Microsatellite instability (MSI) is an emerging actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI ...diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high-throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule molecular inversion probes (smMIPs).
We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material.
Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI-positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care molecular diagnostic for MSI, proved equally robust for colorectal tumors but evidenced multiple false-negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors.
smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.
The combined clinical and molecular heterogeneity of prostate cancer necessitates the use of prognostic, predictive, and diagnostic biomarkers to assist the clinician with treatment selection. The ...pathologist plays a critical role in guiding molecular biomarker testing in prostate cancer and requires a thorough knowledge of the current testing options. In the setting of clinically localized prostate cancer, prognostic biomarkers such as Ki-67 labeling, PTEN loss or mRNA-based genomic signatures can be useful to help determine whether definitive therapy is required. In the setting of advanced disease, predictive biomarkers, such as the presence of DNA repair deficiency mediated by BRCA2 loss or mismatch repair gene defects, may suggest the utility of poly-ADP ribosylase inhibition or immune checkpoint blockade. Finally, androgen receptor–related biomarkers or diagnostic biomarkers indicating the presence of small cell neuroendocrine prostate cancer may help guide the use of androgen receptor signaling inhibitors and chemotherapy. In this review, we examine the current evidence for several prognostic, predictive and diagnostic tissue-based molecular biomarkers in prostate cancer management. For each assay, we summarize a recent survey of the International Society of Urology Pathology (ISUP) members on current testing practices and include recommendations for testing that emerged from the ISUP Working Group on Molecular Pathology of Prostate Cancer and the 2019 Consultation Conference on Molecular Pathology of Urogenital Cancers.
DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ...ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.
Accurate and timely diagnosis of inherited bone marrow failure and inherited myelodysplastic syndromes is essential to guide clinical management. Distinguishing inherited from acquired bone marrow ...failure/myelodysplastic syndrome poses a significant clinical challenge. At present, diagnostic genetic testing for inherited bone marrow failure/myelodysplastic syndrome is performed gene-by-gene, guided by clinical and laboratory evaluation. We hypothesized that standard clinically-directed genetic testing misses patients with cryptic or atypical presentations of inherited bone marrow failure/myelodysplastic syndrome. In order to screen simultaneously for mutations of all classes in bone marrow failure/myelodysplastic syndrome genes, we developed and validated a panel of 85 genes for targeted capture and multiplexed massively parallel sequencing. In patients with clinical diagnoses of Fanconi anemia, genomic analysis resolved subtype assignment, including those of patients with inconclusive complementation test results. Eight out of 71 patients with idiopathic bone marrow failure or myelodysplastic syndrome were found to harbor damaging germline mutations in GATA2, RUNX1, DKC1, or LIG4. All 8 of these patients lacked classical clinical stigmata or laboratory findings of these syndromes and only 4 had a family history suggestive of inherited disease. These results reflect the extensive genetic heterogeneity and phenotypic complexity of bone marrow failure/myelodysplastic syndrome phenotypes. This study supports the integration of broad unbiased genetic screening into the diagnostic workup of children and young adults with bone marrow failure and myelodysplastic syndromes.
Prostate cancer (PC) is initially dependent on androgen receptor (AR) signaling for survival and growth. Therapeutics designed to suppress AR activity serve as the primary intervention for advanced ...disease. However, supraphysiological androgen (SPA) concentrations can produce paradoxical responses leading to PC growth inhibition. We sought to discern the mechanisms by which SPA inhibits PC and to determine if molecular context associates with anti-tumor activity. SPA produced an AR-mediated, dose-dependent induction of DNA double-strand breaks (DSBs), G0/G1 cell cycle arrest and cellular senescence. SPA repressed genes involved in DNA repair and delayed the restoration of damaged DNA which was augmented by PARP1 inhibition. SPA-induced DSBs were accentuated in BRCA2-deficient PCs, and combining SPA with PARP or DNA-PKcs inhibition further repressed growth. Next-generation sequencing was performed on biospecimens from PC patients receiving SPA as part of ongoing Phase II clinical trials. Patients with mutations in genes mediating homology-directed DNA repair were more likely to exhibit clinical responses to SPA. These results provide a mechanistic rationale for directing SPA therapy to PCs with AR amplification or DNA repair deficiency, and for combining SPA therapy with PARP inhibition.
The
tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, ...which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2,
missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS,
p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality.
p.L1363P led to a similar acceleration in the development of
-deficient mammary tumors as
loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in
p.L1363P tumors. Nevertheless,
p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. SIGNIFICANCE: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.
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microRNAs (miRNAs) are fundamental to cellular biology. Although only approximately 22 bases long, miRNAs regulate complex processes in health and disease, including human cancer. Because miRNAs are ...highly stable in circulation when compared with several other classes of nucleic acids, they have generated intense interest as clinical biomarkers in diverse epidemiologic studies. As with other molecular biomarker fields, however, miRNA research has become beleaguered by pitfalls related to terminology and classification; procedural, assay, and study cohort heterogeneity; and methodological inconsistencies. Together, these issues have led to both false-positive and potentially false-negative miRNA associations. In this review, we summarize the biological rationale for studying miRNAs in human disease with a specific focus on circulating miRNAs, which highlight some of the most challenging topics in the field to date. Examples from lung cancer are used to illustrate the potential utility and some of the pitfalls in contemporary miRNA research. Although the field is in its infancy, several important lessons have been learned relating to cohort development, sample preparation, and statistical analysis that should be considered for future studies. The goal of this primer is to equip epidemiologists and clinical researchers with sound principles of study design and analysis when using miRNAs.
At the Advanced Prostate Cancer Consensus Conference 2021, three important areas of controversy in management of advanced prostate cancer were identified and discussed, and experts voted on 107 ...predefined consensus questions. The full report of the results is summarised here.
Innovations in treatments, imaging, and molecular characterisation in advanced prostate cancer have improved outcomes, but various areas of management still lack high-level evidence to inform clinical practice. The 2021 Advanced Prostate Cancer Consensus Conference (APCCC) addressed some of these questions to supplement guidelines that are based on level 1 evidence.
To present the voting results from APCCC 2021.
The experts identified three major areas of controversy related to management of advanced prostate cancer: newly diagnosed metastatic hormone-sensitive prostate cancer (mHSPC), the use of prostate-specific membrane antigen ligands in diagnostics and therapy, and molecular characterisation of tissue and blood. A panel of 86 international prostate cancer experts developed the programme and the consensus questions.
The panel voted publicly but anonymously on 107 pre-defined questions, which were developed by both voting and non-voting panel members prior to the conference following a modified Delphi process.
The voting reflected the opinions of panellists and did not incorporate a standard literature review or formal meta-analysis. The answer options for the consensus questions received varying degrees of support from panellists, as reflected in this article and the detailed voting results reported in the Supplementary material.
These voting results from a panel of experts in advanced prostate cancer can help clinicians and patients to navigate controversial areas of management for which high-level evidence is scant. However, diagnostic and treatment decisions should always be individualised according to patient characteristics, such as the extent and location of disease, prior treatment(s), comorbidities, patient preferences, and treatment recommendations, and should also incorporate current and emerging clinical evidence and logistic and economic constraints. Enrolment in clinical trials should be strongly encouraged. Importantly, APCCC 2021 once again identified salient questions that merit evaluation in specifically designed trials.
The Advanced Prostate Cancer Consensus Conference is a forum for discussing current diagnosis and treatment options for patients with advanced prostate cancer. An expert panel votes on predefined questions focused on the most clinically relevant areas for treatment of advanced prostate cancer for which there are gaps in knowledge. The voting results provide a practical guide to help clinicians in discussing treatment options with patients as part of shared decision-making.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mismatch repair protein (MMR) immunohistochemistry is an important tool in screening for Lynch syndrome in colorectal cancer patients. Unusual staining patterns such as heterogeneous MSH6 staining ...have been reported in colorectal and endometrial cancers. We aim to better understand MSH6 staining heterogeneity in colorectal cancer by comparative sequencing of different tumor areas for MMR and DNA polymerase mutations. Whole-section slides of 1754 colorectal cancers were reviewed for heterogeneous MSH6 staining, defined as discrete tumor areas with abrupt loss of staining juxtaposed to tumor areas with retained staining. Nine cases (0.05%) demonstrated heterogeneous MSH6 staining; none received neoadjuvant therapy prior to the specimen collection. The area of tumor with loss of MSH6 expression ranged from 5% to 60% (average 22%). Four cases had enough tissue remaining in both retained and lost MSH6 areas to perform tumor sequencing on both areas. All 9 cases were negative for MSH6 germline mutation; MSH6 heterogeneous staining was seen in tumors with MLH1 or PMS2 abnormalities (6 cases of MLH1 methylation, 2 PMS2 germline mutation, 1 MLH1 germline mutation). In addition, case 1 also had a somatic POLD1 exonuclease domain mutation (p.Y405C) in the MSH6 loss area but not in the intact area. We recommend reporting MSH6 heterogeneous pattern as MSH6 staining is present with a comment stating that the heterogeneous pattern typically does not indicate germline mutation in MSH6 but is commonly associated with abnormality in another MMR gene such as MLH1 or PMS2, or even other DNA repair genes such as DNA polymerase.
•MSH6 heterogeneous staining was seen in 9 cases of colorectal cancers with MLH1 or PMS2 abnormalities; not MSH6 germline mutation•One case had a somatic POLD1 exonuclease domain mutation (p.Y405C) in the MSH6 loss of expression area but not in the intact area•MSH6 heterogeneous staining pattern is usually due to a secondary MSH6 somatic alteration, most commonly at the genomic locus c.3261•MSH6 heterogeneous staining is associated with an abnormality in another MMR gene or other DNA repair gene, but not MSH6 germline mutation
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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