Neoadjuvant chemotherapy induces phenotypic mast cell changes in high grade serous ovarian cancer.

BACKGROUND: High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy in which patients have still yet to respond meaningfully to clinically available immunotherapies. Hence, novel immune targets are urgently needed. Our past work has identified that mast cells are significantly upregulated at the mRNA level in HGSOC patient tumors following neoadjuvant chemotherapy (NACT) exposure. Therefore, in this current investigation we sought to characterize intratumoral mast cell phenotypic changes as a result of NACT exposure and determine how these adaptations are associated with patient clinical outcomes. METHODS: Hematologic immunohistochemistry was employed to determine mast cell levels in 36 matched pre- and post-NACT HGSOC patient tumors. Fluorescent Immunohistochemistry was utilized to identify Tryptase+(carboxypeptidase A3 (CPA3) + mast cells as well as histamine levels in 29 and 20, respectively, matched pre- and post-NACT HGSOC patient tumors. Finally, human immortalized mast cells, LUVA were stimulated with carboplatin and paclitaxel and genomic changes were analyzed by quantitative PCR. RESULTS: Hematologic labeled intratumoral mast cells were significantly upregulated in the intraepithelial and stromal regions of the tumor, post-NACT. Lower levels of pre-NACT mast cells were significantly associated with an improved progression-free survival (PFS). Histamine, a marker of mast cell degranulation was similarly upregulated in post-NACT exposed tumors. Through the characterization of mast cell specific proteases Tryptase and CPA3, it was found that Tryptase+/ CPA3 + mast cells were significantly upregulated both in the intraepithelial and stromal compartments of the tumor, while Tryptase + cells were significantly upregulated in the stromal regions of the tumor. Lower post-NACT treated levels with Tryptase+/ CPA3 + cells were significantly associated with improved overall survival (OS) and PFS while higher Tryptase + mast cells were associated with improved OS. Finally, following chemotherapy exposure mast cell activating factors AREG and CCL2 were significantly upregulated while TGFB1, an inhibitor of mast cell activation was downregulated in LUVA cells. CONCLUSIONS: Enhanced mast cell numbers, as well as activation and degranulation are a consequence of NACT exposure. Post-NACT mast cells displayed differing associations with survival outcomes that was dependent upon granule classification. Ultimately, mast cells represent a clinically relevant putative HGSOC immune target.

Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study.

OBJECTIVES: K-ras gene product in the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway is critical in the development of certain types of malignancies. K-ras mutation-associated pancreatic and ovarian carcinomas often display mucinous differentiation. Previous studies have shown that k-ras mutation is found in 10% to 30% of endometrial carcinomas. We investigated k-ras mutations in several morphologic subtypes of endometrial carcinomas with particular emphasis on various degrees of mucinous differentiation. METHODS: Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections. Polymerase chain reaction amplification for k-ras codons 12 and 13 were performed, followed by sequencing using capillary electrophoresis. The Fisher exact test is used to compare the prevalent difference of k-ras mutation among the groups. P < 0.05 was considered significant. RESULTS: K-ras mutations were detected in 8 (80%) of 10 mucinous carcinomas, 12 (67%) of 18 endometrioid carcinomas (ECs) with significant mucinous differentiation (ECMD), 4 (25%) of 16 ECs, and 1 (9%) of 11 serous carcinomas. The differences were statistically significant between mucinous carcinomas versus EC (P < 0.01) and ECMD versus EC (P < 0.05). CONCLUSION: The findings suggest that mucinous carcinoma and endometrioid carcinoma with significant mucinous component are more likely to be associated with k-ras mutation. Potential clinical implications of k-ras mutation lies in the management of recurrent or higher-stage endometrial mucinous tumors, which would not be responsive to treatment protocols containing epidermal growth factor receptor inhibitors.

Bridging Anatomy, Histology, and Pathology: A Novel Curriculum Component Incorporating Self-directed Learning for First-Year Medical Students.

During cadaveric dissection or prosection, medical students frequently encounter pathology that can be a springboard to further learning. We designed a novel educational activity linking anatomy, histology, and pathology that incorporated self-directed learning and teamwork, followed by feedback and instruction from pathologists. Post-activity, more than 97% of students rated the activity as useful (Likert scale of 1 to 5), indicating this activity should be continued in the future. Strengths included self-directed learning, content and design, and teamwork, peer, and faculty interactions. Clarity of assignment expectations, range of pathologies, and virtual presentation format remained areas for improvement.

Impact of Drug Exposure on Resistance Selection Following Artemether-Lumefantrine Treatment for Malaria in Children With and Without HIV in Uganda.

Artemisinin-based combination therapies (ACTs) are the primary treatment for malaria. It is essential to characterize the pharmacokinetics (PKs) and pharmacodynamics (PDs) of ACTs in vulnerable populations at risk of suboptimal dosing. We developed a population PK/PD model using data from our previous study of artemether-lumefantrine in HIV-uninfected and HIV-infected children living in a high-transmission region of Uganda. HIV-infected children were on efavirenz-, nevirapine-, or lopinavir-ritonavir-based antiretroviral regimens, with daily trimethoprim-sulfamethoxazole prophylaxis. We assessed selection for resistance in two key parasite transporters, pfcrt and pfmdr1, over 42-day follow-up and incorporated genotyping into a time-to-event model to ascertain how resistance genotype in relation to drug exposure impacts recurrence risk. Two hundred seventy-seven children contributed 364 episodes to the model (186 HIV-uninfected and 178 HIV-infected), with recurrent microscopy-detectable parasitemia detected in 176 episodes by day 42. The final model was a two-compartment model with first-order absorption and an estimated age effect on bioavailability. Systemic lumefantrine exposure was highest with lopinavir-ritonavir, lowest with efavirenz, and equivalent with nevirapine and HIV-uninfected children. HIV status and lumefantrine concentration were significant factors associated with recurrence risk. Significant selection was demonstrated for pfmdr1 N86 and pfcrt K76 in recurrent infections, with no evidence of selection for pfmdr1 Y184F. Less sensitive parasites were able to tolerate lumefantrine concentrations ~ 3.5-fold higher than more sensitive parasites. This is the first population PK model of lumefantrine in HIV-infected children and demonstrates selection for reduced lumefantrine susceptibility, a concern as we confront the threat to ACTs posed by emerging artemisinin resistance in Africa.

Ciliated/tubal-type in-situ and invasive endocervical adenocarcinoma: Report of three cases with limited follow-up and review of the literature.

In situ (AIS) and invasive endocervical adenocarcinoma have two broad categories, HPV-associated (HPV) and HPV-independent groups. (1) These entities show various types of cell morphology. Tubal and tubo-endometrioid type metaplasia of the cervix is a benign finding (Suh and Silverberg, 1990). Tubal metaplasia is also encountered in benign and malignant endometrial lesions. During cervical biopsy interpretations, differentiating the site of origin of the tissue is often tricky. We intend to document three cases of the sparsely reported hrHPV-associated ciliated/tubal-type endocervical AIS and invasive adenocarcinoma and bring it to the attention of readers how to avoid any misinterpretation during routine sign-out. Only three of fifty-three cases of hrHPV-associated AIS and invasive adenocarcinoma were of ciliated/tubal type in our department over a 5-year time. The presence of tubal-type epithelium should not automatically trigger the assumption of endometrial origin of the lesion. These cases are red herrings as tubal/ciliated type dysplasia, and carcinoma is rare and have potential to escape accurate diagnosis.

A Comparative Analysis of Deep Learning Models for Automated Cross-Preparation Diagnosis of Multi-Cell Liquid Pap Smear Images.

Routine Pap smears can facilitate early detection of cervical cancer and improve patient outcomes. The objective of this work is to develop an automated, clinically viable deep neural network for the multi-class Bethesda System diagnosis of multi-cell images in Liquid Pap smear samples. 8 deep learning models were trained on a publicly available multi-class SurePath preparation dataset. This included the 5 best-performing transfer learning models, an ensemble, a novel convolutional neural network (CNN), and a CNN + autoencoder (AE). Additionally, each model was tested on a novel ThinPrep Pap dataset to determine model generalizability across different liquid Pap preparation methods with and without Deep CORAL domain adaptation. All models achieved accuracies >90% when classifying SurePath images. The AE CNN model, 99.80% smaller than the average transfer model, maintained an accuracy of 96.54%. During consecutive training attempts, individual transfer models had high variability in performance, whereas the CNN, AE CNN, and ensemble did not. ThinPrep Pap classification accuracies were notably lower but increased with domain adaptation, with ResNet101 achieving the highest accuracy at 92.65%. This indicates a potential area for future improvement: development of a globally relevant model that can function across different slide preparation methods.

Intratumoral expression analysis reveals that OX40 and TIM-3 are prominently expressed and have variable associations with clinical outcomes in high grade serous ovarian cancer.

Patients with ovarian cancer exhibit low response rates to anti-programmed cell death protein-1 (PD-1) based therapies, despite ovarian tumors demonstrating measurable immune responses. Therefore, the aim of the present study was to comparatively examine expression of notable immune co-stimulatory and co-inhibitory receptors in order identify the most abundant receptors that could potentially serve as therapeutic targets to enhance immunotherapy response in high grade serous ovarian cancer (HGSOC). The Cancer Genome Atlas (TCGA) was employed to compare levels of various HGSOC and pan-cancer cohorts. To confirm these findings at the protein level, immunofluorescence of select receptors was performed in 29 HGSOC patient tissue samples. TCGA and Kaplan Meier analysis was employed to determine the association of highly expressed immune receptors with clinical outcomes. TIM-3 and OX40 exhibited the highest expression in HGSOC at both the gene and protein level, with TIM-3 demonstrating highest levels on both CD8+ and CD4+ T cell subsets. Pan-cancer analysis determined that TIM-3 and OX40 levels were similar to those in immunotherapy-responsive cancers, while PD-1 exhibited much lower expression in HGSOC. Finally, OX40 was most strongly associated with improved patient survival. Overall, the current study suggested that TIM-3 and OX40 are frequently expressed intratumoral immune receptors in HGSOC and thus represent promising immune targets. Furthermore, the present analysis strongly suggested that OX40 was significantly associated with a longer survival and could potentially be utilized as a prognostic factor for improved patient outcomes in HGSOC.

Immune Modeling Analysis Reveals Immunologic Signatures Associated With Improved Outcomes in High Grade Serous Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy worldwide, as patients are typically diagnosed at a late stage and eventually develop chemoresistant disease following front-line platinum-taxane based therapy. Only modest results have been achieved with PD-1 based immunotherapy in ovarian cancer patients, despite the fact that immunological responses are observed in EOC patients. Therefore, the goal of this present study was to identify novel immune response genes and cell subsets significantly associated with improved high grade serous ovarian cancer (HGSOC) patient prognosis. A transcriptomic-based immune modeling analysis was employed to determine levels of 8 immune cell subsets, 10 immune escape genes, and 22 co-inhibitory/co-stimulatory molecules in 26 HGSOC tumors. Multidimensional immune profiling analysis revealed CTLA-4, LAG-3, and Tregs as predictive for improved progression-free survival (PFS). Furthermore, the co-stimulatory receptor ICOS was also found to be significantly increased in patients with a longer PFS and positively correlated with levels of CTLA-4, PD-1, and infiltration of immune cell subsets. Both ICOS and LAG-3 were found to be significantly associated with improved overall survival in The Cancer Genome Atlas (TCGA) ovarian cancer cohort. Finally, PVRL2 was identified as the most highly expressed transcript in our analysis, with immunohistochemistry results confirming its overexpression in HGSOC samples compared to normal/benign. Results were corroborated by parallel analyses of TCGA data. Overall, this multidimensional immune modeling analysis uncovers important prognostic immune factors that improve our understanding of the unique immune microenvironment of ovarian cancer.

The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes.

Epithelial ovarian cancer (EOC) is a highly lethal gynecologic malignancy arising from the fallopian tubes that has a high rate of chemoresistant recurrence and low five-year survival rate. The ovarian cancer biomarker HE4 is known to promote proliferation, metastasis, chemoresistance, and suppression of cytotoxic lymphocytes. In this study, we sought to examine the effects of HE4 on signaling within diverse cell types that compose the tumor microenvironment. HE4 was found to activate STAT3 signaling and promote upregulation of the pro-angiogenic STAT3 target genes IL8 and HIF1A in immune cells, ovarian cancer cells, and endothelial cells. Moreover, HE4 promoted increases in tube formation in an in vitro model of angiogenesis, which was also dependent upon STAT3 signaling. Clinically, HE4 and IL8 levels positively correlated in ovarian cancer patient tissue. Furthermore, HE4 serum levels correlated with microvascular density in EOC tissue and inversely correlated with cytotoxic T cell infiltration, suggesting that HE4 may cause deregulated blood vessel formation and suppress proper T cell trafficking in tumors. Collectively, this study shows for the first time that HE4 has the ability to affect signaling events and gene expression in multiple cell types of the tumor microenvironment, which could contribute to angiogenesis and altered immunogenic responses in ovarian cancer.

Invasive Lobular Carcinoma With Extracellular Mucin: Not All Mucinous Mammary Carcinomas Are Ductal!

Mucinous component in mammary carcinoma indicates ductal phenotype. Although intracytoplasmic mucin and signet ring cell change are frequently described with lobular carcinomas, extracellular mucin is not associated with lobular phenotype. We report 4 cases of invasive lobular carcinoma with extracellular mucin (ILCEM) and review findings of previously published cases of this rare ILC variant. Variable amount of extracellular mucin and pseudoglandular architectural pattern may lead to diagnosis of ILCEM as ductal mucinous carcinoma. Pathologists should be aware of this rare variant of ILC that will help identify more cases of this entity and clarify relevance of extracellular mucin production in ILC.

Molecular diagnosis and monitoring in the clinical management of patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors.

The well-established molecular pathogenesis of chronic myelogenous leukemia (CML) and its consequences for laboratory testing and clinical management illustrate a classic paradigm for the importance of molecular diagnostics in targeted drug therapy. The success of the tyrosine kinase inhibitor (TKI), imatinib, as the currently recommended first-line treatment of early chronic phase CML has both fueled the need for timely and reproducible molecular testing of the BCR-ABL1 fusion transcript in diagnosis and monitoring as well as necessitated the detection of kinase domain mutations that confer resistance to this agent. As, ongoing research continues to refine guidelines for monitoring residual disease in patients undergoing TKI therapy, an understanding of molecular technologies and their interpretation is critical. This review summarizes the molecular strategies that are currently employed in the initial diagnosis and subsequent management of CML patients maintained on TKI therapy.

Routine histologic features in complex atypical hyperplasia can predict the presence of endometrial carcinoma: a clinicopathological study of 222 cases.

There is a wide range of finding endometrial adenocarcinoma (ADCA) in the uterus after a diagnosis of complex atypical hyperplasia (CAH), likely due to a poor diagnostic reproducibility and an inherent heterogeneity in CAH. We evaluated whether histologic subtyping of CAH would help predict ADCA. Our study consisted of 222 cases of CAH diagnosed by endometrial biopsy or curettage. ADCA was seen in 38.3% of these cases at hysterectomy. We divided CAH into 2 subtypes: type A was defined as back-to-back glands in a focus smaller than 2.1 mm, and type B as crowded glands with cytologic atypia but with still-intervening stroma regardless of lesional size. Type A was associated with a significantly higher frequency of ADCA (75.9%) compared with type B (26.2%). Lesions containing neutrophilic/cellular debris showed a higher association of ADCA (60.0%) compared with those without neutrophilic/cellular debris (35.5%). CAH present outside endometrial polyp was associated with a higher frequency of ADCA (42.5%) than that confined to endometrial polyp (19.5%). Within type B cases, lesions greater than 3 mm had a higher association of ADCA (34.3%) than did smaller ones (13.6%). Patients older than 50 years were more likely to have ADCA in the uterus compared with younger women with a preoperative diagnosis of CAH (43.2% versus 28.3%). CAH made on office biopsy showed a higher association of ADCA (46.6%) compared with a diagnosis made on curettage (31.1%). Recognition of these clinicopathological features in CAH may prove useful in predicting the likelihood of ADCA in the uterus.

High-risk human papillomavirus infection involving multiple anatomic sites of the female lower genital tract: a multiplex real-time polymerase chain reaction-based study.

High-risk human papillomavirus infection usually is seen at one anatomic site in an individual. Rarely, infection at multiple anatomic sites of the female lower genital tract in the same individual is encountered either simultaneously and/or at a later date. The current study identifies the various subtypes of high-risk human papillomavirus infection in these scenarios and analyzes the potential significance of these findings. High-risk human papillomavirus infection involving 22 anatomic sites from 7 individuals was identified after institutional review board approval. Residual paraffin-embedded tissue samples were retrieved, and all 15 high-risk human papillomavirus were identified and viral load quantified using multiplex real-time polymerase chain reaction-based method. Multiple high-risk human papillomavirus subtypes were identified in 32% of the samples and as many as 5 different subtypes of high-risk human papillomavirus infection in a single anatomic site. In general, each anatomic site has unique combination of viral subtypes, although one individual showed overlapping subtypes in the vagina, cervix, and vulvar samples. Higher viral load and rare subtypes are more frequent in younger patients and in dysplasia compared with carcinoma. Follow-up ranging from 3 to 84 months revealed persistent high-risk human papillomavirus infection in 60% of cases.

Advanced glycation endproduct precursor alters intracellular amyloid-beta/A beta PP carboxy-terminal fragment aggregation and cytotoxicity.

Carbonyl stress from products of lipid peroxidation, such as 4-hydroxynonenal (HNE), and products of sugars in diabetes mellitus, such as methylglyoxal (MG) and glyoxal (G), may contribute to neurodegeneration in Alzheimer's disease (AD). We tested the hypothesis that these carbonyls alter the proposed central pathogenic mechanism of AD, intracellular amyloid-beta (A beta)-mediated cytotoxicity, using a human neuroblastoma cell line that conditionally expresses carboxy-terminal fragments (CTFs) of the amyloid precursor protein. HNE was a potent cytotoxin, whereas G was mildly cytotoxic; cytotoxicity from each was independent of A beta/CTF expression and not altered by alpha-tocopherol. In contrast, MG cytotoxicity was enhanced by the induced expression of A beta/CTFs and suppressed by alpha-tocopherol. alpha-tocopherol cytoprotection was accompanied by decreased A beta/CTF aggregation. G also promoted beta/CTF aggregation but by mechanisms unaffected by alpha-tocopherol treatment. Our findings showed that A beta/CTF aggregation and cytotoxicity may be profoundly altered by aldehydes associated with diabetes and that in the case of MG, this process is suppressed by alpha-tocopherol. Moreover, our results suggest that while intracellular aggregation of A beta/CTFs may be necessary for the development of toxicity attributable to their expression in this model, the presence of high-molecular weight aggregated A beta/CTFs does not invariably lead to cytotoxicity.

Utility of ProExC and IMP3 immunocytochemical staining in atypical glandular cells of undetermined significance in liquid-based cervical cytology.

The diagnosis of atypical glandular cells of undetermined significance (AGUS) in liquid-based cervical cytology specimens shows significant underlying pathology in only 30% of cases, while the remaining cases are found to be benign (reactive, reparative/metaplastic). Previous studies have reported positive ProExC and IMP3 staining in neoplastic glandular lesions of the uterine cervix and corpus. We present our experience with the utility of these markers in the evaluation of AGUS cases in liquid-based cervical cytology. The case cohort included 34 cases diagnosed as AGUS. ProExC and IMP3 immunocytochemical (ICC) stains were performed on ThinPrep® slides and the results correlated with subsequent biopsy findings. Positive expression was classified as strong diffuse nuclear immunostaining for ProExC and granular cytoplasmic for IMP3. The presence of AGUS cells on the ICC stained slides was confirmed in all cases. IMP3 was positive in 80% of glandular neoplasms and negative in 93% non-glandular lesions/cases negative for squamous intraepithelial lesion (SIL). ProExC was positive in 60% of glandular neoplasms and negative in 83% non-glandular lesions/cases negative for SIL. When used as a panel (ProExC + IMP3), at least one stain was positive in 100% of glandular neoplasm cases and they were both negative in 83% of non-glandular lesions/cases negative for SIL. Based on this study, both ProExC and IMP3, when used as an immuno panel, can predict the presence of glandular lesions on subsequent biopsies and can serve as an aid in the diagnosis and management of AGUS cases.

Liesegang-like rings in lactational changes in the breast.

Liesagang-like rings (LR) are periodic structures with equally spaced radial striations formed by a process that involves diffusion, nucleation, flocculation or precipitation, and supersaturation. Being more common in vitro, on rare occasions also reported in vivo in association with inflammatory or cystic lesions and confused with parasites or calcification on needle aspirates. The current paper documents that LRs may be seen in noncystic and noninflammatory changes of the breast.

The presence and location of epithelial implants and implants with epithelial proliferation may predict a higher risk of recurrence in serous borderline ovarian tumors: a clinicopathologic study of 188 cases.

Serous borderline ovarian tumors have a favorable prognosis, and recurrences are uncommon. The factors influencing recurrence are not fully understood. Epithelial inclusions are identified in serous borderline ovarian tumors and are traditionally referred to as epithelial implants, which often show epithelial proliferation. We investigated whether the presence of epithelial implant and epithelial proliferation portends a higher risk for recurrence of serous borderline ovarian tumors in patients who underwent surgical removal of these tumors. Also examined was whether the anatomical site of epithelial implant and epithelial proliferation was associated with a higher risk of recurrence. One hundred eighty-eight cases of pure serous or predominantly serous borderline ovarian tumors were studied for the presence of epithelial implant and epithelial proliferation, and subsequent recurrences were recorded. The anatomical sites of epithelial implant and epithelial proliferation were compared between serous borderline ovarian tumors with or without recurrence. Statistical analysis was performed using the χ(2) test. Epithelial implant was noted in 106 cases (56%), and epithelial proliferation, in 26 cases (14%). Recurrence was identified in 10.4% cases with epithelial implant and 23% cases with epithelial proliferation. Statistical analyses of patients with recurrence showed significant differences in the following groups: epithelial implant versus no epithelial implant (P < .025) and epithelial proliferation versus no epithelial implant (P < .001). Recurrence rates were higher in the epithelial implant and epithelial proliferation groups as compared with no epithelial implant or epithelial proliferation groups. Epithelial implant and epithelial proliferation appear to pose a statistically significantly higher risk of recurrence in serous borderline ovarian tumors as compared with the absence of epithelial implant. Although the anatomical location of such implants was not significantly associated with a higher risk, the presence of epithelial proliferation at multiple sites was more frequently seen in recurrent serous borderline ovarian tumors.

A Rapidly Enlarging Squamous Inclusion Cyst in an Axillary Lymph Node following Core Needle Biopsy.

A 73-year-old woman was found to have a 1.7 cm axillary mass, for which a core needle biopsy was performed. The specimen revealed fragmented squamous epithelium surrounded by lymphoid tissue consistent with a squamous inclusion cyst in a lymph node, but a metastatic squamous cell carcinoma could not be excluded. Within one month, the lesion enlarged to 5 cm and was excised. Touch preparation cytology during intraoperative consultation displayed numerous single and sheets of atypical epithelioid cells with enlarged nuclei and occasional mitoses, suggesting a carcinoma. However, multinucleated giant cells and neutrophils in the background indicated reactive changes. We interpreted the touch preparation as atypical and recommended conservative surgical management. Permanent sections revealed a ruptured squamous inclusion cyst in a lymph node with extensive reactive changes. Retrospectively, the atypical epithelioid cells on touch preparation corresponded to reactive histiocytes. This is the first case report of a rapidly enlarging ruptured squamous inclusion cyst in an axillary lymph node following core needle biopsy. Our case demonstrates the diagnostic challenges related to a ruptured squamous inclusion cyst and serves to inform the readers to consider this lesion in the differential diagnosis for similar situations.