A color-based tumor segmentation method for clinical ex vivo breast tissue assessment utilizing a multi-contrast brightfield imaging strategy.

We demonstrate an automated two-step tumor segmentation method leveraging color information from brightfield images of fresh core needle biopsies of breast tissue. Three different color spaces (HSV, CIELAB, YCbCr) were explored for the segmentation task. By leveraging white-light and green-light images, we identified two different types of color transformations that could separate adipose from benign and tumor or cancerous tissue. We leveraged these two distinct color transformation methods in a two-step process where adipose tissue segmentation was followed by benign tissue segmentation thereby isolating the malignant region of the biopsy. Our tumor segmentation algorithm and imaging probe could highlight suspicious regions on unprocessed biopsy tissue to guide selection of areas most similar to malignant tissues for tissue pathology whether it be formalin fixed or frozen sections, expedite tissue selection for molecular testing, detect positive tumor margins, or serve an alternative to tissue pathology, in countries where these services are lacking.

After neoadjuvant therapy, axillary sentinel lymph node frozen sections from breast cancer patients are accurately diagnosed using telepathology.

Context: Telepathology is a digital, microscope-independent method of diagnosing pathology from scanned slides. Frozen sections (FS) can be performed and read by a pathologist at any site. At our institution, telepathology is used for diagnosis of frozen sections of sentinel lymph nodes (SLN) in patients who have undergone neoadjuvant chemotherapy and are enrolled in a clinical trial. Objective: We investigated the accuracy of diagnosing SLN frozen sections in the neoadjuvant setting using telepathology. Design: SLN were entirely submitted for frozen section. A pathology assistant prepared the frozen and scanned the slides using VisionTek M6 digital microscope ecosystem (East Dundee, IL). Cases were interpreted by trained, board-certified pathologists. All frozen sections remnants were submitted for formalin-fixed paraffin-embedded permanent sections. Frozen section diagnoses using telepathology were compared to final pathology. Turn-around time from specimen collection to frozen section diagnosis was recorded. Results: 54 SLN from 22 breast neoadjuvant cases were diagnosed via telepathology from March 2017 to July 2019. 95% of SLNs interpreted as negative on frozen section and on permanents. A definitive diagnosis could not be rendered on six SLNs; diagnosed "atypical" at frozen. Sensitivity and specificity were 80% and 100% respectively with accuracy of 95.8%. The false-negative rate was 5%. There were no false positives. The average turn-around time was over an hour. Conclusions: Telepathology is an accurate method of diagnosing SLN frozen sections in the neoadjuvant setting, but lobular carcinomas and treatment effect pose diagnostic challenges and the time to report results is increased compared to standard microscopy.

A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

Novel temporal and spatial patterns of metastatic colonization from breast cancer rapid-autopsy tumor biopsies.

BACKGROUND: Metastatic breast cancer is a deadly disease with a low 5-year survival rate. Tracking metastatic spread in living patients is difficult and thus poorly understood. METHODS: Via rapid autopsy, we have collected 30 tumor samples over 3 timepoints and across 8 organs from a triple-negative metastatic breast cancer patient. The large number of sites sampled, together with deep whole-genome sequencing and advanced computational analysis, allowed us to comprehensively reconstruct the tumor's evolution at subclonal resolution. RESULTS: The most unique, previously unreported aspect of the tumor's evolution that we observed in this patient was the presence of "subclone incubators," defined as metastatic sites where substantial tumor evolution occurs before colonization of additional sites and organs by subclones that initially evolved at the incubator site. Overall, we identified four discrete waves of metastatic expansions, each of which resulted in a number of new, genetically similar metastasis sites that also enriched for particular organs (e.g., abdominal vs bone and brain). The lung played a critical role in facilitating metastatic spread in this patient: the lung was the first site of metastatic escape from the primary breast lesion, subclones at this site were likely the source of all four subsequent metastatic waves, and multiple sites in the lung acted as subclone incubators. Finally, functional annotation revealed that many known drivers or metastasis-promoting tumor mutations in this patient were shared by some, but not all metastatic sites, highlighting the need for more comprehensive surveys of a patient's metastases for effective clinical intervention. CONCLUSIONS: Our analysis revealed the presence of substantial tumor evolution at metastatic incubator sites in a patient, with potentially important clinical implications. Our study demonstrated that sampling of a large number of metastatic sites affords unprecedented detail for studying metastatic evolution.

Histology to 3D in vivo MR registration for volumetric evaluation of MRgFUS treatment assessment biomarkers.

Advances in imaging and early cancer detection have increased interest in magnetic resonance (MR) guided focused ultrasound (MRgFUS) technologies for cancer treatment. MRgFUS ablation treatments could reduce surgical risks, preserve organ tissue and function, and improve patient quality of life. However, surgical resection and histological analysis remain the gold standard to assess cancer treatment response. For non-invasive ablation therapies such as MRgFUS, the treatment response must be determined through MR imaging biomarkers. However, current MR biomarkers are inconclusive and have not been rigorously evaluated against histology via accurate registration. Existing registration methods rely on anatomical features to directly register in vivo MR and histology. For MRgFUS applications in anatomies such as liver, kidney, or breast, anatomical features that are not caused by the treatment are often insufficient to drive direct registration. We present a novel MR to histology registration workflow that utilizes intermediate imaging and does not rely on anatomical MR features being visible in histology. The presented workflow yields an overall registration accuracy of 1.00 ± 0.13 mm. The developed registration pipeline is used to evaluate a common MRgFUS treatment assessment biomarker against histology. Evaluating MR biomarkers against histology using this registration pipeline will facilitate validating novel MRgFUS biomarkers to improve treatment assessment without surgical intervention. While the presented registration technique has been evaluated in a MRgFUS ablation treatment model, this technique could be potentially applied in any tissue to evaluate a variety of therapeutic options.

Spatially-resolved quantification of proteins in triple negative breast cancers reveals differences in the immune microenvironment associated with prognosis.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype. Recent studies have shown that MHC class II (MHCII) expression and tumor infiltrating lymphocytes are important prognostic factors in patients with TNBC, although the relative importance of lymphocyte subsets and associated protein expression is incompletely understood. NanoString Digital Spatial Profiling (DSP) allows for spatially resolved, highly multiplexed quantification of proteins in clinical samples. In this study, we sought to determine if DSP could be used to characterize expression of MHCII and other immune related proteins in tumor epithelial versus stromal compartments of patient-derived TNBCs (N = 10) using a panel of 39 markers. We confirmed that a subset of TNBCs have elevated expression of HLA-DR in tumor epithelial cells; HLA-DR expression was also significantly higher in the tumors of patients with long-term disease-free survival when compared to patients that relapsed. HLA-DR expression in the epithelial compartment was correlated with high expression of CD4 and ICOS in the stromal compartment of the same tumors. We also identified candidate protein biomarkers with significant differential expression between patients that relapsed versus those that did not. In conclusion, DSP is a powerful method that allows for quantification of proteins in the immune microenvironment of TNBCs.

A Multigene Assay Determines Risk of Recurrence in Patients with Triple-Negative Breast Cancer.

Approximately 40% of patients with stage I-III triple-negative breast cancer (TNBC) recur after standard treatment, whereas the remaining 60% experience long-term disease-free survival (DFS). There are currently no clinical tests to assess the risk of recurrence in TNBC patients. We previously determined that TNBC patients with MHC class II (MHCII) pathway expression in their tumors experienced significantly longer DFS. To translate this discovery into a clinical test, we developed an MHCII Immune Activation assay, which measures expression of 36 genes using NanoString technology. Preanalytical testing confirmed that the assay is accurate and reproducible in formalin-fixed paraffin-embedded (FFPE) tumor specimens. The assay measurements were concordant with RNA-seq, MHCII protein expression, and tumor-infiltrating lymphocyte counts. In a training set of 44 primary TNBC tumors, the MHCII Immune Activation Score was significantly associated with longer DFS (HR = 0.17; P = 0.015). In an independent validation cohort of 56 primary FFPE TNBC tumors, the Immune Activation Score was significantly associated with longer DFS (HR = 0.19; P = 0.011) independent of clinical stage. An Immune Activation Score threshold for identifying patients with very low risk of relapse in the training set provided 100% specificity in the validation cohort. The assay format enables adoption as a standardized clinical prognostic test for identifying TNBC patients with a low risk of recurrence. Correlative data support future studies to determine if the assay can identify patients in whom chemotherapy can be safely deescalated and patients likely to respond to immunotherapy. SIGNIFICANCE: The MHCII Immune Activation assay identifies TNBC patients with a low risk of recurrence, addressing a critical need for prognostic biomarker tests that enable precision medicine for TNBC patients.

The FDA-Approved Breast Cancer HER2 Evaluation Kit (HercepTest; Dako) May Miss Some HER2-Positive Breast Cancers.

OBJECTIVES: Accurate evaluation of human epidermal growth factor receptor 2 (HER2) in breast cancer is critical. METHODS: HER2 fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) tests were performed on 52 cases using a US Food and Drug Administration (FDA)-approved kit (HercepTest, FDA kit) and a laboratory-developed test (LDT) with the HercepTest antibody and a Leica Bond automated stainer. RESULTS: By FISH, 22 were HER2 positive, 29 were negative, and one was equivocal. Of the 22 HER2 FISH-positive cases, five were negative by the FDA kit and none by LDT. The five discrepant cases were retested using the same FDA kit in another Clinical Laboratory Improvement Amendments-certified laboratory, and all five cases were still negative. None of the 29 HER2 FISH-negative cases were positive by the FDA kit or LDT. The overall IHC-FISH concordance rate was 90.4% for the FDA kit and 100% for the LDT. CONCLUSIONS: The FDA kit may miss some HER2-positive cases. The LDT has a higher sensitivity and a higher concordance rate with FISH results.

Differences in molecular features of triple-negative breast cancers based on the age at diagnosis.

BACKGROUND: Although the proportion of triple-negative breast cancers (TNBCs) diagnosed among older women is low, the number of TNBC cases is substantial because of the high incidence of breast cancer after the age of 65 years. The molecular features of TNBC in this age group have not been well described. METHODS: This study examined a population-based cohort of women with stage I to III TNBC diagnosed between the ages of 25 and 91 years with the PAM50 gene expression subtyping assay. The concordance between the TNBC classification by immunohistochemistry and the gene expression classification by PAM50, the expression of individual genes, and 5-year recurrence and breast cancer mortality in older women (≥65 years old) and younger women (<50 years old) was assessed. RESULTS: The molecular subtype distribution in TNBC was significantly different according to the age at diagnosis. TNBC was more likely to be classified as basal-like in women younger than 50 years (sensitivity, 0.91; 95% confidence interval, 0.77-0.97) than women 65 years old or older (sensitivity, 0.72; 95% confidence interval, 0.48-0.87); 35% of clinical TNBC cases in the latter group were the human epidermal growth factor receptor 2 (HER2)-enriched subtype by molecular classification. Older women with TNBC also had significantly higher expression of ERBB2 and lower expression of all 10 proliferation-associated genes tested (P < .01). The risk of breast cancer death within 5 years was significantly higher in women with TNBC in comparison with women with hormone receptor-positive cancers in all age groups. CONCLUSIONS: This study revealed differences in molecular subtypes among clinical TNBC cases based on patient age. A potentially targetable HER2-enriched group raises the possible need for intrinsic subtyping in older women with TNBC.

Rapid On-Site Evaluation of Fine-Needle Aspiration by Non-Cytopathologists: A Systematic Review and Meta-Analysis of Diagnostic Accuracy Studies for Adequacy Assessment.

OBJECTIVE: Rapid on-site evaluation (ROSE) has been shown to improve adequacy rates and reduce needle passes. ROSE is often performed by cytopathologists who have limited availability and may be costlier than alternatives. Several recent studies examined the use of alternative evaluators (AEs) for ROSE. A summary of this information could help inform guidelines regarding the use of AEs. The objective was to assess the accuracy of AEs compared to cytopathologists in assessing the adequacy of specimens during ROSE. STUDY DESIGN: This was a systematic review and meta-analysis. Reporting and study quality were assessed using the STARD guidelines and QUADAS-2. All steps were performed independently by two evaluators. Summary estimates were obtained using the hierarchal method in Stata v14. Heterogeneity was evaluated using Higgins' I2 statistic. RESULTS: The systematic review identified 13 studies that were included in the meta-analysis. Summary estimates of sensitivity and specificity for AEs were 97% (95% CI: 92-99%) and 83% (95% CI: 68-92%). There was wide variation in accuracy statistics between studies (I2 = 0.99). CONCLUSIONS: AEs sometimes have accuracy that is close to cytopathologists. However, there is wide variability between studies, so it is not possible to provide a broad guideline regarding the use of AEs.

Evaluation of the SharkCore® needle for EUS-guided core biopsy of pancreatic neuroendocrine tumors.

BACKGROUND AND OBJECTIVES: EUS guided core biopsy was once rarely performed but is now entering mainstream practice. Neuroendocrine tumors often warrant core biopsy as sufficient tissue must be obtained to allow for special staining to ensure a correct diagnosis. Traditionally these lesions were sampled with FNA needles. We performed a retrospective pilot study to evaluate the clinical value and efficacy of the a new EUS core needle biopsy needle as compared to a standard EUS FNA needle in the evaluation of patients with known or suspected neuroendocrine tumors. METHODS: A retrospective analysis of the first 10 patients (between January 2015 and April 2016) to undergo EUS-FNA with the SharkCore® needle at the University of Utah School of Medicine/Huntsman Cancer Center with neuroendocrine tumors. Each case was retrospectively reviewed by a board certified cytopathologist (BLW) for the following cytologic parameters on the aspirate smears or touch/squash preparations: overall cellularity [1 (low) to 3 (high)], percentage of obtained cells that were lesional/representative (<25%, 26%-50%, and >50%), relative ease of interpretation [1 (difficult) to 3 (easy)]. Pathologic material and reporting records were also reviewed for each case to confirm the number of needle passes to achieve diagnostic adequacy, the presence or absence diagnostic material on H&E slide (from cell block, if prepared), whether a definitive diagnosis was able to be rendered, and the presence or absence of a true core/core fragments (within the cell block, if prepared). RESULTS: A total of 20 patients underwent EUS-FNA for suspected neuroendocrine lesions. Ten patients underwent either transgastric or transduodenal EUS-FNA with the 22 gauge SharkCore® needle. The comparison cohort of 10 patients underwent either transgastric or transduodenal EUS-FNA with the standard 22 gauge Echotip® needle. The SharkCore® needle required a fewer mean number of needle passes to obtain diagnostic adequacy than the Echotip® (P=0.0074). For cases with cell blocks, the SharkCore® needle produced diagnostic material in 100% of cases, whereas Echotip® produced diagnostic material in 60% of cases. There was no significant difference between specimen cellularity, percentage of lesional material, or ease of interpretation between the two needle types. CONCLUSION: Our pilot investigation targeting patients with known or suspected pancreatic NETs indicates that the SharkCore® needle shows promise in obtaining suitable tissue for ancillary testing that can allow for more definitive pathologic interpretations on EUS FNA specimens. Fewer passes were needed with the core needle when compared to a standard needle.

Publisher Correction: Combating subclonal evolution of resistant cancer phenotypes.

The originally published version of this Article contained an error in Figure 4. In panel a, grey boxes surrounding the subclones associated with patients #2 and #4 obscured adjacent portions of the heatmap. This error has now been corrected in both the PDF and HTML versions of the Article.

BRCA1 through Its E3 Ligase Activity Regulates the Transcription Factor Oct1 and Carbohydrate Metabolism.

The tumor suppressor BRCA1 regulates the DNA damage response (DDR) and other processes that remain incompletely defined. Among these, BRCA1 heterodimerizes with BARD1 to ubiquitylate targets via its N-terminal E3 ligase activity. Here, it is demonstrated that BRCA1 promotes oxidative metabolism by degrading Oct1 (POU2F1), a transcription factor with proglycolytic and tumorigenic effects. BRCA1 E3 ubiquitin ligase mutation skews cells toward a glycolytic metabolic profile while elevating Oct1 protein. CRISPR-mediated Oct1 deletion reverts the glycolytic phenotype. RNA sequencing (RNAseq) confirms deregulation of metabolic genes downstream of Oct1. BRCA1 mediates Oct1 ubiquitylation and degradation, and mutation of two ubiquitylated Oct1 lysines insulates the protein against BRCA1-mediated destabilization. Oct1 deletion in MCF-7 breast cancer cells does not perturb growth in standard culture, but inhibits growth in soft agar and xenograft assays. In primary breast cancer clinical specimens, Oct1 protein levels correlate positively with tumor aggressiveness and inversely with BRCA1. These results identify BRCA1 as an Oct1 ubiquitin ligase that catalyzes Oct1 degradation to promote oxidative metabolism and restrict tumorigenicity. Mol Cancer Res; 16(3); 439-52. ©2018 AACR.

X-Ray of Excised Cancerous Breast Tissue Does Not Affect Clinical Biomarker Expression.

CONTEXT: College of American Pathologists (CAP) and the American Society of Clinical Oncology have emphasized the need to reduce preanalytic variables for evaluating predictive biomarker expression in breast cancer. Postoperative x-ray of excised breast tissue is commonplace, yet is a variable that has not been investigated previously. We asked whether such radiation affects expression of relevant biomarkers. DESIGN: A previous study found that human breast cancers grown in mice demonstrate the same immunohistochemical and molecular profiles as the original tumors. Thirteen patient-derived xenografts were harvested fresh and divided for specimen radiography and a matched nonirradiated control, while following CAP/ASCO guidelines for cold ischemia time and fixation. Samples were processed in a tissue microarray for immunohistochemistry. Estrogen receptor (ER), progesterone receptor (PR), p53, and Ki67 staining was evaluated using an optimized scoring algorithm performed on digitally scanned slides. Samples were also scored manually by a blinded pathologist using the H-score method, and HER2 by the CAP/ASCO 2013 protocol. Histologic scores were compared by analysis of variance. RESULTS: There was no significant difference in quantity or intensity of staining between irradiated and nonirradiated samples for estrogen receptor (P=0.28), p53 (P=0.96), and Ki67 (P=0.94). A small but statistically significant difference was observed for PR (P=0.0058). HER2 staining was similarly unchanged in the 1 tumor exhibiting 3+ staining. CONCLUSIONS: Our study demonstrates that x-ray of breast carcinomas does not significantly affect the expression of predictive biomarkers, with the exception of PR for unclear reasons. It also highlights the utility of the patient-derived xenograft model for biomarker studies.

Combating subclonal evolution of resistant cancer phenotypes.

Metastatic breast cancer remains challenging to treat, and most patients ultimately progress on therapy. This acquired drug resistance is largely due to drug-refractory sub-populations (subclones) within heterogeneous tumors. Here, we track the genetic and phenotypic subclonal evolution of four breast cancers through years of treatment to better understand how breast cancers become drug-resistant. Recurrently appearing post-chemotherapy mutations are rare. However, bulk and single-cell RNA sequencing reveal acquisition of malignant phenotypes after treatment, including enhanced mesenchymal and growth factor signaling, which may promote drug resistance, and decreased antigen presentation and TNF-α signaling, which may enable immune system avoidance. Some of these phenotypes pre-exist in pre-treatment subclones that become dominant after chemotherapy, indicating selection for resistance phenotypes. Post-chemotherapy cancer cells are effectively treated with drugs targeting acquired phenotypes. These findings highlight cancer's ability to evolve phenotypically and suggest a phenotype-targeted treatment strategy that adapts to cancer as it evolves.

HER2 immunohistochemical and fluorescence in situ hybridization discordances in invasive breast carcinoma with micropapillary features.

The 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations for HER2 testing contain a recommendation for pathologists with respect to invasive micropapillary carcinoma. The guidelines suggest that HER2 immunohistochemical staining that is intense but incomplete and would be considered 1+ may actually be HER2-amplified by fluorescence in situ hybridization. Thus, pathologists should consider reporting the immunohistochemistry as equivocal (2+) and employ an alternative testing methodology. This recommendation is based largely on one paper wherein the authors tested a series of 22 micropapillary carcinomas that were considered 1+ by immunohistochemistry and identified HER2 amplification in one case (5%). In order to assess for a possible discordance between HER2 immunohistochemistry and fluorescence in situ hybridization, we evaluated a series of invasive carcinomas with micropapillary features using both methodologies. As described by the WHO, invasive carcinomas with micropapillary features have small, hollow, or morula-like clusters of cells surrounded by clear stromal spaces. All cases had HER2 immunohistochemistry and fluorescence in situ hybridization performed, and for cases with equivocal fluorescence in situ hybridization results, an alternative Chromosome 17 probe (RAI1) was employed. All assays were scored according to the 2013 ASCO/CAP guidelines. Specifically for this study, immunohistochemistry was scored irrespective of the presence of micropapillary features. Overall, we identified HER2 amplification in 21 (47%) of the cases assayed, with the corresponding immunohistochemistry being 1+ (n=9), 2+ (n=11), and 3+ (n=1). The ASCO/CAP recommendation that this morphology may deviate from the typical staining pattern is highlighted, as we found that 43% of cases with micropapillary features and HER2 staining that would otherwise be scored as 1+ were HER2-amplified by fluorescence in situ hybridization. This study supports the ASCO/CAP recommendation that pathologists should consider reporting immunohistochemistry in this morphology as equivocal and perform reflex testing using in situ hybridization.

Window-of-Opportunity Study of Valproic Acid in Breast Cancer Testing a Gene Expression Biomarker.

PURPOSE: The anticancer activity of valproic acid (VPA) is attributed to the inhibition of histone deacetylase. We previously published the genomically derived sensitivity signature for VPA (GDSS-VPA), a gene expression biomarker that predicts breast cancer sensitivity to VPA in vitro and in vivo. We conducted a window-of-opportunity study that examined the tolerability of VPA and the ability of the GDSS-VPA to predict biologic changes in breast tumors after treatment with VPA. PATIENTS AND METHODS: Eligible women had untreated breast cancer with breast tumors larger than 1.5 cm. After a biopsy, women were given VPA for 7 to 12 days, increasing from 30 mg/kg/d orally divided into two doses per day to a maximum of 50 mg/kg/d. After VPA treatment, serum VPA level was measured and then breast surgery or biopsy was performed. Tumor proliferation was assessed by using Ki-67 immunohistochemistry. Histone acetylation of peripheral blood mononuclear cells was assessed by Western blot. Dynamic contrast-enhanced magnetic resonance imaging scans were performed before and after VPA treatment. RESULTS: Thirty women were evaluable. The median age was 54 years (range, 31-73 years). Fifty-two percent of women tolerated VPA at 50 mg/kg/d, but 10% missed more than two doses as a result of adverse events. Grade 3 adverse events included vomiting and diarrhea (one patient) and fatigue (one patient). The end serum VPA level correlated with a change in histone acetylation of peripheral blood mononuclear cells (ρ = 0.451; P = .024). Fifty percent of women (three of six) with triple-negative breast cancer had a Ki-67 reduction of at least 10% compared with 17% of other women. Women whose tumors had higher GDSS-VPA were more likely to have a Ki-67 decrease of at least 10% (area under the curve, 0.66). CONCLUSION: VPA was well tolerated and there was a significant correlation between serum VPA levels and histone acetylation. VPA treatment caused a decrease in proliferation of breast tumors. The genomic biomarker correlated with decreased proliferation. Inhibition of histone deacetylase is a valid strategy for drug development in triple-negative breast cancer using gene expression biomarkers.

Statistical Literacy Among Academic Pathologists: A Survey Study to Gauge Knowledge of Frequently Used Statistical Tests Among Trainees and Faculty.

CONTEXT: -Statistical literacy can be defined as understanding the statistical tests and terminology needed for the design, analysis, and conclusions of original research or laboratory testing. Little is known about the statistical literacy of clinical or anatomic pathologists. OBJECTIVE: -To determine the statistical methods most commonly used in pathology studies from the literature and to assess familiarity and knowledge level of these statistical tests by pathology residents and practicing pathologists. DESIGN: -The most frequently used statistical methods were determined by a review of 1100 research articles published in 11 pathology journals during 2015. Familiarity with statistical methods was determined by a survey of pathology trainees and practicing pathologists at 9 academic institutions in which pathologists were asked to rate their knowledge of the methods identified by the focused review of the literature. RESULTS: -We identified 18 statistical tests that appear frequently in published pathology studies. On average, pathologists reported a knowledge level between "no knowledge" and "basic knowledge" of most statistical tests. Knowledge of tests was higher for more frequently used tests. Greater statistical knowledge was associated with a focus on clinical pathology versus anatomic pathology, having had a statistics course, having an advanced degree other than an MD degree, and publishing research. Statistical knowledge was not associated with length of pathology practice. CONCLUSIONS: -An audit of pathology literature reveals that knowledge of about 12 statistical tests would be sufficient to provide statistical literacy for pathologists. On average, most pathologists report they can interpret commonly used tests but are unable to perform them. Most pathologists indicated that they would benefit from additional statistical training.

The molecular basis of breast cancer pathological phenotypes.

The histopathological evaluation of morphological features in breast tumours provides prognostic information to guide therapy. Adjunct molecular analyses provide further diagnostic, prognostic and predictive information. However, there is limited knowledge of the molecular basis of morphological phenotypes in invasive breast cancer. This study integrated genomic, transcriptomic and protein data to provide a comprehensive molecular profiling of morphological features in breast cancer. Fifteen pathologists assessed 850 invasive breast cancer cases from The Cancer Genome Atlas (TCGA). Morphological features were significantly associated with genomic alteration, DNA methylation subtype, PAM50 and microRNA subtypes, proliferation scores, gene expression and/or reverse-phase protein assay subtype. Marked nuclear pleomorphism, necrosis, inflammation and a high mitotic count were associated with the basal-like subtype, and had a similar molecular basis. Omics-based signatures were constructed to predict morphological features. The association of morphology transcriptome signatures with overall survival in oestrogen receptor (ER)-positive and ER-negative breast cancer was first assessed by use of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset; signatures that remained prognostic in the METABRIC multivariate analysis were further evaluated in five additional datasets. The transcriptomic signature of poorly differentiated epithelial tubules was prognostic in ER-positive breast cancer. No signature was prognostic in ER-negative breast cancer. This study provided new insights into the molecular basis of breast cancer morphological phenotypes. The integration of morphological with molecular data has the potential to refine breast cancer classification, predict response to therapy, enhance our understanding of breast cancer biology, and improve clinical management. This work is publicly accessible at www.dx.ai/tcga_breast. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.