Collective invasion in breast cancer requires a conserved basal epithelial program.

Carcinomas typically invade as a cohesive multicellular unit, a process termed collective invasion. It remains unclear how different subpopulations of cancer cells contribute to this process. We developed three-dimensional (3D) organoid assays to identify the most invasive cancer cells in primary breast tumors. Collective invasion was led by specialized cancer cells that were defined by their expression of basal epithelial genes, such as cytokeratin-14 (K14) and p63. Furthermore, K14+ cells led collective invasion in the major human breast cancer subtypes. Importantly, luminal cancer cells were observed to convert phenotypically to invasive leaders following induction of basal epithelial genes. Although only a minority of cells within luminal tumors expressed basal epithelial genes, knockdown of either K14 or p63 was sufficient to block collective invasion. Our data reveal that heterotypic interactions between epithelial subpopulations are critical to collective invasion. We suggest that targeting the basal invasive program could limit metastatic progression.

Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

Neoadjuvant PD-1 Blockade in Resectable Lung Cancer.

BACKGROUND: Antibodies that block programmed death 1 (PD-1) protein improve survival in patients with advanced non-small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, a condition in which little progress has been made during the past decade. METHODS: In this pilot study, we administered two preoperative doses of PD-1 inhibitor nivolumab in adults with untreated, surgically resectable early (stage I, II, or IIIA) NSCLC. Nivolumab (at a dose of 3 mg per kilogram of body weight) was administered intravenously every 2 weeks, with surgery planned approximately 4 weeks after the first dose. The primary end points of the study were safety and feasibility. We also evaluated the tumor pathological response, expression of programmed death ligand 1 (PD-L1), mutational burden, and mutation-associated, neoantigen-specific T-cell responses. RESULTS: Neoadjuvant nivolumab had an acceptable side-effect profile and was not associated with delays in surgery. Of the 21 tumors that were removed, 20 were completely resected. A major pathological response occurred in 9 of 20 resected tumors (45%). Responses occurred in both PD-L1-positive and PD-L1-negative tumors. There was a significant correlation between the pathological response and the pretreatment tumor mutational burden. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in eight of nine patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a complete response on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab. CONCLUSIONS: Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced a major pathological response in 45% of resected tumors. The tumor mutational burden was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. (Funded by Cancer Research Institute-Stand Up 2 Cancer and others; ClinicalTrials.gov number, NCT02259621 .).

Chemotherapy induces enrichment of CD47+/CD73+/PDL1+ immune evasive triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is treated with cytotoxic chemotherapy and is often characterized by early relapse and metastasis. To form a secondary (recurrent and/or metastatic) tumor, a breast cancer cell must evade the innate and adaptive immune systems. CD47 enables cancer cells to evade killing by macrophages, whereas CD73 and PDL1 mediate independent mechanisms of evasion of cytotoxic T lymphocytes. Here, we report that treatment of human or murine TNBC cells with carboplatin, doxorubicin, gemcitabine, or paclitaxel induces the coordinate transcriptional induction of CD47, CD73, and PDL1 mRNA and protein expression, leading to a marked increase in the percentage of CD47+CD73+PDL1+ breast cancer cells. Genetic or pharmacological inhibition of hypoxia-inducible factors (HIFs) blocked chemotherapy-induced enrichment of CD47+CD73+PDL1+ TNBC cells, which were also enriched in the absence of chemotherapy by incubation under hypoxic conditions, leading to T cell anergy or death. Treatment of mice with cytotoxic chemotherapy markedly increased the intratumoral ratio of regulatory/effector T cells, an effect that was abrogated by HIF inhibition. Our results delineate an HIF-dependent transcriptional mechanism contributing to TNBC progression and suggest that combining chemotherapy with an HIF inhibitor may prevent countertherapeutic induction of proteins that mediate evasion of innate and adaptive antitumor immunity.

Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): Moving from targeted therapy to immunotherapy.

Recent large scale genomic studies from the Clinical Lung Cancer Genome Project have identified different driver gene mutations in the subtypes of non-small cell lung carcinoma (NSCLC). These findings not only lead to remarkable progress in targeted therapies for lung cancer patients, but also provide fundamental knowledge for the subclassification of NSCLC. More recently, the advancement and clinical application of immunotherapy have reinforced the need for the accurate subclassification of NSCLC. In 2015, the World Health Organization (WHO) and the International Association for the Study of Lung Cancer (IASLC) updated their guidelines for the subclassification of lung cancers. These guidelines emphasize: (1) the subclassification of NSCLC, (2) the critical role of molecular characterization of tumors for targeted therapy, (3) the unique terminology for subclassifying NSCLC using small biopsy specimens, and (4) the utility of IHC biomarkers in the accurate diagnosis and subclassification of lung cancer. The guidelines have significant prognostic impact on oncologic practice and patient care. In this review, we summarize the current WHO guidelines for the classification of lung cancer, discuss advancements of targeted therapy and immunotherapy, and address the utility and limitation of immunomarkers in the subclassification of NSCLC, as well as the prospective future of the field.

De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.

Oncogenic Kras mutations are one of the most common alterations in non-small cell lung cancer and are associated with poor response to treatment and reduced survival. Driver oncogenes, such as Kras are now appreciated for their ability to promote tumor growth via up-regulation of anabolic pathways. Therefore, we wanted to identify metabolic vulnerabilities in Kras-mutant lung cancer. Using the Kras LSL-G12D lung cancer model, we show that mutant Kras drives a lipogenic gene-expression program. Stable-isotope analysis reveals that mutant Kras promotes de novo fatty acid synthesis in vitro and in vivo. The importance of fatty acid synthesis in Kras-induced tumorigenesis was evident by decreased tumor formation in Kras LSL-G12D mice after treatment with a fatty acid synthesis inhibitor. Importantly, with gain and loss of function models of mutant Kras, we demonstrate that mutant Kras potentiates the growth inhibitory effects of several fatty acid synthesis inhibitors. These studies highlight the potential to target mutant Kras tumors by taking advantage of the lipogenic phenotype induced by mutant Kras.-Singh, A., Ruiz, C., Bhalla, K., Haley, J. A., Li, Q. K., Acquaah-Mensah, G., Montal, E., Sudini, K. R., Skoulidis, F., Wistuba, I. I., Papadimitrakopoulou, V., Heymach, J. V., Boros, L. G., Gabrielson, E., Carretero, J., Wong, K.-k., Haley, J. D., Biswal, S., Girnun, G. D. De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.

Prognostic Impact of Phosphorylated Discoidin Domain Receptor-1 in Esophageal Cancer.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is common in East Asia and also is often deadly. We sought to determine whether measuring the discoidin domain receptor-1 (DDR1)-both total and phosphorylated proteins-could improve our ability to predict recurrence in ESCC. MATERIALS AND METHODS: Total DDR1 and phosphorylated DDR1 (pDDR1) were measured using semiquantitative immunohistochemistry in a cohort of 60 patients with ESCC. Association between these immunohistochemical measurements and standard clinical-pathological variables such as patient recurrence-free survival was examined using univariate and multivariate analyses. RESULTS: Six patients (10.0%) had regional recurrence and eight patients (13.3%) had distant recurrence. In univariate analysis, early disease recurrence correlated with intense staining of total DDR1 (P = 0.03) as well as intense staining of pDDR1 (P < 0.001). On multivariate analysis, only regional lymph node metastasis (P = 0.04, HR = 4.20) and intensity of pDDR1 immunohistochemistry (P = 0.03, HR = 4.27) emerged as significant independent prognostic factors for recurrence. CONCLUSIONS: This study suggests that immunohistochemical measurements of both the DDR1 protein and pDDR1 can provide prognostic value in ESCC, even when other clinical and pathological factors are also being considered.

A Randomized Phase II Study of Metformin plus Paclitaxel/Carboplatin/Bevacizumab in Patients with Chemotherapy-Naïve Advanced or Metastatic Nonsquamous Non-Small Cell Lung Cancer.

BACKGROUND: In the absence of a targeted oncogenic driver mutation or high programmed death-ligand 1 expression, systemic therapy with platinum-based doublet chemotherapy with or without bevacizumab has been the standard treatment in advanced or metastatic non-small cell lung cancer (NSCLC). Metformin has been shown to have antitumor effects via a variety of insulin-dependent and insulin-independent mechanisms and to be potentially synergistic with chemotherapy. MATERIALS AND METHODS: This open-label single-center phase II study (NCT01578551) enrolled patients with chemotherapy-naïve advanced or metastatic nonsquamous NSCLC and randomized them (3:1) to receive carboplatin, paclitaxel, and bevacizumab with (Arm A) or without (Arm B) concurrent metformin for four to six cycles followed by maintenance therapy with bevacizumab ± metformin continued until disease progression, intolerable toxicity, or study withdrawal. The primary outcome was 1-year progression free survival (PFS). Secondary outcomes included overall survival, response to therapy, and toxicity. RESULTS: A total of 25 patients were enrolled from August 2012 to April 2015, of whom 24 received at least one cycle of therapy administration. The study was stopped early due to slow accrual and changes in standard first-line therapy of advanced NSCLC. The 1-year PFS on Arm A (n = 18) was 47% (95% confidence interval [CI]: 25%-88%), which exceeded the historical control 1-year PFS of 15%. Median overall survival of patients treated on Arm A was 15.9 months (95% CI: 8.4-not available [NA]) and 13.9 months (95% CI: 12.7-NA) on Arm B. There were no significant differences in toxicity between the study arms. CONCLUSION: To the authors' knowledge, this is the first study to show a significant benefit in PFS with the use of metformin in this patient population and is a signal of efficacy for metformin in advanced NSCLC. IMPLICATIONS FOR PRACTICE: The anticancer effects of metformin continue to be elucidated. To the authors' knowledge, this is the first trial in nondiabetic advanced non-small cell lung cancer patients to show a significant change in outcome with the addition of metformin to standard first-line chemotherapy. Well tolerated and widely available, metformin is a drug that should be considered for further study in the lung cancer treatment landscape.

Tumor and serum DNA methylation in women receiving preoperative chemotherapy with or without vorinostat in TBCRC008.

BACKGROUND: Methylated gene markers have shown promise in predicting breast cancer outcomes and treatment response. We evaluated whether baseline and changes in tissue and serum methylation levels would predict pathological complete response (pCR) in patients with HER2-negative early breast cancer undergoing preoperative chemotherapy. METHODS: The TBCRC008 trial investigated pCR following 12 weeks of preoperative carboplatin and albumin-bound paclitaxel + vorinostat/placebo (n = 62). We measured methylation of a 10-gene panel by quantitative multiplex methylation-specific polymerase chain reaction and expressed results as cumulative methylation index (CMI). We evaluated association between CMI level [baseline, day 15 (D15), and change] and pCR using univariate and multivariable logistic regression models controlling for treatment and hormone receptor (HR) status, and performed exploratory subgroup analyses. RESULTS: In univariate analysis, one log unit increase in tissue CMI levels at D15 was associated with 40% lower chance of obtaining pCR (odds ratio, OR 0.60, 95% CI 0.37-0.97; p = 0.037). Subgroup analyses suggested a significant association between tissue D15 CMI levels and pCR in vorinostat-treated [OR 0.44 (0.20, 0.93), p = 0.03], but not placebo-treated patients. CONCLUSION: In this study investigating the predictive roles of tissue and serum CMI levels in patients with early breast cancer for the first time, we demonstrate that high D15 tissue CMI levels may predict poor response. Larger studies and improved analytical procedures to detect methylated gene markers in early stage breast cancer are needed. TBCRC008 is registered on ClinicalTrials.gov (NCT00616967).

Added Value of Computer-aided CT Image Features for Early Lung Cancer Diagnosis with Small Pulmonary Nodules: A Matched Case-Control Study.

Purpose To test whether computer-aided diagnosis (CAD) approaches can increase the positive predictive value (PPV) and reduce the false-positive rate in lung cancer screening for small nodules compared with human reading by thoracic radiologists. Materials and Methods A matched case-control sample of low-dose computed tomography (CT) studies in 186 participants with 4-20-mm noncalcified lung nodules who underwent biopsy in the National Lung Screening Trial (NLST) was selected. Variables used for matching were age, sex, smoking status, chronic obstructive pulmonary disease status, body mass index, study year of the positive screening test, and screening results. Studies before lung biopsy were randomly split into a training set (70 cancers plus 70 benign controls) and a validation set (20 cancers plus 26 benign controls). Image features from within and outside dominant nodules were extracted. A CAD algorithm developed from the training set and a random forest classifier were applied to the validation set to predict biopsy outcomes. Receiver operating characteristic analysis was used to compare the prediction accuracy of CAD with the NLST investigator's diagnosis and readings from three experienced and board-certified thoracic radiologists who used contemporary clinical practice guidelines. Results In the validation cohort, the area under the receiver operating characteristic curve for CAD was 0.9154. By default, the sensitivity, specificity, and PPV of the NLST investigators were 1.00, 0.00, and 0.43, respectively. The sensitivity, specificity, PPV, and negative predictive value of CAD and the three radiologists' combined reading were 0.95, 0.88, 0.86, and 0.96 and 0.70, 0.69, 0.64, and 0.75, respectively. Conclusion CAD could increase PPV and reduce the false-positive rate in the early diagnosis of lung cancer. © RSNA, 2017 Online supplemental material is available for this article.

Correction to: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

[This corrects the article DOI: 10.1186/s12014-018-9197-x.].

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. METHODS: In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. RESULTS: We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. CONCLUSIONS: Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

Unique pulmonary immunotoxicological effects of urban PM are not recapitulated solely by carbon black, diesel exhaust or coal fly ash.

BACKGROUND: Exposure to particulate matter (PM) is increasing worldwide as a result of increased human activity, the rapid industrialization of developing countries, and effects of climate change. Adverse effects of PM on human health are well documented, and because PM exposure occurs mostly through the airways, PM has especially deleterious impact on the lungs. OBJECTIVE: We investigated whether surrogate PM particles like carbon black (CB), diesel exhaust particle (DEP), coal fly ash (CFA) can recapitulate the allergic airway inflammatory response induced by urban particulate matter. METHODS: We compared the pro-inflammatory potential of urban PM collected from New York (NYC) and Baltimore (Balt) with CB, DEP and CFA surrogate PM particles. Eight to ten weeks old BALB/cJ mice were exposed through the airways to particulate material, and markers of airway inflammation were determined. Specifically, we assessed cellular influx, mucus production, lung function, cytokine levels as well as immune cell profiling of the lungs. RESULTS: Herein, we demonstrate that exposure to equivalent mass of stand-alone surrogate PM particles like CB, DEP and CFA, fails to induce significant airway inflammatory response seen after similar exposure to urban PMs. Specifically, we observe that PM collected from New York (NYC) and Baltimore city (Balt) triggers a mixed Th2/Th17 response accompanied by eosinophilic and neutrophilic influx, mucus production and airway hyperresponsiveness (AHR). Although the immune profile of NYC and Baltimore PMs are similar, they demonstrate considerable differences in their potency. Baltimore PM induced more robust airway inflammation, AHR, and Th2 cytokine production, possibly due to the greater metal content in Baltimore PM. CONCLUSIONS: Urban particulate matter with its unique physiochemical properties and heterogeneous composition elicits a mixed Th2/Th17 allergic airway response that is not seen after similar exposures to surrogate PM particles.

Heterogeneous expression of PD-L1 in pulmonary squamous cell carcinoma and adenocarcinoma: implications for assessment by small biopsy.

Predicting response to checkpoint blockade therapy for lung cancer has largely focused on measuring programmed death-ligand 1 (PD-L1) expression on tumor cells. PD-L1 expression is geographically heterogeneous within many tumors, however, and we questioned whether small tissue samples, such as biopsies, might be sufficiently representative of PD-L1 expression for evaluating this marker in lung cancer tumors. To evaluate the extent of variability of PD-L1 expression in small tissue samples, and how that variability affects accuracy of overall assessment of PD-L1 in lung cancer, we scored immunohistochemical staining for PD-L1 in tissue microarray cores from a series of 79 squamous cell lung cancers and 71 pulmonary adenocarcinomas. Our study found substantial inconsistencies for the percentages of cells staining positive for PD-L1 among different tissue microarray cores in many cases of both adenocarcinoma and squamous cell carcinoma. This variable scoring was seen at both high levels and low levels of PD-L1 expression, and by further evaluation of cases with discordant results on full-face sections to assess geographic distribution of staining, we found that discordant results among different tissue microarray cores reflected geographic variation of PD-L1 expression in those tumors. Moreover, we found that as a result of heterogeneous expression, the sensitivity of a single small tissue sample can be as low as 85% for detecting PD-L1 expression at scoring thresholds commonly used in clinical practice. Based on these studies, we conclude that many cases of lung cancer could be inaccurately or variably scored for PD-L1 expression with a single biopsy sample. Accordingly, lung cancer patients can be inconsistently classified for PD-L1 expression status, particularly when a threshold for the percentage of positive cells is used to determine eligibility for checkpoint blockade therapy.

Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers.

Long interspersed element-1 protein expression is a hallmark of many human cancers.

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen.

Intraoperative Assessment of Final Margins with a Handheld Optical Imaging Probe During Breast-Conserving Surgery May Reduce the Reoperation Rate: Results of a Multicenter Study.

BACKGROUND: A multicenter, prospective, blinded study was performed to test the feasibility of using a handheld optical imaging probe for the intraoperative assessment of final surgical margins during breast-conserving surgery (BCS) and to determine the potential impact on patient outcomes. METHODS: Forty-six patients with early-stage breast cancer (one with bilateral disease) undergoing BCS at two study sites, the Johns Hopkins Hospital and Anne Arundel Medical Center, were enrolled in this study. During BCS, cavity-shaved margins were obtained and the final margins were examined ex vivo in the operating room with a probe incorporating optical coherence tomography (OCT) hardware and interferometric synthetic aperture microscopy (ISAM) image processing. Images were interpreted after BCS by three physicians blinded to final pathology-reported margin status. Individual and combined interpretations were assessed. Results were compared to conventional postoperative histopathology. RESULTS: A total of 2,191 images were collected and interpreted from 229 shave margin specimens. Of the eight patients (17 %) with positive margins (0 mm), which included invasive and in situ diseases, the device identified all positive margins in five (63%) of them; reoperation could potentially have been avoided in these patients. Among patients with pathologically negative margins (>0 mm), an estimated mean additional tissue volume of 10.7 ml (approximately 1% of overall breast volume) would have been unnecessarily removed due to false positives. CONCLUSIONS: Intraoperative optical imaging of specimen margins with a handheld probe potentially eliminates the majority of reoperations.

High levels of the Mps1 checkpoint protein are protective of aneuploidy in breast cancer cells.

Most human cancers are aneuploid and have chromosomal instability, which contrasts to the inability of human cells to normally tolerate aneuploidy. Noting that aneuploidy in human breast cancer correlates with increased expression levels of the Mps1 checkpoint gene, we investigated whether these high levels of Mps1 contribute to the ability of breast cancer cells to tolerate this aneuploidy. Reducing Mps1 levels in cultured human breast cancer cells by RNAi resulted in aberrant mitoses, induction of apoptosis, and decreased ability of human breast cancer cells to grow as xenografts in nude mice. Remarkably, breast cancer cells that survive reductions in levels of Mps1 have relatively less aneuploidy, as measured by copies of specific chromosomes, compared with cells that have constitutively high levels of Mps1. Thus, high levels of Mps1 in breast cancer cells likely contribute to these cells tolerating aneuploidy.

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells.

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

A Large Postmortem Database of COVID-19 Patients Can Inform Disease Research and Public Policy Decision Making.

CONTEXT.­: Autopsies performed on COVID-19 patients have provided critical information about SARS-CoV-2's tropism, mechanisms of tissue injury, and the spectrum of disease. OBJECTIVE.­: To provide an updated database of postmortem disease in COVID-19 patients, assess relationships among clinical and pathologic variables, evaluate the accuracy of death certification, and correlate disease variables to causes of death. DESIGN.­: The 272 postmortem examinations reported in this paper were submitted by 14 pathologists from 9 medical or forensic institutions across the United States. The study spans the eras of the 3 principal COVID-19 strains and incorporates surveyed demographic, clinical, and postmortem data from decedents infected with SARS-CoV-2, including primary and contributing causes of death. It is the largest database of its kind to date. RESULTS.­: Demographics of the decedents reported here correspond well to national statistics. Primary causes of death as determined by autopsy and official death certificates were significantly correlated. When specifically cited disease conditions found at autopsy were correlated with COVID-19 versus non-COVID-19 death, only lung findings characteristic of SARS-CoV-2 infection or the absence of lung findings were significantly associated. CONCLUSIONS.­: Changes in hospitalization and disease likely stem from longer lifespans after COVID-19 diagnosis and alteration in treatment approaches. Although Omicron variants preferentially replicate in the upper airways, autopsied patients who died of COVID-19 in that time period showed the same lung damage as earlier decedents. Most importantly, findings suggest that there are still unelucidated risk factors for death from COVID-19 including possibly genetic susceptibility.