Immunohistochemical analysis of IDH2 R172 hotspot mutations in breast papillary neoplasms: applications in the diagnosis of tall cell carcinoma with reverse polarity.

Tall cell carcinoma with reverse polarity is a rare subtype of breast carcinoma with solid and papillary growth and nuclear features reminiscent of those of the tall cell variant of papillary thyroid carcinoma. These tumors harbor recurrent IDH2 R172 hotspot mutations or TET2 mutations, co-occurring with mutations in PI3K pathway genes. Diagnosis of tall cell carcinomas with reverse polarity is challenging in view of their rarity and the range of differential diagnosis. We sought to determine the sensitivity and specificity of IDH2 R172 immunohistochemistry for the detection of IDH2 R172 hotspot mutations in this entity. We evaluated 14 tall cell carcinomas with reverse polarity (ten excision and five core needle biopsy specimens), 13 intraductal papillomas, 16 solid papillary carcinomas, and 5 encapsulated papillary carcinomas by Sanger sequencing of the IDH2 R172 hotspot locus and of exons 9 and 20 of PIK3CA, and by immunohistochemistry using monoclonal antibodies (11C8B1) to the IDH2 R172S mutation. The 14 tall cell carcinomas with reverse polarity studied harbored IDH2 R172 hotspot mutations, which co-occurred with PIK3CA hotspot mutations in 50% of cases. None of the other papillary neoplasms analyzed displayed IDH2 R172 mutations, however PIK3CA hotspot mutations were detected in 54% of intraductal papillomas, 6% of solid papillary carcinomas, and 20% of encapsulated papillary carcinomas tested. Immunohistochemical analysis with anti-IDH2 R172S antibodies (11C8B1) detected IDH2 R172 mutated protein in 93% (14/15) of tall cell carcinomas with reverse polarity samples including excision (n = 9/10) and core needle biopsy specimens (n = 5), whereas the remaining papillary neoplasms (n = 34) were negative. Our findings demonstrate that immunohistochemical analysis of IDH2 R172 is highly sensitive and specific for the detection of IDH2 R172 hotspot mutations, and likely suitable as a diagnostic tool in the evaluation of excision and core needle biopsy material of tall cell carcinomas with reverse polarity.

Inflammatory Pain Promotes Increased Opioid Self-Administration: Role of Dysregulated Ventral Tegmental Area µ Opioid Receptors.

Pain management in opioid abusers engenders ethical and practical difficulties for clinicians, often resulting in pain mismanagement. Although chronic opioid administration may alter pain states, the presence of pain itself may alter the propensity to self-administer opioids, and previous history of drug abuse comorbid with chronic pain promotes higher rates of opioid misuse. Here, we tested the hypothesis that inflammatory pain leads to increased heroin self-administration resulting from altered mu opioid receptor (MOR) regulation of mesolimbic dopamine (DA) transmission. To this end, the complete Freund's adjuvant (CFA) model of inflammation was used to assess the neurochemical and functional changes induced by inflammatory pain on MOR-mediated mesolimbic DA transmission and on rat intravenous heroin self-administration under fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement. In the presence of inflammatory pain, heroin intake under an FR schedule was increased for high, but attenuated for low, heroin doses with concomitant alterations in mesolimbic MOR function suggested by DA microdialysis. Consistent with the reduction in low dose FR heroin self-administration, inflammatory pain reduced motivation for a low dose of heroin, as measured by responding under a PR schedule of reinforcement, an effect dissociable from high heroin dose PR responding. Together, these results identify a connection between inflammatory pain and loss of MOR function in the mesolimbic dopaminergic pathway that increases intake of high doses of heroin. These findings suggest that pain-induced loss of MOR function in the mesolimbic pathway may promote opioid dose escalation and contribute to opioid abuse-associated phenotypes. SIGNIFICANCE STATEMENT: This study provides critical new insights that show that inflammatory pain alters heroin intake through a desensitization of MORs located within the VTA. These findings expand our knowledge of the interactions between inflammatory pain and opioid abuse liability, and should help to facilitate the development of novel and safer opioid-based strategies for treating chronic pain.

Low levels of copper disrupt brain amyloid-ß homeostasis by altering its production and clearance.

Whereas amyloid-ß (Aß) accumulates in the brain of normal animals dosed with low levels of copper (Cu), the mechanism is not completely known. Cu could contribute to Aß accumulation by altering its clearance and/or its production. Because Cu homeostasis is altered in transgenic mice overexpressing Aß precursor protein (APP), the objective of this study was to elucidate the mechanism of Cu-induced Aß accumulation in brains of normal mice and then to explore Cu's effects in a mouse model of Alzheimer's disease. In aging mice, accumulation of Cu in brain capillaries was associated with its reduction in low-density lipoprotein receptor-related protein 1 (LRP1), an Aß transporter, and higher brain Aß levels. These effects were reproduced by chronic dosing with low levels of Cu via drinking water without changes in Aß synthesis or degradation. In human brain endothelial cells, Cu, at its normal labile levels, caused LRP1-specific down-regulation by inducing its nitrotyrosination and subsequent proteosomal-dependent degradation due in part to Cu/cellular prion protein/LRP1 interaction. In APP(sw/0) mice, Cu not only down-regulated LRP1 in brain capillaries but also increased Aß production and neuroinflammation because Cu accumulated in brain capillaries and, unlike in control mice, in the parenchyma. Thus, we have demonstrated that Cu's effect on brain Aß homeostasis depends on whether it is accumulated in the capillaries or in the parenchyma. These findings should provide unique insights into preventative and/or therapeutic approaches to control neurotoxic Aß levels in the aging brain.

Changes in the Diagnoses of Breast Core Needle Biopsies on Second Review at a Tertiary Care Center: Implications for Surgical Management.

Core needle biopsy (CNB) of breast lesions is routine for diagnosis and treatment planning. Despite refinement of diagnostic criteria, the diagnosis of breast lesions on CNB can be challenging. At many centers, including ours, confirmation of diagnoses rendered in other laboratories is required before treatment planning. We identified CNBs first diagnosed elsewhere that were reviewed in our department over the course of 1 year because the patients sought care at our center and in which a change in diagnosis had been recorded. The outside and in-house CNB diagnoses were then classified based on Breast WHO Fifth Edition diagnostic categories. The impact of the change in diagnosis was estimated based on the subsequent surgical management. Findings in follow-up surgical excisions (EXCs) were used for validation. In 2018, 4950 outside cases with CNB were reviewed at our center. A total of 403 CNBs diagnoses were discrepant. Of these, 147 had a change in the WHO diagnostic category: 80 (54%) CNBs had a more severe diagnosis and 44 (30%) a less severe diagnosis. In 23 (16%) CNBs, the change of diagnostic category had no impact on management. Intraductal proliferations (n=54), microinvasive carcinoma (n=18), and papillary lesions (n=35) were the most disputed diagnoses. The in-house CNB diagnosis was confirmed in most cases with available excisions. Following CNB reclassification, 22/147 (15%) lesions were not excised. A change affecting the surgical management at our center occurred in 2.5% of all CNBs. Our results support routine review of outside breast CNB as a clinically significant practice before definitive treatment.

NRE: a tool for exploring neutral loci in the human genome.

BACKGROUND: Analyzing regions of the genome where genetic variation is free from the confounding effects of natural selection is essential for many population genetic studies. Several recent studies in humans have stressed the large effect of natural selection at linked neutral sites and have shown that the choice of putatively neutral regions can have a marked effect on estimates of demographic history. RESULTS: NRE (Neutral Region Explorer) provides a mechanism for the easy extraction and analysis of nearly neutral regions from the human genome. It can combine many genomic filters, including filters for selection, recombination rate, genetic distance to the nearest gene, percent overlap with annotated regions, and user-provided loci. The program implements a two-step filtering process for greater versatility, allowing users to compile a basic set of neutrality criteria, explore their effect, and use this knowledge to refine filtering. Results can be instantly downloaded in standard formats, along with summary and ranking statistics, or exported to genome browsers such as those from the 1000 Genomes and UCSC. The applicability and value of NRE are demonstrated through an example in the estimation of the ratio of chromosome X-to-autosomal effective population size using different strategies for the selection of neutral regions. CONCLUSIONS: The combined features of NRE make possible the sort of flexible, rigorous mining and analysis of neutral loci increasingly demanded by population genetic studies. NRE is available at http://nre.cb.bscb.cornell.edu.

Expression of novel neuroendocrine markers in breast carcinomas: a study of INSM1, ASCL1, and POU2F3.

INSM1, ASCL1, and POU2F3 are novel transcription factors involved in neuroendocrine (NE) differentiation of neoplasms in several organs, but data on their expression in breast carcinomas (BCs) are limited. We retrospectively evaluated the expression of these markers in a series of 97 BCs (58 with NE morphology and 39 with otherwise uncommon morphology) tested prospectively using immunohistochemistry (IHC). Nuclear staining in >50% of the cells was used as the positive cut-off. Thirty-two of the 97 BCs (33%) were INSM1-positive. INSM1-positivity correlated significantly with histologic type and presence of stromal mucin. INSM1 also correlated with synaptophysin and chromogranin, established markers of NE differentiation (P < .0001 and P = .0023, respectively). In BC with NE morphology, the expression of INSM1 supported NE differentiation, and INSM1 was more specific than synaptophysin and more sensitive and specific than chromogranin. INSM1 was the most expressed NE marker in 17 BCs. INSM1-positive BCs included 56% of solid papillary BCs, 88% of BCs with solid papillary features, and 75% of high-grade NE carcinomas. Of 35 BCs tested for POU2F3 and ASCL1, only 1 and 4 cases were positive, respectively. Our results show that INSM1 is a sensitive marker of NE differentiation in BC and should be included with synaptophysin and chromogranin in the IHC panel used to evaluate NE differentiation in BC with NE morphology. ASCL1 and POU2F3 are uncommon in BC and their routine assessment is not warranted.

Expression Analysis of GD2 by Immunohistochemistry in Invasive Breast Carcinoma: Clinical and Pathologic Correlation.

The glycosphingolipid disialoganglioside GD2 is a cell surface-associated antigen expressed on tumors of neuroectodermal origin that serves as a target of immunotherapy in select cancer types. Information about the expression of GD2 in breast cancer is limited. In the present study, we investigate the utility of GD2 as a potential biomarker for targeted treatment. The study cohort consists of 386 breast carcinomas of several histologic types. GD2 expression was assessed in both whole tumor sections and tissue microarrays with anti-GD2 3F8 monoclonal antibody immunohistochemistry and correlated with clinicopathologic features and survival outcomes. A total of 134 (35%) breast carcinomas were positive for GD2, with a median H-score of 100. 3F8 staining displayed granular and predominantly cytoplasmic or perinuclear patterns, which was confined to the neoplastic tissue in nearly all cases. GD2 positivity was significantly associated with tumor histologic type (P=0.0015), low grade (P<0.0001), estrogen receptor positivity (P<0.0001), low stage (P=0.0014), and multifocality (P=0.022). Event-free survival and overall survival of patients with GD2-positive and GD2-negative tumors were not significantly different. Our results support further assessment of GD2 using the 3F8 antibody as a predictive and prognostic biomarker in breast cancer.

Postconvalescent SARS-CoV-2 IgG and Neutralizing Antibodies are Elevated in Individuals with Poor Metabolic Health.

PURPOSE: Comorbidities making up metabolic syndrome (MetS), such as obesity, type 2 diabetes, and chronic cardiovascular disease can lead to increased risk of coronavirus disease-2019 (COVID-19) with a higher morbidity and mortality. SARS-CoV-2 antibodies are higher in severely or critically ill COVID-19 patients, but studies have not focused on levels in convalescent patients with MetS, which this study aimed to assess. METHODS: This retrospective study focused on adult convalescent outpatients with SARS-CoV-2 positive serology during the COVID-19 pandemic at NewYork Presbyterian/Weill Cornell. Data collected for descriptive and correlative analysis included SARS-COV-2 immunoglobin G (IgG) levels and history of MetS comorbidities from April 17, 2020 to May 20, 2020. Additional data, including SARS-CoV-2 IgG levels, body mass index (BMI), hemoglobin A1c (HbA1c) and lipid levels were collected and analyzed for a second cohort from May 21, 2020 to June 21, 2020. SARS-CoV-2 neutralizing antibodies were measured in a subset of the study cohort. RESULTS: SARS-CoV-2 IgG levels were significantly higher in convalescent individuals with MetS comorbidities. When adjusted for age, sex, race, and time duration from symptom onset to testing, increased SARS-CoV-2 IgG levels remained significantly associated with obesity (P < 0.0001). SARS-CoV-2 IgG levels were significantly higher in patients with HbA1c ≥6.5% compared to those with HbA1c <5.7% (P = 0.0197) and remained significant on multivariable analysis (P = 0.0104). A positive correlation was noted between BMI and antibody levels [95% confidence interval: 0.37 (0.20-0.52) P < 0.0001]. Neutralizing antibody titers were higher in COVID-19 individuals with BMI ≥ 30 (P = 0.0055). CONCLUSION: Postconvalescent SARS-CoV-2 IgG and neutralizing antibodies are elevated in obese patients, and a positive correlation exists between BMI and antibody levels.

Papillary Lesions of the Male Breast: A Study of 117 Cases and Brief Review of the Literature Demonstrate a Broad Clinicopathologic Spectrum.

Papillary lesions of the male breast (PLMB) are uncommon. To date, PLMB have been reported as individual case reports and in relatively small series. We reviewed cases of PLMB diagnosed at our medical center over a 19-year (2000-2019) period. A total of 117 cases were identified, with an age range of 7 months to 88 years. These cases included 3 of papillary ductal hyperplasia, 5 intraductal papillomas, 1 adenomyoepithelioma, 5 atypical papillomas (ie, papillomas with atypia), 51 papillary ductal carcinoma in situ, 14 encapsulated papillary carcinomas, 38 solid papillary carcinomas, and 8 invasive papillary carcinomas. Malignant papillary neoplasms, including invasive and noninvasive ones, had a mean size of 1.3 cm (range: 0.3 to 4.4 cm), and all were ER and HER2. Fifty-four percent (19/35) of carcinomas were treated with excision alone, 46% (16/35) underwent mastectomy, and 63% (22/35) had axillary lymph node sampling. Only one case had metastatic involvement of axillary lymph nodes. Of the cases with follow-up, no (0/8) invasive carcinoma showed distant metastasis or proved fatal, and no (0/23) noninvasive papillary carcinoma recurred. Two notable cases of PLMB were encountered: one of a 7-month-old boy with NF1 mutation and florid papillary hyperplasia, and another of a 57-year-old man with Klippel-Feil syndrome and bilateral solid papillary carcinoma, invasive and oligometastatic on one side and noninvasive on the other. On the basis of this study of PLMB cases, the largest to date, and review of literature, we conclude that PLMB span a broad clinicopathologic spectrum, and that both invasive and noninvasive papillary carcinomas have relatively good prognosis.

Apocrine Variant of Pleomorphic Lobular Carcinoma In Situ: Further Clinical, Histopathologic, Immunohistochemical, and Molecular Characterization of an Emerging Entity.

To date, the apocrine variant of lobular carcinoma in situ (AP-LCIS) has been cursorily described as a subtype of lobular carcinoma in situ (LCIS). We retrospectively reviewed 34 cases of AP-LCIS (including 23 associated with invasive lobular carcinoma) to fully characterize it. AP-LCIS typically presented with screen-detected calcifications in older women (mean age: 65 y) and was characterized by distended terminal duct lobular units with relatively large "pleomorphic" cells, central necrosis, and calcifications. AP-LCIS cells exhibited abundant eosinophilic occasionally granular cytoplasm, hyperchromatic nuclei, and prominent nucleoli. Synchronous classic and/or florid LCIS was identified in 24/34 (70%) AP-LCIS, and in 9/11 (82%) pure AP-LCIS. Most (68%) cases of AP-LCIS were estrogen receptor-positive (50% strongly), 35% were progesterone receptor-positive, 26% were human epidermal growth factor 2-positive, 18% demonstrated high-proliferation rate (Ki67: >15%), and 90% were androgen receptor-positive. Aurora kinase A, immunoreactive in 38% of AP-LCIS cases, was not significantly associated with recurrence, development of invasion, or nodal positivity (P>0.05). Compared with conventional (nonapocrine) pleomorphic lobular carcinoma in situ (P-LCIS), aurora kinase A was expressed in a significantly greater proportion of P-LCIS (100%). AP-LCIS and P-LCIS did not otherwise differ in clinicopathologic features. Next-generation sequencing utilizing the Oncomine Comprehensive Panel v2, performed on 27 AP-LCIS cases, showed no specific molecular findings. In a mean follow-up of 57 months, 2 (of 11, 18%) pure AP-LCIS cases recurred (2 both in situ and invasive) and none metastasized or proved fatal. AP-LCIS should be regarded as another high-grade LCIS similar to P-LCIS in many respects, and pending additional studies should be managed similarly.

SARS-CoV-2 antibody characterization in emergency department, hospitalized and convalescent patients by two semi-quantitative immunoassays.

BACKGROUND: In the ongoing COVID-19 pandemic, there is an urgent need for comprehensive performance evaluation and clinical utility assessment of serological assays to understand the immune response to SARS-CoV-2. METHODS: IgM/IgG and total antibodies against SARS-CoV-2 were measured by a cyclic enhanced fluorescence assay (CEFA) and a microsphere immunoassay (MIA), respectively. Independent performance evaluation included imprecision, reproducibility, specificity and cross-reactivity (CEFA n = 320, MIA n = 364). Clinical utility was evaluated by both methods in 87 patients at initial emergency department visit, 28 during subsequent hospitalizations (106 serial samples), and 145 convalescent patients. Totally 916 patients and 994 samples were evaluated. RESULTS: Agreement of CEFA and MIA was 90.4%-94.5% (Kappa: 0.81-0.89) in 302 samples. CEFA and MIA detected SARS-CoV-2 antibodies in 26.2% and 26.3%, respectively, of ED patients. Detection rates increased over time reaching 100% after 21 days post-symptom onset. Longitudinal antibody kinetic changes by CEFA and MIA measurements correlated well and exhibited three types of seroconversion. Convalescent sera showed a wide range of antibody levels. CONCLUSION: Rigorously validated CEFA and MIA assays are reliable for detecting antibodies to SARS-CoV-2 and show promising clinical utility when evaluating immune response in hospitalized and convalescent patients, but are not useful for early screening at patient's initial ED visit.

Juvenile granulosa cell tumor associated with Maffucci syndrome in pregnancy: A case report.

Juvenile granulosa cell tumor (JGCT) is an extremely rare ovarian tumor that has been associated with Maffucci syndrome. It both secretes hormone and has been postulated to grow in response to hormone. We present a case of a 33-year-old G1P0 asymptomatic woman with a history of Maffucci syndrome found to have a left adnexal mass on routine ultrasonography at 13 weeks gestation. This case demonstrates the sonographic and magnetic resonance imaging (MRI) features of JGCT, as well as the natural progression of the tumor during pregnancy. A follow-up ultrasound 3 weeks after initial diagnosis demonstrated marked growth in size and vascularity of the tumor, prompting unilateral salpingo-oophorectomy. Histopathological findings confirmed the diagnosis of JGCT.

Breast Tumor Resembling the Tall Cell Variant of Papillary Thyroid Carcinoma: Molecular Characterization by Next-Generation Sequencing and Histopathological Comparison With Tall Cell Papillary Carcinoma of Thyroid.

Recent publications have brought attention to the histopathological, immunohistochemical, and molecular aspects of the rare breast tumor resembling the tall cell variant of papillary thyroid carcinoma (BrTC). Nine archived cases of this entity were retrieved, reviewed, and compared with randomly selected tall cell variants of papillary thyroid carcinoma (ThTC). Seven of the BrTC cases as well as 5 cases of solid papillary carcinoma of breast were analyzed by Oncomine next-generation sequencing. BrTC and ThTC were histologically distinguishable by the presence of solid architecture, luminal histiocytes, and reverse polarity in the former, and psammoma bodies, giant cells, and optically clear nuclei in the latter. Sequencing revealed IDH2 R172 single-nucleotide variants in all 7 BrTCs, 6 of which had concurrent PIK3CA mutations. None of the conventional solid papillary carcinomas demonstrated IDH2 mutation. BrTC bears superficial resemblance to other papillary tumors but is unique in terms of histology and molecular profile.

Hotspot enumeration of CD8+ tumor-infiltrating lymphocytes using digital image analysis in triple-negative breast cancer yields consistent results.

Tumor-infiltrating lymphocytes (TILs) have emerged as prognostic in triple-negative breast cancer (TNBC). We aimed to assess the consistency of hotspot placement and TIL enumeration among multiple pathologists. Additionally, we assessed hotspot TIL count consistency by comparing hotspot counts in 3 separate locations within a single whole-tissue section. Anti-CD8 immunohistochemistry was performed on a representative section from 66 cases of primary TNBC, which were then scanned as whole-slide images. Quantification of the tissue area and combined stromal and intratumoral CD8+ TILs was performed using digital image analysis (DIA) within 2.2 mm-diameter circle hotspots. TIL counts were quantified as absolute counts and densities (absolute count/tissue area in micrometers2). For each case, 6 pathologists placed a single hotspot, defined as an area with the subjectively highest CD8+ immunoreactivity, within the tumor bed. Separately for each case, a single pathologist placed hotspots in 3 different locations within a single tumor section. Intraclass correlation coefficients (ICCs) were generated following TIL enumeration via DIA. ICCs for single hotspot placement by 6 pathologists were 0.96 for density and 0.97 for absolute counts, respectively. In 32% of cases (21/66), all the hotspots placed by the 6 pathologists were in the same location. When evaluating hotspots in 3 different locations within a tumor, the ICC was 0.95 for both density and absolute counts. Hotspot evaluation by DIA is a reproducible method for CD8+ TIL quantification, and the use of hotspots may reduce TIL count variation caused by intratumoral TIL heterogeneity.