The 1000 Mitoses Project: A Consensus-Based International Collaborative Study on Mitotic Figures Classification.

Introduction. The identification of mitotic figures is essential for the diagnosis, grading, and classification of various different tumors. Despite its importance, there is a paucity of literature reporting the consistency in interpreting mitotic figures among pathologists. This study leverages publicly accessible datasets and social media to recruit an international group of pathologists to score an image database of more than 1000 mitotic figures collectively. Materials and Methods. Pathologists were instructed to randomly select a digital slide from The Cancer Genome Atlas (TCGA) datasets and annotate 10-20 mitotic figures within a 2 mm2 area. The first 1010 submitted mitotic figures were used to create an image dataset, with each figure transformed into an individual tile at 40x magnification. The dataset was redistributed to all pathologists to review and determine whether each tile constituted a mitotic figure. Results. Overall pathologists had a median agreement rate of 80.2% (range 42.0%-95.7%). Individual mitotic figure tiles had a median agreement rate of 87.1% and a fair inter-rater agreement across all tiles (kappa = 0.284). Mitotic figures in prometaphase had lower percentage agreement rates compared to other phases of mitosis. Conclusion. This dataset stands as the largest international consensus study for mitotic figures to date and can be utilized as a training set for future studies. The agreement range reflects a spectrum of criteria that pathologists use to decide what constitutes a mitotic figure, which may have potential implications in tumor diagnostics and clinical management.

The Ansa Hypoglossi: Quantifying Axonal Density of a Donor Nerve for Facial Reinnervation.

Background: There are a number of nerve grafting options for facial reanimation and the ansa hypoglossi (AH) may be considered in select situations. Objective: To compare axonal density, area, and diameter of AH with other nerves more usually used for facial reanimation. Methods: AH specimens from patients undergoing neck dissections were submitted in formalin. Proximal to distal cross sections, nerve diameters, and the number of axons per nerve, proximally and distally, were measured and counted. Results: Eighteen nerve specimens were analyzed. The average manual axon count for the distal and proximal nerve sections was 1378 ± 333 and 1506 ± 306, respectively. The average QuPath counts for the proximal and distal nerve sections were 1381 ± 325 and 1470 ± 334, respectively. The mean nerve area of the proximal and distal nerve sections was 0.206 ± 0.01 and 0.22 ± 0.064 mm2, respectively. The mean nerve diameter for the proximal and distal nerve sections were 0.498 ± 0.121 and 0.526 ± 0.75 mm, respectively. Conclusion: The histological characteristics of the AH support clinical examination of outcomes as a promising option in facial reanimation.

Impact of COVID-19-related health care disruptions on pathologic cancer staging during the first pandemic year: a retrospective cohort study from March 2018 to March 2021.

BACKGROUND: The COVID-19 pandemic has created major disruptions in cancer care, with reductions in diagnostic tests and treatments. We evaluated the impact of these health care-related changes on cancer staging by comparing cancers staged before and during the pandemic. METHODS: We performed a retrospective cohort study at London Health Sciences Centre and St. Joseph's Health Care London, London, Ontario, Canada. We evaluated all pathologically staged breast, colorectal, prostate, endometrial and lung cancers (the 5 most common cancers by site, excluding nonmelanoma skin cancer) over a 3-year period (Mar. 15, 2018-Mar. 14, 2021). The pre-COVID-19 group included procedures performed between Mar. 15, 2018, and Mar. 14, 2020, and the COVID-19 group included procedures performed between Mar. 15, 2020, and Mar. 14, 2021. The primary outcome was cancer stage group, based on the pathologic tumour, lymph node, metastasis system. We performed univariate analyses to compare demographic characteristics, pathologic features and cancer stage between the 2 groups. We performed multivariable ordinal regression analyses using the proportional odds model to evaluate the association between stage and timing of staging (before v. during the pandemic). RESULTS: There were 4055 cases across the 5 cancer sites. The average number of breast cancer staging procedures per 30 days increased during the pandemic compared to the yearly average in the pre-COVID-19 period (41.3 v. 39.6), whereas decreases were observed for endometrial cancer (15.9 v. 16.4), colorectal cancer (21.8 v. 24.3), prostate cancer (13.6 v. 18.5) and lung cancer (11.5 v. 15.9). For all cancer sites, there were no statistically significant differences in demographic characteristics, pathologic features or cancer stage between the 2 groups (p > 0.05). In multivariable regression analysis, for all cancer sites, cases staged during the pandemic were not associated with higher stage (breast: odds ratio [OR] 1.071, 95% confidence interval [CI] 0.826-1.388; colorectal: OR 1.201, 95% CI 0.869-1.661; endometrium: OR 0.792, 95% CI 0.495-1.252; prostate: OR 1.171, 95% CI 0.765-1.794; and lung: OR 0.826, 95% CI 0.535-1.262). INTERPRETATION: Cancer cases staged during the first year of the COVID-19 pandemic were not associated with higher stage; this likely reflects the prioritization of cancer procedures during times of reduced capacity. The impact of the pandemic period on staging procedures varied between cancer sites, which may reflect differences in clinical presentation, detection and treatment.

Impact of COVID-19 pandemic on neuropathology service: Experience at one Canadian center.

The COVID-19 pandemic has had a significant impact on medical services. Many countries postponed nonemergent procedures to preserve hospital resources for the unprecedented situation. Surgical backlogs caused by the COVID-19 pandemic have been evaluated by different groups. However, the impact of this pandemic on pathology and specifically neuropathology (NP) services has received limited attention. In this study, we reviewed all NP reports of the London Health Sciences Centre from January 2018 (2 years before the pandemic declaration) until the end of the year 2021. Demographic information and pathology details were collected. For tumors, site, histopathology types, and WHO grading were analyzed. In nontumoral specimens, pathological diagnoses were compared in pre- and postpandemic time. The total number of NP samples reached its lowest in April 2020, corresponding to the first Ontario provincial lockdown, and fluctuated throughout the studied period. Among the different types of NP surgical specimens, muscle and epilepsy-related specimens showed a more significant reduction, compared to neoplastic specimens. In 2020, the proportion of tumor specimens from patients older than 40 years of age increased. Similarly, the proportion of high-grade glioma and brain metastasis diagnoses also increased. Lastly, we observed a marked increase in biopsies for temporal arteritis and other inflammatory lesions.

Explainable artificial intelligence to predict the risk of side-specific extraprostatic extension in pre-prostatectomy patients.

INTRODUCTION: We aimed to develop an explainable machine learning (ML) model to predict side-specific extraprostatic extension (ssEPE) to identify patients who can safely undergo nerve-sparing radical prostatectomy using preoperative clinicopathological variables. METHODS: A retrospective sample of clinicopathological data from 900 prostatic lobes at our institution was used as the training cohort. Primary outcome was the presence of ssEPE. The baseline model for comparison had the highest performance out of current biopsy-derived predictive models for ssEPE. A separate logistic regression (LR) model was built using the same variables as the ML model. All models were externally validated using a testing cohort of 122 lobes from another institution. Models were assessed by area under receiver-operating-characteristic curve (AUROC), precision-recall curve (AUPRC), calibration, and decision curve analysis. Model predictions were explained using SHapley Additive exPlanations. This tool was deployed as a publicly available web application. RESULTS: Incidence of ssEPE in the training and testing cohorts were 30.7 and 41.8%, respectively. The ML model achieved AUROC 0.81 (LR 0.78, baseline 0.74) and AUPRC 0.69 (LR 0.64, baseline 0.59) on the training cohort. On the testing cohort, the ML model achieved AUROC 0.81 (LR 0.76, baseline 0.75) and AUPRC 0.78 (LR 0.75, baseline 0.70). The ML model was explainable, well-calibrated, and achieved the highest net benefit for clinically relevant cutoffs of 10-30%. CONCLUSIONS: We developed a user-friendly application that enables physicians without prior ML experience to assess ssEPE risk and understand factors driving these predictions to aid surgical planning and patient counselling (https://share.streamlit.io/jcckwong/ssepe/main/ssEPE_V2.py).

Evaluation of an open-source machine-learning tool to quantify bone marrow plasma cells.

AIMS: The objective of this study was to develop and validate an open-source digital pathology tool, QuPath, to automatically quantify CD138-positive bone marrow plasma cells (BMPCs). METHODS: We analysed CD138-scanned slides in QuPath. In the initial training phase, manual positive and negative cell counts were performed in representative areas of 10 bone marrow biopsies. Values from the manual counts were used to fine-tune parameters to detect BMPCs, using the positive cell detection and neural network (NN) classifier functions. In the testing phase, whole-slide images in an additional 40 cases were analysed. Output from the NN classifier was compared with two pathologist's estimates of BMPC percentage. RESULTS: The training set included manual counts ranging from 2403 to 17 287 cells per slide, with a median BMPC percentage of 13% (range: 3.1%-80.7%). In the testing phase, the quantification of plasma cells by image analysis correlated well with manual counting, particularly when restricted to BMPC percentages of <30% (Pearson's r=0.96, p<0.001). Concordance between the NN classifier and the pathologist whole-slide estimates was similarly good, with an intraclass correlation of 0.83 and a weighted kappa for the NN classifier of 0.80 with the first rater and 0.90 with the second rater. This was similar to the weighted kappa between the two human raters (0.81). CONCLUSIONS: This represents a validated digital pathology tool to assist in automatically and reliably counting BMPC percentage on CD138-stained slides with an acceptable error rate.

Performance of different criteria for refractory myasthenia gravis.

BACKGROUND AND PURPOSE: Defining refractory myasthenia gravis is important, as this can drive clinical decision making, for example, by escalating treatments in refractory individuals. There are several definitions of refractory myasthenia, and their performances have not been compared. Having valid and reliable criteria can help select patients in whom more aggressive treatments may be needed. METHODS: We applied five different refractory myasthenia criteria (Drachman, Mantegazza, Suh, the International Consensus Guideline (ICG), and the randomised controlled trial of eculizumab in refractory, anti-acetylcholine receptor positive, generalised myasthenia gravis (REGAIN), to a cohort of 237 patients. We compared the proportion of refractory patients among different criteria and their scores on disease severity, fatigue, and quality-of-life (QoL) scales. We also assessed the agreement for each criterion between two trained assessors. RESULTS: The Drachman, Mantegazza, and Suh criteria resulted in high proportions of refractory individuals (40.1%, 39.2%, and 38.8%, respectively), compared with the ICG and REGAIN criteria (9.7% and 3.0%, respectively). Refractory patients by the ICG and REGAIN criteria had worse disease severity, QoL, and fatigue scores, compared with patients classified as refractory by other criteria. All criteria had high agreement between raters (between 70% and 100%). CONCLUSIONS: There is high variability in the proportion of refractory myasthenia gravis patients depending on the criteria used, with ICG and REGAIN criteria capturing patients with worse disease severity. This reflects conceptual differences as to what refractory means. Further multicenter studies are needed to determine appropriate criteria for refractory myasthenia gravis.

Patient-acceptable symptom states in myasthenia gravis.

OBJECTIVES: To estimate patient-acceptable symptom state (PASS) cut points for myasthenia gravis (MG) health scales. METHODS: We conducted an electronic survey that included the Myasthenia Gravis Impairment Index (MGII), EuroQol 5-Dimension (EQ5D), and a simple PASS question. PASS-anchored thresholds were estimated for the MGII questionnaire through receiver operating characteristic curves. We used the MGII PASS cut point in a validation cohort of 257 patients to estimate PASS thresholds for other clinically relevant health scales such as the Quantitative Myasthenia Gravis Scale (QMGS), Myasthenia Gravis Activities of Daily Living (MG-ADL), Myasthenia Gravis Composite (MGC), and Myasthenia Quality of Life (MG-QoL15). RESULTS: One hundred twenty-four of ≈250 invited patients answered the electronic survey (49% response rate), and 80 considered their current symptom state acceptable (PASS-positive). They had lower MGII scores than PASS-negative patients (7.76 ± 9.37 vs 25.0 ± 13.7, p < 0.0001) and better EQ5D scores (0.86 ± 0.17 vs 0.69 ± 0.18, p < 0.0001). The MGII questionnaire threshold for PASS was ≤10 points. With the use of this threshold in an independent dataset of 257 patients, all patients in remission or minimal manifestation status were PASS-positive. In addition, some patients in Classes I, II, and IIIA also achieved PASS status. PASS thresholds for the QMGS, MG-ADL, MGC, and MG-QoL15 were ≤7, 2, 3, and 8 points, respectively. CONCLUSIONS: We have estimated thresholds for commonly used myasthenia health scales reflecting patient-acceptable states in patients with MG. These thresholds indicate a global state of well being, rather than a change in scores, or being better. Therefore, PASS thresholds can be used as secondary endpoints for myasthenia research.

Evaluating the impact of lymph node resampling on colorectal cancer nodal stage.

AIMS: Nodal staging in colorectal cancer (CRC) informs prognosis and guides adjuvant treatment decisions. A standard minimum of 12 lymph nodes is widely used, with additional sampling being performed as required. However, there are few data on how lymph node resampling in this context has an impact on nodal stage. The aims of this study were to evaluate the effectiveness of resampling in detecting metastases and tumour deposits, and the impact on stage. METHODS AND RESULTS: A retrospective cohort analysis was performed on CRC resections that underwent resampling because of an initial yield of <12 lymph nodes, from 2008 to 2018. Data relating to patient demographics, specimen, malignancy and prosection were collected. Slides were reviewed to quantify nodal metastases and tumour deposits before and after resampling. Among ≥pN1 cases, logistic regression analysis was performed to evaluate factors that predicted the finding of additional metastases and tumour deposits. The cohort comprised 395 cases: resampling identified nodal metastases and/or tumour deposits in 30 (7.6%) cases; nodal upstaging occurred in 20 (5.1%) cases; and eight (2.0%) cases changed from pN0 to ≥pN1. No factors predicted resampling of positive lymph nodes or tumour deposits, and pN upstaging occurred across a variety of cases. A subgroup analysis was performed to assess the impact of resampling on high-risk features in stage II cases (n = 117). There were 33 (8.5%) patients who no longer had any high-risk features after resampling. CONCLUSIONS: Lymph node resampling has an impact on nodal staging and possible treatment decisions in a considerable proportion of patients, and is recommended in all cases with <12 lymph nodes.

The Tumor Suppressor BAP1 Regulates the Hippo Pathway in Pancreatic Ductal Adenocarcinoma.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk for mesothelioma and melanocytic tumors. Here, we show that pancreatic intraepithelial neoplasia driven by oncogenic mutant KrasG12D progressed to pancreatic adenocarcinoma in the absence of BAP1. The Hippo pathway was deregulated in BAP1-deficient pancreatic tumors, with the tumor suppressor LATS exhibiting enhanced ubiquitin-dependent proteasomal degradation. Therefore, BAP1 may limit tumor progression by stabilizing LATS and thereby promoting activity of the Hippo tumor suppressor pathway. SIGNIFICANCE: BAP1 is mutated in a broad spectrum of tumors. Pancreatic Bap1 deficiency causes acinar atrophy but combines with oncogenic Ras to produce pancreatic tumors. BAP1-deficient tumors exhibit deregulation of the Hippo pathway.See related commentary by Brekken, p. 1624.

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAFV600E ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.

Fatigue is a relevant outcome in patients with myasthenia gravis.

INTRODUCTION: Patients with myasthenia gravis often experience fatigue, but its effect on quality of life (QoL) is underestimated, and fatigue is rarely measured in clinical trials. METHODS: Two hundred fifty-seven myasthenic patients completed the Neuro-QoL-Fatigue and measures of disease severity and QoL. We studied the relationship between fatigue and clinical and demographic variables. Finally, we studied the responsiveness of the Neuro-QoL-Fatigue in 95 patients receiving treatments for myasthenia and estimated the minimal important difference (MID). RESULTS: Fatigue correlated with greater disease severity (r = 0.52-0.69, P < 0.0001) and worse QoL (r = 0.65-0.75, P < 0.0001). Patients in remission, with minimal manifestations, and pure ocular symptoms reported minimal fatigue. Regression modeling showed that, in addition to its relationship with disease severity, fatigue was worse in females, patients with generalized disease, and those with anxiety/depression. Fatigue improved after immunomodulation (P < 0.0001), and the MID was 5.3 points. DISCUSSION: Fatigue in myasthenia correlates with disease severity, affects QoL, and can improve after treatment. Muscle Nerve 58: 197-203, 2018.

Kras mutant genetically engineered mouse models of human cancers are genomically heterogeneous.

KRAS mutant tumors are largely recalcitrant to targeted therapies. Genetically engineered mouse models (GEMMs) of Kras mutant cancer recapitulate critical aspects of this disease and are widely used for preclinical validation of targets and therapies. Through comprehensive profiling of exomes and matched transcriptomes of >200 KrasG12D-initiated GEMM tumors from one lung and two pancreatic cancer models, we discover that significant intratumoral and intertumoral genomic heterogeneity evolves during tumorigenesis. Known oncogenes and tumor suppressor genes, beyond those engineered, are mutated, amplified, and deleted. Unlike human tumors, the GEMM genomic landscapes are dominated by copy number alterations, while protein-altering mutations are rare. However, interspecies comparative analyses of the genomic landscapes demonstrate fidelity between genes altered in KRAS mutant human and murine tumors. Genes that are spontaneously altered during murine tumorigenesis are also among the most prevalent found in human indications. Using targeted therapies, we also demonstrate that this inherent tumor heterogeneity can be exploited preclinically to discover cancer-specific and genotype-specific therapeutic vulnerabilities. Focusing on Kras allelic imbalance, a feature shared by all three models, we discover that MAPK pathway inhibition impinges uniquely on this event, indicating distinct susceptibility and fitness advantage of Kras-mutant cells. These data reveal previously unknown genomic diversity among KrasG12D-initiated GEMM tumors, places them in context of human patients, and demonstrates how to exploit this inherent tumor heterogeneity to discover therapeutic vulnerabilities.

Thyroid-stimulating hormone acutely increases monocyte gene expression in vivo.

OBJECTIVES: Thyroid-stimulating hormone (TSH) acts in an extra-thyroidal fashion and induces a pro-inflammatory, pro-coagulant state. Blood monocytes can be activated by vascular stress, but it is not known if this occurs upon TSH administration. Our aim was to determine if recombinant human (rh) TSH, administered acutely to patients being screened for thyroid cancer recurrence, alters blood monocyte gene expression. DESIGN AND SETTING: Patients (14 women, 1 man) had a mean (±SD) age of 48±10 years, a body mass index of 26±6 kg/m2, a history of total thyroidectomy and radioablation for thyroid cancer, and were on L-thyroxine therapy at a university teaching hospital. They received 2 intramuscular doses of rhTSH (0.9 mg), administered on days 1 and 2. Blood samples were obtained at baseline on day1, and on days 3 and 5. RESULTS: Monocyte MCP-1 mRNA (mean±SE) increased significantly by 1.7±0.3 fold on day 5 following rhTSH stimulation (p=0.03, n=15). IL-1ß and CD36 mRNA expression also increased on day 5 (1.9±0.4 fold, p=0.07, n=14) and 2.5±0.4 fold, p=0.1, n=10), respectively, although did not quite reach statistical significance. Significant correlations were detected between the BMI of patients and their TSH-stimulated monocyte mRNA responses at day 5 for CD11a, (r=0.66, n=14, p=0.01); CD14 (r=0.638, n=13, p=0.019), and CD16, r=0.84, n=13, p=0.0003). CONCLUSION: TSH administration increases pro-atherogenic monocyte gene expression.

High economic burden of caring for patients with suspected extraesophageal reflux.

OBJECTIVES: Extraesophageal symptoms are common manifestations of gastroesophageal reflux disease (GERD). Lack of a definitive diagnostic or treatment standards complicate management, which often leads to multiple specialty consultations, procedures, pharmaceuticals and diagnostic tests. The aim of this study was to determine the economic burden associated with extraesophageal reflux (EER). METHODS: Direct costs of evaluation were estimated for patients referred with symptoms attributed to EER between 2007 and 2011. Medicare payment for evaluation and management and pharmaceutical prices was used to calculate first year and overall costs of evaluating and treating extraesophageal symptoms attributed to reflux. RESULTS: Overall, 281 patients were studied (cough (50%), hoarseness (23%), globus/post-nasal drainage (15%), asthma (9%), and sore throat (3%)). Over a median (interquartile range) of 32 (16-46) months follow-up, patients had a mean (95% confidence interval) of 10.1 (9.4-10.9) consultations with specialists and underwent 6.4 (3-9) diagnostic procedures. Overall, the mean initial year direct cost was $5,438 per patient being evaluated for EER. Medical and non-medical components contributed $5,154 and $283. Of the overall cost, 52% were attributable to the use of proton pump inhibitors. During the initial year, direct costs were 5.6 times higher than those reported for typical GERD ($971). A total of 54% of patients reported improvement of symptoms. Overall cost per improved patient was $13,700. CONCLUSIONS: EER contributes substantially to health-care expenditures. In this cohort, the cost for initial year's evaluation and treatment of EER symptoms was quintuple that of typical GERD. Prescription costs and, in particular, proton pump inhibitors were the single greatest contributor to the cost of EER management.

Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF-induced neutrophil recruitment.

Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.