the rhenium complexes are amenable to chemical modification and that the morpholine targeted derivatives can be used for live cell confocal fluorescence imaging of endosomes-lysosomes.

Exploring the role of sporadic BRAF and KRAS mutations during colorectal cancer pathogenesis: A spotlight on the contribution of the endosome-lysosome system.

The highly heterogenous nature of colorectal cancer can significantly hinder its early and accurate diagnosis, eventually contributing to high mortality rates. The adenoma-carcinoma sequence and serrated polyp-carcinoma sequence are the two most common sequences in sporadic colorectal cancer. Genetic alterations in adenomatous polyposis coli (APC), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and tumour protein 53 (TP53) genes are critical in adenoma-carcinoma sequence, whereas v-Raf murine sarcoma viral oncogene homolog B (BRAF) and MutL Homolog1 (MLH1) are driving oncogenes in the serrated polyp-carcinoma sequence. Sporadic mutations in these genes contribute differently to colorectal cancer pathogenesis by introducing distinct alterations in several signalling pathways that rely on the endosome-lysosome system. Unsurprisingly, the endosome-lysosome system plays a pivotal role in the hallmarks of cancer and contributes to specialised colon function. Thus, the endosome-lysosome system might be distinctively influenced by different mutations and these alterations may contribute to the heterogenous nature of sporadic colorectal cancer. This review highlights potential connections between major sporadic colorectal cancer mutations and the diverse pathogenic mechanisms driven by the endosome-lysosome system in colorectal carcinogenesis.

Fluid and Bubble Flow Detach Adherent Cancer Cells to Form Spheroids on a Random Positioning Machine.

In preparing space and microgravity experiments, the utilization of ground-based facilities is common for initial experiments and feasibility studies. One approach to simulating microgravity conditions on Earth is to employ a random positioning machine (RPM) as a rotary bioreactor. Combined with a suitable low-mass model system, such as cell cultures, these devices simulating microgravity have been shown to produce results similar to those obtained in a space experiment under real microgravity conditions. One of these effects observed under real and simulated microgravity is the formation of spheroids from 2D adherent cancer cell cultures. Since real microgravity cannot be generated in a laboratory on Earth, we aimed to determine which forces lead to the detachment of individual FTC-133 thyroid cancer cells and the formation of tumor spheroids during culture with exposure to random positioning modes. To this end, we subdivided the RPM motion into different static and dynamic orientations of cell culture flasks. We focused on the molecular activation of the mechanosignaling pathways previously associated with spheroid formation in microgravity. Our results suggest that RPM-induced spheroid formation is a two-step process. First, the cells need to be detached, induced by the cell culture flask's rotation and the subsequent fluid flow, as well as the presence of air bubbles. Once the cells are detached and in suspension, random positioning prevents sedimentation, allowing 3D aggregates to form. In a comparative shear stress experiment using defined fluid flow paradigms, transcriptional responses were triggered comparable to exposure of FTC-133 cells to the RPM. In summary, the RPM serves as a simulator of microgravity by randomizing the impact of Earth's gravity vector especially for suspension (i.e., detached) cells. Simultaneously, it simulates physiological shear forces on the adherent cell layer. The RPM thus offers a unique combination of environmental conditions for in vitro cancer research.

Insights into Melanoma Clinical Practice: A Perspective for Future Research.

BACKGROUND: Early diagnosis is the key to improving outcomes for patients with melanoma, and this requires a standardized histological assessment approach. The objective of this survey was to understand the challenges faced by clinicians when assessing melanoma cases, and to provide a perspective for future studies. METHODS: Between April 2022 and February 2023, national and international dermatologists, pathologists, general practitioners, and laboratory managers were invited to participate in a six-question online survey. The data from the survey were assessed using descriptive statistics and qualitative responses. RESULTS: A total of 54 responses were received, with a 51.4% (n = 28) full completion rate. Of the respondents, 96.4% reported ambiguity in their monthly melanoma diagnosis, and 82.1% routinely requested immunohistochemistry (IHC) testing to confirm diagnosis. SOX10 was the most frequently requested marker, and most respondents preferred multiple markers over a single marker. Diagnostic and prognostic tests, as well as therapeutic options and patient management, were all identified as important areas for future research. CONCLUSIONS: The respondents indicated that the use of multiple IHC markers is essential to facilitate diagnostic accuracy in melanoma assessment. Survey responses indicate there is an urgent need to develop new biomarkers for clinical decision making at multiple critical intervention points.

Distinct patterns of biomarker expression for atypical intraductal proliferations in prostate cancer.

High-grade prostatic intraepithelial neoplasia (HGPIN) is a well-characterised precursor lesion in prostate cancer. The term atypical intraductal proliferations (AIP) describes lesions with features that are far too atypical to be considered HGPIN, yet insufficient to be diagnosed as intraductal carcinoma of the prostate (IDCP). Here, a panel of biomarkers was assessed to provide insights into the biological relationship between IDCP, HGPIN, and AIP and their relevance to current clinicopathological recommendations. Tissue samples from 86 patients with prostate cancer were assessed by routine haematoxylin and eosin staining and immunohistochemistry (IHC) with a biomarker panel (Appl1/Sortilin/Syndecan-1) and a PIN4 cocktail (34ßE12+P63/P504S). Appl1 strongly labelled atypical secretory cells, effectively visualising intraductal lesions. Sortilin labelling was moderate-to-strong in > 70% of cases, while Syndecan-1 was moderate-to-strong in micropapillary HGPIN/AIP lesions (83% cases) versus flat/tufting HGPIN (≤ 20% cases). Distinct biomarker labelling patterns for atypical intraductal lesions of the prostate were observed, including early atypical changes (flat/tufting HGPIN) and more advanced atypical changes (micropapillary HGPIN/AIP). Furthermore, the biomarker panel may be used as a tool to overcome the diagnostic uncertainty surrounding AIP by supporting a definitive diagnosis of IDCP for such lesions displaying the same biomarker pattern as cribriform IDCP.

Dynamic interplay between sortilin and syndecan-1 contributes to prostate cancer progression.

Prostate cancer (PCa) development and progression relies on the programming of glucose and lipid metabolism, and this involves alterations in androgen receptor expression and signalling. Defining the molecular mechanism that underpins this metabolic programming will have direct significance for patients with PCa who have a poor prognosis. Here we show that there is a dynamic balance between sortilin and syndecan-1, that reports on different metabolic phenotypes. Using tissue microarrays, we demonstrated by immunohistochemistry that sortilin was highly expressed in low-grade cancer, while syndecan-1 was upregulated in high-grade disease. Mechanistic studies in prostate cell lines revealed that in androgen-sensitive LNCaP cells, sortilin enhanced glucose metabolism by regulating GLUT1 and GLUT4, while binding progranulin and lipoprotein lipase (LPL) to limit lipid metabolism. In contrast, in androgen-insensitive PC3 cells, syndecan-1 was upregulated, interacted with LPL and colocalised with ß3 integrin to promote lipid metabolism. In addition, androgen-deprived LNCaP cells had decreased expression of sortilin and reduced glucose-metabolism, but increased syndecan-1 expression, facilitating interactions with LPL and possibly ß3 integrin. We report a hitherto unappreciated molecular mechanism for PCa, which may have significance for disease progression and how androgen-deprivation therapy might promote castration-resistant PCa.

Altered endosomal-lysosomal biogenesis in melanoma.

Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is currently reliant upon melanin visualisation, research into melanosome biogenesis, as a key driver of pathogenesis, has not yielded technology that can reliably distinguish between atypical benign, amelanotic and melanotic lesions. The endosomal-lysosomal system has important regulatory roles in cancer cell biology, including a specific functional role in melanosome biogenesis. Herein, the involvement of the endosomal-lysosomal system in melanoma was examined by pooled secondary analysis of existing gene expression datasets. A set of differentially expressed endosomal-lysosomal genes was identified in melanoma, which were interconnected by biological function. To illustrate the protein expression of the dysregulated genes, immunohistochemistry was performed on samples from patients with cutaneous melanoma to reveal candidate markers. This study demonstrated the dysregulation of Syntenin-1, Sortilin and Rab25 may provide a differentiating feature between cutaneous melanoma and squamous cell carcinoma, while IGF2R may indicate malignant propensity in these skin cancers.

Appl1, Sortilin and Syndecan-1 immunohistochemistry on intraductal carcinoma of the prostate provides evidence of retrograde spread.

The presence of intraductal carcinoma of the prostate (IDCP) correlates with late-stage disease and poor outcomes for patients with prostatic adenocarcinoma, but the accurate and reliable staging of disease severity remains challenging. Immunohistochemistry (IHC) has been utilised to overcome problems in assessing IDCP morphology, but the current markers have only demonstrated limited utility in characterising the complex biology of this lesion. In a retrospective study of a cohort of patients who had been diagnosed with IDCP, we utilised IHC on radical prostatectomy sections with a biomarker panel of Appl1, Sortilin and Syndecan-1, to interpret different architectural patterns and to explore the theory that IDCP occurs from retrograde spread of high-grade invasive prostatic adenocarcinoma. Cribriform IDCP displayed strong Appl1, Sortilin and Syndecan-1 labelling patterns, while solid IDCP architecture had high intensity Appl1 and Syndecan-1 labelling, but minimal Sortilin labelling. Notably, the expression pattern of the biomarker panel in regions of IDCP was similar to that of adjacent invasive prostatic adenocarcinoma, and also comparable to prostate cancer showing perineural and vascular invasion. The Appl1, Sortilin, and Syndecan-1 biomarker panel in IDCP provides evidence for the model of retrograde spread of invasive prostatic carcinoma into ducts/acini, and supports the inclusion of IDCP into the five-tier Gleason grading system.

Prediction of Prostate Cancer Biochemical and Clinical Recurrence Is Improved by IHC-Assisted Grading Using Appl1, Sortilin and Syndecan-1.

Gleason scoring is used within a five-tier risk stratification system to guide therapeutic decisions for patients with prostate cancer. This study aimed to compare the predictive performance of routine H&E or biomarker-assisted ISUP (International Society of Urological Pathology) grade grouping for assessing the risk of biochemical recurrence (BCR) and clinical recurrence (CR) in patients with prostate cancer. This retrospective study was an assessment of 114 men with prostate cancer who provided radical prostatectomy samples to the Australian Prostate Cancer Bioresource between 2006 and 2014. The prediction of CR was the primary outcome (median time to CR 79.8 months), and BCR was assessed as a secondary outcome (median time to BCR 41.7 months). The associations of (1) H&E ISUP grade groups and (2) modified ISUP grade groups informed by the Appl1, Sortilin and Syndecan-1 immunohistochemistry (IHC) labelling were modelled with BCR and CR using Cox proportional hazard approaches. IHC-assisted grading was more predictive than H&E for BCR (C-statistic 0.63 vs. 0.59) and CR (C-statistic 0.71 vs. 0.66). On adjusted analysis, IHC-assisted ISUP grading was independently associated with both outcome measures. IHC-assisted ISUP grading using the biomarker panel was an independent predictor of individual BCR and CR. Prospective studies are needed to further validate this biomarker technology and to define BCR and CR associations in real-world cohorts.

Pathways of Individuals Experiencing Serious Illness While Homeless: An Exploratory 4-Point Typology from the RASCAL-UP Study.

The shifting age demographics of those experiencing homelessness in the United States expose shortcomings and barriers within homelessness response services and safety-net healthcare to address serious illness. The purpose of this study is to describe the common trajectories of patients concurrently experiencing homelessness and serious illness. As a part of the Research, Action, and Supportive Care at Later-life for Unhoused People (RASCAL-UP) study, the study uses patient charts (n = 75) from the only specialty palliative care program in the U.S. specifically for people experiencing homelessness. Through a thematic mixed-method analysis, a four-point typology of care pathways taken by people experiencing homelessness while seriously ill is introduced: (1) aging and dying-in-place within the housing care system; (2) frequent transitions during serious illness; (3) healthcare institutions as housing; and (4) housing as palliation. Implications of this exploratory typology include targeted, site-specific interventions for supporting goal-concordant patient care and assisting researchers and policy makers in appreciating heterogeneity in experience and need among older and chronically ill people experiencing homelessness and housing precarity.

Accuracy of diagnostic tests in cardiac injury after blunt chest trauma: a systematic review and meta-analysis.

INTRODUCTION: The diagnosis of cardiac contusion, caused by blunt chest trauma, remains a challenge due to the non-specific symptoms it causes and the lack of ideal tests to diagnose myocardial damage. A cardiac contusion can be life-threatening if not diagnosed and treated promptly. Several diagnostic tests have been used to evaluate the risk of cardiac complications, but the challenge of identifying patients with contusions nevertheless remains. AIM OF THE STUDY: To evaluate the accuracy of diagnostic tests for detecting blunt cardiac injury (BCI) and its complications, in patients with severe chest injuries, who are assessed in an emergency department or by any front-line emergency physician. METHODS: A targeted search strategy was performed using Ovid MEDLINE and Embase databases from 1993 up to October 2022. Data on at least one of the following diagnostic tests: electrocardiogram (ECG), serum creatinine phosphokinase-MB level (CPK-MB), echocardiography (Echo), Cardiac troponin I (cTnI) or Cardiac troponin T (cTnT). Diagnostic tests for cardiac contusion were evaluated for their accuracy in meta-analysis. Heterogeneity was assessed using the I2 and the QUADAS-2 tool was used to assess bias of the studies. RESULTS: This systematic review yielded 51 studies (n = 5,359). The weighted mean incidence of myocardial injuries after sustaining a blunt force trauma stood at 18.3% of cases. Overall weighted mean mortality among patients with blunt cardiac injury was 7.6% (1.4-36.4%). Initial ECG, cTnI, cTnT and transthoracic echocardiography TTE all showed high specificity (> 80%), but lower sensitivity (< 70%). TEE had a specificity of 72.1% (range 35.8-98.2%) and sensitivity of 86.7% (range 40-99.2%) in diagnosing cardiac contusion. CK-MB had the lowest diagnostic odds ratio of 3.598 (95% CI: 1.832-7.068). Normal ECG accompanied by normal cTnI showed a high sensitivity of 85% in ruling out cardiac injuries. CONCLUSION: Emergency physicians face great challenges in diagnosing cardiac injuries in patients following blunt trauma. In the majority of cases, joint use of ECG and cTnI was a pragmatic and cost-effective approach to rule out cardiac injuries. In addition, TEE may be highly accurate in identifying cardiac injuries in suspected cases.

Pitfalls in Cutaneous Melanoma Diagnosis and the Need for New Reliable Markers.

Cutaneous melanoma is one of the most aggressive forms of skin cancer, with the development of advanced stage disease resulting in a high rate of patient mortality. Accurate diagnosis of melanoma at an early stage is essential to improve patient outcomes, as this enables treatment before the cancer has metastasised. Histopathologic analysis is the current gold standard for melanoma diagnosis, but this can be subjective due to discordance in interpreting the morphological heterogeneity in melanoma and other skin lesions. Immunohistochemistry (IHC) is sometimes employed as an adjunct to conventional histology, but it remains occasionally difficult to distinguish some benign melanocytic lesions and melanoma. Importantly, the complex morphology and lack of specific biomarkers that identify key elements of melanoma pathogenesis can make an accurate confirmation of diagnosis challenging. We review the diagnostic constraints of melanoma heterogeneity and discuss issues with interpreting routine histology and problems with current melanoma markers. Innovative approaches are required to find effective biomarkers to enhance patient management.

Aberrant protein expression of Appl1, Sortilin and Syndecan-1 during the biological progression of prostate cancer.

Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occurring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop biomarkers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient samples, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC-assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy sample cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient samples. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the potential to transform clinical pathology practice by standardising the interpretation of the tissue morphology.

Matched Molecular Profiling of Cell-Free DNA and Tumor Tissue in Patients With Advanced Clear Cell Renal Cell Carcinoma.

PURPOSE: The clinical utility of cell-free DNA (cfDNA) as a biomarker for advanced clear cell renal cell carcinoma (ccRCC) remains unclear. We evaluated the validity of cfDNA-based genomic profiling in a large cohort of patients with ccRCC with matched next-generation sequencing (NGS) from primary tumor tissues. MATERIALS AND METHODS: We performed paired NGS of tumor DNA and plasma cfDNA using the MSK-IMPACT platform in 110 patients with metastatic ccRCC. Tissues were profiled for variants and copy number alterations with germline comparison. Manual cross-genotyping between cfDNA and tumor tissue was performed. Deep sequencing with a higher sensitivity platform, MSK-ACCESS, was performed on a subset of cfDNA samples. Clinical data and radiographic tumor volumes were assessed to correlate cfDNA yield with treatment response and disease burden. RESULTS: Tumor tissue MSK-IMPACT testing identified 582 genomic alterations (GAs) across the cohort. Using standard thresholds for de novo variant calling in cfDNA, only 24 GAs were found by MSK-IMPACT in cfDNA in 7 of 110 patients (6%). With manual cross-genotyping, 210 GAs were detectable below thresholds in 74 patients (67%). Intrapatient concordance with tumor tissue was limited, including VHL (31.6%), PBRM1 (24.1%), and TP53 (52.9%). cfDNA profiling did not identify 3p loss because of low tumor fractions. Tumor volume was associated with cfDNA allele frequency, and VHL concordance was superior for patients with greater disease burden. CONCLUSION: cfDNA-based NGS profiling yielded low detection rates in this metastatic ccRCC cohort. Concordance with tumor profiling was low, even for truncal mutations such as VHL, and some findings in peripheral blood may represent clonal hematopoiesis. Routine cfDNA panel testing is not supported, and its application in biomarker efforts must account for these limitations.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Acute Radiation-Induced Hematopoietic Depletion Does Not Alter the Onset or Severity of Pneumonitis in Mice.

Survival from partial-body irradiation (PBI) may be limited by the development of the late lung injury response of pneumonitis. Herein we investigated the hypothesis that acute hematopoietic depletion alters the onset and severity of lung disease in a mouse model. To establish depletion, C3H/HeJ mice received 8 Gy PBI with shielding of only the tibiae, ankles and feet. One week after irradiation, blood lymphocyte and neutrophil counts were each significantly reduced (P < 0.04) in these mice compared to levels in untreated controls or in mice receiving 16 Gy to the whole thorax only. All 8 Gy PBI mice survived to the experimental end point of 16 weeks postirradiation. To determine whether the hematopoietic depletion affects lung disease, groups of mice received 8 Gy PBI plus 8 Gy whole-thorax irradiation (total lung dose of 16 Gy) or 16 Gy whole-thorax irradiation only. The weight loss, survival to onset of respiratory distress (P = 0.17) and pneumonitis score (P = 0.96) of mice that received 8 Gy PBI plus 8 Gy whole-thorax irradiation were not significantly different from those of mice receiving 16 Gy whole-thorax irradiation only. Mice in respiratory distress from PBI plus whole-thorax irradiation had significantly reduced (P = 0.02) blood monocyte counts compared to levels in distressed, whole-thorax irradiated mice, and symptomatic pneumonitis was associated with increased blood neutrophil counts (P = 0.04) relative to measures from irradiated, non-distressed mice. In conclusion, survivable acute hematopoietic depletion by partial-body irradiation did not alter the onset or severity of lethal pneumonitis in the C3H/HeJ mouse model.