Miro1 is a mitochondrial outer membrane protein that regulates mitochondrial and peroxisome trafficking, endoplasmic reticulum (ER) association, and mitophagy. Prior studies suggest a role for Miro1 in cell migration and proliferation in both normal and tumor cells. High Miro1 expression is associated with poor overall survival in breast cancer patients. To investigate the role of Miro1 in breast cancer tumorigenesis and metastasis, we established stable Miro1 knockdown (KD) in MDA-MB-231 human triple-negative breast cancer cells using shRNA. Miro1 KD significantly impaired cell proliferation, migration, and invasion in vitro . When implanted into the mammary fat pad of SCID mice, MDA-MB-231 cells formed tumors, whereas Miro1 KD cells showed markedly reduced tumorigenesis. Additionally, we generated a novel transgenic mouse model with inducible, tissue-specific Miro1 deletion in mammary epithelial cells, alongside polyomavirus middle T-antigen (PyVMT) oncogene activation. In this model, wild-type (WT) mice formed tumors at all mammary gland sites, with frequent lung metastases. However, Miro1-deficient mice failed to develop tumors, while heterozygous mice exhibited reduced tumor growth and metastasis. Additionally, these findings identify Miro1 as a key regulator of breast cancer onset and metastatic potential, positioning it as a potential biomarker and therapeutic target.

Miro1 is an outer mitochondrial membrane protein that links mitochondria to motor protein complexes to support subcellular mitochondrial trafficking. Preliminary observations and published work has established that metastatic breast cancer cells have increased expression of Miro1 in comparison to breast cancer cells with low metastatic potential. Furthermore, tumors from breast cancer patients with high Miro1 expression have poorer survival rates compared to patients harboring tumors with low Miro1 expression. This data suggests that Miro1 has a functional role in aggressive breast cancers although this has not been widely studied or confirmed in advanced preclinical models. The objective of our research is to investigate the role of Miro1 in breast cancer tumorigenesis and metastasis. We hypothesize that that Miro1-mediated mitochondrial dynamics support subcellular signaling events driving cell migration, invasion, and tumorigenesis. To examine the function of Miro1 in promoting metastatic phenotypes in vitro, we generated human breast cancer cell lines (MDA-MB-231, MDA-MB-468, T-47d, and MCF7) with Miro1 knockdown using adenoviral shRNA. Knockdown of Miro1 to levels greater than 50% of that in control cells resulted in mitochondria sequestered around the nucleus and reduced cell migration. Additionally, MDA-MB-231 cells with ≥50% Miro1 knockdown resulted in a significant decrease in cell invasion through Matrigel coated membranes in transwell invasion assays. Our research has also uncovered altered phosphorylation of key cell migration and stress-response proteins including vinculin, focal adhesion kinase and ERK ½ when Miro1 is knocked down. To investigate the role of Miro1 in vivo, we generated a novel transgenic mouse model with inducible, tissue specific Miro1 deletion in mammary epithelial cells with concurrent polyomavirus middle T-antigen oncogene activation. We found that when breast cancer formation is induced utilizing the transgenic middle-T antigen system, our control mice form tumors at all mammary gland sites and have metastasis to the lungs. Conversely, mice with concurrent Miro1 deletion and middle T- antigen activation fail to develop tumors. This data suggests that Miro1 has a functional role in both mammary tumor onset and tumor cell migration and invasion, providing a possible new biomarker of tumor status and pathway for therapeutic intervention. Citation Format: Randi Gravelle, Margaret McCoy, Nathaniel Shannon, Martin Chang, Brian Cunniff. The role of miro1-mediated mitochondrial positioning in the development and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 87.

The RAD51D gene codes a protein-paralog of the RAD51 DNA recombinase, which catalyzes DNA strand exchange during homologous recombination. Similar to BRCA1 / BRCA2 , mutations in RAD51D both predispose to ovarian carcinoma and impart sensitivity to poly (ADP-ribose) polymerase inhibitors in preclinical studies. Based on cancer risk prediction models, RAD51D mutations pose a moderate-to-high risk for ovarian cancer warranting consideration for risk-reducing surgery. We report a case of serous tubal intraepithelial carcinoma in a patient undergoing risk-reducing total hysterectomy with bilateral salpingo-oophorectomy for a RAD51D pathogenic variant. The histopathologic and p53-immunophenotypic features of this lesion are similar to those reported previously in BRCA1 / BRCA2 mutation carriers and those of serous tubal intraepithelial carcinoma associated with sporadic high-grade serous carcinomas. These features include marked increase in nuclear-to-cytoplasmic ratio, loss of cell polarity, absence of ciliation, prominent nucleoli, mitotic activity, epithelial stratification, surface exfoliative changes, and complete loss of p53 staining. Although familial ovarian cancers with mutations in RAD51D -or other genes in the Fanconi anemia pathway-are much less common those with BRCA1 / BRCA2 mutations, our findings support a common phenotype for early serous cancers in this pathway.

Supplemental Figure 1: Study Schema Supplemental Table 1: Tissue Insulin Receptor Staining

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error ( p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections ( p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

NTRK-rearranged uterine sarcomas are rare spindle cell neoplasms that typically arise in the uterine cervix of young women. Some tumors recur or metastasize, but features which predict behavior have not been identified to date. Distinguishing these tumors from morphologic mimics is significant because patients with advanced stage disease may be treated with TRK inhibitors. Herein, we present 15 cases of NTRK-rearranged uterine sarcomas, the largest series to date. Median patient age was 35 years (range: 16 to 61). The majority arose in the uterine cervix (n=14) and all but 2 were organ-confined at diagnosis. Tumors were composed of an infiltrative, fascicular proliferation of spindle cells and most showed mild-to-moderate cytologic atypia. All were pan-TRK positive by immunohistochemistry (13/13); S100 (11/13) and CD34 (6/10) were usually positive. RNA or DNA sequencing found NTRK1 (10/13) and NTRK3 (3/13) fusions with partners TPR, TPM3, EML4, TFG, SPECC1L, C16orf72, and IRF2BP2. Unusual morphology was seen in 2 tumors which were originally diagnosed as unclassifiable uterine sarcomas, 1 of which also harbored TP53 mutations. Follow up was available for 9 patients, of whom 3 died of disease. By incorporating outcome data of previously reported tumors, adverse prognostic features were identified, including a mitotic index ≥8 per 10 high-power fields, lymphovascular invasion, necrosis, and NTRK3 fusion. Patients with tumors which lacked any of these 4 features had an excellent prognosis. This study expands the morphologic spectrum of NTRK-rearranged uterine sarcomas and identifies features which can be used for risk stratification.

Abstract Background and Rationale: Biomarker evaluation on breast tumor tissue is an important component of clinical research. There is a concern that tissues collected at different times (pre- vs intra-op) and with different techniques (core biopsy vs. surgical excision) produce different results that may confound comparisons of patient samples. Our objective is to address the difference between tumor tissue from the same patient collected pre- vs intra-op and by core biopsy (Core Bx) vs surgical excision, with an emphasis on ER/PgR/HER2/Ki-67 and biomarkers related to insulin metabolism. Design: Following a protocol approved by the IRB, patients with Core Bx proven invasive breast cancer (BC) >1.5 cm in size by imaging underwent a peri-operative Core Bx followed by surgical excision. No neoadjuvant therapy was administered. Formalin-fixed paraffin-embedded tumor sections of the diagnostic and peri-operative Core Bx and surgical excision were immunohistochemically (IHC) stained for ER, PgR, HER2, Ki-67, insulin receptor (IR), phospho-AKT (pAKT), and phospho-AMPK (pAMPK) using standardized protocols on the same platforms. A pathologist (MCC) scored all sections in blinded order to yield H-Scores (which combine percentage of stain-positive cells and staining intensity). The level of agreement for each assay between specimens was assessed by using pairwise models based on normal theory. Results: 18 women (mean age = 66.8 years, 16/18 postmenopausal) provided all specimens. Mean (± SD) invasive tumor size was 2.7 (± 1.2) cm. 1 BC was Nottingham Grade 1 (6%), 9 Grade 2 (50%), and 8 Grade 3 (44%). An in situ component was present in 4/18 (22%) cases (all non-extensive). Lymphovascular invasion was present in 6/18 (33%) cases. 10/18 were node-negative (N0, 56%), 2/18 had isolated tumor cells (pN0[i+], 22%), 5/18 had 1 to 3 nodes (pN1, 28%), 1/18 had >9 nodes involved (pN3, 6%).There was high concordance between diagnostic Core Bx, peri-op Core Bx, and excisions for the standard prognostic markers ER, PgR, and HER2. ER was concordant in all samples including 16/18 (89%) ER-positive and 2/19 (11%) ER-negative BCs. PgR was concordant in 16/18 (89%) of cases. In the 2 discordant cases, PgR was negative in 2 of 3 tissue samples with low expression in the 3rd sample (1 diagnostic Core Bx and 1 excision). HER2 was concordant in all samples in 16/18 (89%) HER2-negative cases and in 2/18 (11%) HER2-positive cases including 1/18 cases (6%) positive by IHC, and 1/18 cases (6%) positive by ISH. Table 1 summarizes the agreement between samples for Ki-67, IR, pAKT, and pAMPK. Ki-67 scores were statistically similar between diagnostic and peri-op Core Bx and excision samples. The IR, pAKT, and pAMPK H-Scores were statistically similar between diagnostic and peri-op Core Bx, but significantly different between Core Bxs versus excisional specimens. There was a systematic tendency towards lower IHC H-Scores in the excisional specimen for IR, pAKT, and pAMPK. Conclusion: Tissue from surgical excisions are susceptible to reduced IHC staining for metabolic markers such as IR, and phosphorylated kinases, when compared to core biopsies. When evaluating non-standard biomarkers for research, core biopsies should be used when possible. Funding: This study was funded by the Hold'Em For Life Charity and the Breast Cancer Research Foundation Table 1: Concordance Between IHC Scores for Ki-67, Insulin Receptor, pAKT, and pAMPKScore: Mean ± Standard DeviationPeri-Operative Core Bx versus Diagnostic Core BxExcision versus Diagnostic Core BxDiagnostic Core BxPeri-Operative Core BxExcisionDifferenceP-valueDifferenceP-valueKi-67 (% Positive)33.5 ± 28.235.9 ± 31.034.2 ± 29.1Mean ± SD: 2.4 ± 5.60.09Mean ± SD: 0.6 ± 6.30.68Range: -7.7 to 12.3Range: -11.5 to 12.2Insulin Receptor (H-Score**)136 ± 102124 ± 9986 ± 78Mean ± SD: -13 ± 530.32Mean ± SD: -51 ± 540.001*Range: -215 to 40Range: -150 to 5Phospho-AKT (H-Score**)102 ± 70100 ± 8641 ± 41Mean ± SD: -1 ± 750.95Mean ± SD: -61 ± 520.0001*Range: -170 to 170Range: -144 to 0Phospho-AMPK (H-Score**)201 ± 79185 ± 78157 ± 86Mean ± SD: -15 ± 760.41Mean ± SD: -44 ± 690.016*Range: -225 to 120Range: -215 to 70* Statistically significant difference in pairwise values based on t-test** H-Score is the percentage of stain-positive cells multiplied by the average intensity score (0 = absent, 1 = faint, 2 = moderate, 3 = strong) Citation Format: Martin C. Chang, Marguerite Ennis, Jaime Escallon, Karina Bukhanov, Ryan JO Dowling, Vuk Stambolic, Pamela J Goodwin. Concordance of immunohistochemical assays between peri-operative and post-operative breast tumor specimens: A prospective observational study of 18 cases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-25.

Crown-like structures of the breast (CLS-B), defined by the clustering of macrophages (identified using CD68 immunohistochemical staining) to surround a dying adipocyte, are a sign of adipose-tissue inflammation. In human cohorts, CLS-B positively correlates with older age, obesity, dyslipidemia and higher levels of glucose, insulin, C-reactive protein and IL-6. In an existing cohort of early-stage breast cancer patients, CLS-B were identified using H&E stained histologic sections (hCLS-B), and by CD68 immunohistochemistry (CD68 + CLS-B). We examined associations of H&E and CD68-detected CLS-B with clinicopathologic features using χ 2 tests, with metabolic factors using Wilcoxon rank sum tests and with disease free and overall survival using Cox regression models. hCLS-B were detected in 59 of 163 patients with slides (36.2%) and CD68 + CLS-B in 37 of 119 patients with paraffin blocks (31.1%). hCLS-B were positively correlated with higher weight ( p = 0.003), BMI ( p = 0.0008) and C-reactive protein ( p = 0.045). CD68 + CLS-B were positively correlated with higher weight ( p = 0.006), BMI p = 0.001), leptin ( p = 0.034), insulin ( p = 0.008) and Homeostasis Model Assessment ( p = 0.027). CD68 + CLS-B were associated with poor distant disease-free with a hazard ratio (HR) of 2.81, 95% confidence interval (CI) 1.20–6.57, and overall survival with HR 3.97 (1.66–9.48), while hCLS-B were not associated with either: HR for distant recurrence 0.59 (0.26–1.30); HR for death 1.04 (0.50–2.16). The presence of hCLS-B and of CD68 + CLS-B were associated with obesity; CD68 + CLS-B were associated with insulin resistance and adverse prognosis. Similar patterns were not seen for hCLS-B. Research is needed to understand the biologic basis for these differences.

Abstract Background: The nuclear proliferation biomarker Ki67 has multiple potential roles in breast cancer, including aiding decisions based on prognosis, but has unacceptable between-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) Assess inter-laboratory reproducibility of automated Ki67 measurement among 17 participating labs and compare those with standardized pathologist-based visual scoring. (ii) Investigate the comparability of Ki67 measurement across corresponding core biopsy and whole section cases. (iii) Test prognostic potential of the built Ki67 scoring algorithms on an independent cohort. Methods: Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding whole tumor sections from 30 ER+ breast cancer cases were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and stained for Ki67 using the Mib-1 antibody. The QuPath (open-source software) DIA platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed in our previous study (Acs et al, Lab Invest 2019). A detailed guideline for building an automated Ki67 scoring algorithm was sent to the participating labs. Visual scoring of average Ki67 expression was performed by pathologists according to published standardized methods (Leung et al, NPJ Br Cancer 2016; Leung et al, Histopath 2019). Locked down DIA Ki67 scoring algorithms were applied to a validation cohort: 222 breast cancer cases from the Karolinska University Hospital in whole section format. Sufficient reproducibility to declare analytical validity was defined as an Intra Class Correlation (ICC) with lower limit of 95% credible interval (CI) >0.80. Markov Chain Monte Carlo routines for generalized linear mixed models were used to estimate ICCs and calculate corresponding CIs. Results: The same-section ICC was 0.902 (CI: 0.852-0.949) across 17 labs using calibrated DIA platform on core biopsy slides and 0.845 (CI: 0.778-0.912) on whole sections. The different-section ICC across the 17 labs was 0.873 (CI: 0.806-0.932) scoring on core biopsy slides and 0.777 (CI: 0.670-0.874) on whole sections. The pathologist-based visual Ki67 scoring showed ICC of 0.860 for all comparisons, respectively (CI: 0.795-0.927). Similar to what was observed for visual Ki67 scoring, the DIA scores are higher for core biopsy slides compared to paired whole sections (p≤0.001; median difference: 5.31%; IQR: 11.50%). Ki67 scores of all locked down DIA algorithms correlates significantly (p≤0.023) with outcome on the validation cohort (observed hazard ratios range: 2.518-2.922). Conclusions: Automated Ki67 evaluation using a calibrated, open-source DIA platform (QuPath) met the pre-specified criterion of success on core biopsies but not on whole sections in the multi-institutional setting. The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation) and intratumor heterogeneity. We found that different algorithms built according to calibrated DIA methods had similar prognostic potential. Assessment of clinical utility is planned. Citation Format: Balazs Acs, Samuel C.Y. Leung, Kelley M. Kidwell, Indu Arun, Renaldas Augulis, Sunil S. Badve, Yalai Bai, Anita L. Bane, John M.S. Bartlett, Jane Bayani, Gilbert Bigras, Annika Blank, Signe Borgquist, Henk Buikema, Martin C. Chang, Robin L. Dietz, Andrew Dodson, Anna Ehinger, Susan Fineberg, Cornelia M. Focke, Dongxia Gao, Allen M. Gown, Carolina Gutierrez, Johan Hartman, Judith C. Hugh, Zuzana Kos, Anne-Vibeke Lænkholm, Arvydas Laurinavicius, Richard M. Levenson, Rustin Mahboubi-Ardakani, Mauro G. Mastropasqua, Takuya Moriya, Sharon Nofech-Mozes, C. Kent Osborne, Liron Pantanowitz, Frédérique M. Penault-Llorca, Tammy Piper, Mary Anne Quintayo, Tilman T. Rau, Stefan Reinhard, Stephanie Robertson, Takashi Sakatani, Roberto Salgado, Melanie Spears, Jane Starczynski, Tomoharu Sugie, Bert van der Vegt, Giuseppe Viale, Shakeel Virk, Lila A. Zabaglo, Daniel F. Hayes, Mitch Dowsett, Torsten O. Nielsen, David L. Rimm, International Ki67 in Breast Cancer Working Group, BIG-NABCG. Analytical validation and prognostic potential of an automated digital scoring protocol for Ki67: An International Ki67 Working Group study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-01.

Abstract Introduction: The association of breast cancer (BC) with obesity is complex and multifactorial, depending partly on diet and related metabolic imbalances. Previous reports have shown that local inflammation in adipocytes can be seen in tissue sections as rings of macrophages of macrophages around necrotic adipocytes (“Crown-Like Structures of the Breast”, CLS-B). We and others have shown that CLS-B have higher prevalence and density within the adipose tissue of obese patients. Our goal was to examine the association of CLS-B with survival in participants enrolled at one center (Mount Sinai Hospital, Toronto) of a multicenter prospective cohort study designed to investigate obesity-related prognostic factors in early stage BC. Design: Archived H&E sections of breast adipose tissue were retrieved from Mount Sinai (Toronto) participants (N=163). All specimens were from breast excisions for invasive carcinoma. A CLS-B was defined as a ring of macrophages surrounding an adipocyte in otherwise normal breast tissue. In a subset (N=119), immunostains for CD68 were performed on a representative block to highlight macrophages. Sections containing tumor, fat necrosis, and mastitis were excluded. Slides were pathologist-reviewed, recording the number of CLS-B, slides containing CLS-B, and slides with normal tissue. Serologic markers of metabolism and inflammation were previously performed in a central laboratory. Correlations with clinical and serologic markers were examined using descriptive statistics and Wilcoxon tests. Survival analysis was performed with Cox proportional hazards models. Results: CLS-B were identified in routine H&E sections of normal fat in 59 out of 163 cases (36%). In the representative subset stained for CD68 (N=119), 22 cases were identified as positive for CLS-B by both H&E and CD68, 15 cases by CD68 alone and 24 by H&E alone, giving a CLS-B prevalence of 39% as detected by H&E, 31% by CD68 and 51% when combining the two. Both i) CD68-positive and ii) H&E-positive CLS-B showed similar positive relationships with BMI (median BMI 27 versus 24 kg/m2, P<0.01 for CLS-B present versus absent); however the relationship with insulin was stronger for CD68-positive CLS-B (median insulin 49 vs 35 pmol/L, P=0.009) than for H&E-positive CLS-B (insulin 39 vs 35 pmol/L, P=0.39). Univariable Cox model hazard ratios (HRs) for CD68-positive CLS-B were 2.79 (95% confidence interval (CI) 1.31-5.95) and 3.74 (CI 1.73-8.07) for disease-free and overall survival respectively, and after adjustment for age, nodal status, tumour grade, stage, ER/PR status, adjuvant treatment and BMI, HRs were 2.43 (CI 1.01-5.81) and 3.19 (CI 1.3-7.79). In the same subset, H&E-positive CLS-B had univariable HRs of 0.66 (CI 0.29-1.52) and 1.18 (CI 0.55-2.54) respectively and adjusted HRs of 0.31 (CI 0.11-0.82) and 0.57 (CI 0.23-1.39). Combining H&E and CD68 did not improve prediction over using CD68 alone. Conclusion: CLS-B visualized by the macrophage immunostain CD68 were associated with poor outcome, independent of patient BMI and tumour characteristics. Detection of CLS-B using H&E alone was not associated with poor outcome. Our results demonstrate that although CLS-B are a phenotype of obesity, they may also reflect a tissue-specific risk of adverse BC outcome. This risk may relate to localized tissue inflammation with higher prevalence in obese individuals. Although we do not advocate the clinical use of the CD68 stain, further work is warranted to clarify the connection between tissue inflammation and poor breast cancer outcome. Acknowledgement: The authors wish to acknowledge the Hold'Em For Life Charity Challenge for Cancer Research for their generous support. Citation Format: Martin C. Chang, Marguerite Ennis, Zohreh Eslami, Pamela J. Goodwin. CD68-positive crown-like structures of the breast are independently associated with adverse survival: A retrospective analysis of cases from a prospective cohort [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-02.

Advances in digital image analysis have the potential to transform the practice of breast pathology. In the near future, a move to a digital workflow offers improvements in efficiency. Coupled with artificial intelligence (AI), digital pathology can assist pathologist interpretation, automate time-consuming tasks, and discover novel morphologic patterns. Opportunities for digital enhancements abound in breast pathology, from increasing reproducibility in grading and biomarker interpretation, to discovering features that correlate with patient outcome and treatment. Our objective is to review the most recent developments in digital pathology with clear impact to breast pathology practice. Although breast pathologists currently undertake limited adoption of digital methods, the field is rapidly evolving. Care is needed to validate emerging technologies for effective patient care.

Low-grade endometrial stromal sarcoma (LGESS) represents a morphologically and genetically heterogenous mesenchymal neoplasm. Previous work has shown that approximately half of LGESS are characterized by JAZF1::SUZ12 gene fusions, while a smaller proportion involves rearrangement of other genes. However, a subset of cases has no known genetic abnormalities. To better characterize the genomic landscape of LGESS, we interrogated a cohort with targeted RNA sequencing (RNA-Seq). Cases previously diagnosed as low-grade endometrial stromal neoplasia (n=51) were identified and re-reviewed for morphology and subjected to RNA-Seq, of which 47 were successfully sequenced. The median patient age was 49 years (range: 19 to 85). The most commonly detected fusions were JAZF1::SUZ12 (n=26, 55%) and BRD8::PHF1 (n=3, 6%). In addition to the usual/typical LGESS morphology, some JAZF1::SUZ12 fusion tumors showed other morphologies, including fibrous, smooth muscle, sex-cord differentiation, and myxoid change. Novel translocations were identified in 2 cases: MEAF6::PTGR2 and HCFC1::PHF1 . Ten tumors (21%) had no identifiable fusion, despite a similar morphology and immunophenotype to fusion-positive cases. This suggests that a subset of cases may be attributable to fusion products among genes that are not covered by the assay, or perhaps altogether different molecular mechanisms. In all, these findings confirm that RNA-Seq is a potentially useful ancillary test in the diagnosis of endometrial stromal neoplasms and highlight their diverse morphology.

Osteoarthritis (OA) is the most prevalent disabling disease, affecting quality of life and contributing to morbidity, particularly during aging. Current treatments for OA are limited to palliation: pain management and surgery for end-stage disease. Innovative approaches and animal models are needed to develop curative treatments for OA. Here, we investigated the naked mole-rat (NMR) as a potential model of OA resistance. NMR is a small rodent with the maximum lifespan of over 30 years, resistant to a wide range of age-related diseases. NMR tissues accumulate large quantities of unique, very high molecular weight, hyaluronan (HA). HA is a major component of cartilage and synovial fluid. Importantly, both HA molecular weight and cartilage stiffness decline with age and progression of OA. As increased polymer length is known to result in stiffer material, we hypothesized that NMR high molecular weight HA contributes to stiffer cartilage. Our analysis of biomechanical properties of NMR cartilage revealed that it is significantly stiffer than mouse cartilage. Furthermore, NMR chondrocytes were highly resistant to traumatic damage. In vivo experiments using an injury-induced model of OA revealed that NMRs were highly resistant to OA. While similarly treated mice developed severe cartilage degeneration, NMRs did not show any signs of OA. Our study shows that NMRs are remarkably resistant to OA, and this resistance is likely conferred by high molecular weight HA. This work suggests that NMR is a useful model to study OA resistance and NMR high molecular weight HA may hold therapeutic potential for OA treatment.

To study the association between number and positions of mutations with MICs of fluoroquinolone non‐susceptible Haemophilus influenzae.

Synovial sarcoma most commonly occurs in the extremities but has rarely been described in the female genital tract. In this series, we describe the clinical, morphologic, immunohistochemical, and molecular features of 7 cases of vulvovaginal synovial sarcoma (vulva, n=6; vagina, n=1). We emphasize their wide morphologic spectrum, which can overlap significantly with other more common tumors at these sites, as highlighted by 2 cases initially diagnosed as other entities (endometrioid carcinoma and malignant peripheral nerve sheath tumor). The average patient age was 41 (range: 23 to 62) years and tumor size ranged from 0.8 to 7 cm. Histologically, the tumors were biphasic (n=6) and monophasic (n=1). All cases were confirmed with fluorescence in situ hybridization or sequencing, and 5/5 cases were positive for the novel immunohistochemical markers SSX and SS18-SSX. In 3 cases with follow-up, 2 patients died of disease and 1 was alive with no evidence of disease. Previously described cases arising in the female genital tract are also reviewed. Vulvovaginal monophasic synovial sarcoma raises a broad differential diagnosis, including smooth muscle tumors, spindled carcinomas, and melanoma. Biphasic synovial sarcoma may mimic Müllerian carcinosarcoma, endometrioid carcinoma with spindled, corded, and hyalinized elements, and mesonephric-like adenocarcinoma. Awareness that synovial sarcoma can occur in the female genital tract with a wide variety of histologic appearances is critical for correctly diagnosing this rare entity. In particular, synovial sarcoma should be considered for any deeply situated "adenocarcinoma" in the vulva, with attention to subtle spindle cell differentiation.

Abstract Background/Goal: Ki67 expression has been a valuable prognostic marker in breast cancer, but has not seen broad adoption due to lack of standardization between institutions. Automation could represent a solution. Here we tested 3 automated digital image analysis (DIA) platforms including an open source platform to: (i) Investigate the reproducibility of Ki67 measurement across platforms with supervised classifiers performed by the same operator and by multiple operators. (ii) Compare accuracy of the 3 DIA platforms against outcome (prognostic potential). (iii) Assess inter-laboratory reproducibility of a calibrated DIA tool to evaluate Ki67 in breast cancer among 10 participating labs of the International Ki67 in Breast Cancer Working Group (IKWG). Methods: The Mib-1 antibody (Dako) was used to detect Ki67 (dilution 1:100). HALO (H) (IndicaLabs), QuantCenter (QC) (3DHistech), QuPath (QP) (open-source software) digital image analysis (DIA) platforms were used to evaluate Ki67 expression. As a ground truth, we evaluated Ki67 LI with meticulous manual tissue segmentation using the Spectrum Webscope (SW) (Aperio). Calibration was performed using 30 ER+ breast cancer cases from phase 3 of the IKWG initiative where blocks were centrally cut and stained for Ki67. The inter-laboratory analysis was done with 10 participating laboratories divided into 2 groups where members within the same group were given the same set of images. The outcome cohort consisted of 149 breast cancer cases from the Yale Pathology archives in tissue microarray format. Intra-class correlation coefficient (ICC) was used to measure reproducibility with the pre-specified criterion for success being to exceed 0.80. Kaplan-Meier analysis supported with log-rank test was performed to assess prognostic potential. Results: All 3 DIA platforms showed excellent inter-platform reproducibility (ICC: 0.933, CI: 0.879-0.966). Also, excellent reproducibility was found between all DIA platforms and the reference standard Ki67 values of SW (QP ICC: 0.970, CI: 0.936-0.986; H ICC: 0.968, CI: 0.933-0.985; QC ICC: 0.964, CI: 0.919-0.983). The intra-DIA reproducibility was also excellent for all platforms (QP ICC: 0.992, CI: 0.986-0.996; H ICC: 0.972, CI: 0.924-0.988; QC ICC: 0.978, CI: 0.932-0.991). Comparing each DIA against outcome, the hazard ratios were similar (QP=3.309, H=3.077, QC=3.731). The inter-operator reproducibility was particularly high (ICC: 0.962-0.995). As QP is open source software and also showed the lowest intra-DIA platform variability, we selected the QP platform to investigate inter-laboratory reproducibility among 10 IKWG labs. The different-section ICC across the 10 labs was 0.974 (CI: 0.954 - 0.986). The same-section ICC estimate was 0.984 (CI: 0.971-0.992) for group 1 and 0.978 (CI: 0.956-0.989) for group 2. Conclusions: Our results showed outstanding reproducibility both within and between DIA platforms. We also found the platforms essentially indistinguishable with respect to prediction of breast cancer patient outcome. Automated Ki67 evaluation using a calibrated, open-source DIA platform (QuPath) met the pre-specified criterion of success in the multi-institutional setting. Assessment of clinical utility is planned. Citation Format: Acs B, Leung SC, Pelekanou V, Bai Y, Martinez-Morilla S, Toki M, Chang MC, Gholap A, Jadhav A, Hugh JC, Bigras G, Laurinavicius A, Augulis R, Levenson R, Todd A, Piper T, Virk S, van der Vegt B, Hayes DF, Dowsett M, Nielsen TO, Rimm DL. Analytical validation of an automated digital scoring protocol for Ki67: International multicenter collaboration study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-02-01.