Single-cell analysis of menstrual endometrial tissues defines phenotypes associated with endometriosis.

BACKGROUND: Endometriosis is a common, complex disorder which is underrecognized and subject to prolonged delays in diagnosis. It is accompanied by significant changes in the eutopic endometrial lining. METHODS: We have undertaken the first single-cell RNA-sequencing (scRNA-Seq) comparison of endometrial tissues in freshly collected menstrual effluent (ME) from 33 subjects, including confirmed endometriosis patients (cases) and controls as well as symptomatic subjects (who have chronic symptoms suggestive of endometriosis but have not been diagnosed). RESULTS: We identify a unique subcluster of proliferating uterine natural killer (uNK) cells in ME-tissues from controls that is almost absent from endometriosis cases, along with a striking reduction of total uNK cells in the ME of cases (p < 10-16). In addition, an IGFBP1+ decidualized subset of endometrial stromal cells are abundant in the shed endometrium of controls when compared to cases (p < 10-16) confirming findings of compromised decidualization of cultured stromal cells from cases. By contrast, endometrial stromal cells from cases are enriched in cells expressing pro-inflammatory and senescent phenotypes. An enrichment of B cells in the cases (p = 5.8 × 10-6) raises the possibility that some may have chronic endometritis, a disorder which predisposes to endometriosis. CONCLUSIONS: We propose that characterization of endometrial tissues in ME will provide an effective screening tool for identifying endometriosis in patients with chronic symptoms suggestive of this disorder. This constitutes a major advance, since delayed diagnosis for many years is a major clinical problem in the evaluation of these patients. Comprehensive analysis of ME is expected to lead to new diagnostic and therapeutic approaches to endometriosis and other associated reproductive disorders such as female infertility.

BORIS Expression in Ovarian Cancer Precursor Cells Alters the CTCF Cistrome and Enhances Invasiveness through GALNT14.

High-grade serous carcinoma (HGSC) is the most aggressive and predominant form of epithelial ovarian cancer and the leading cause of gynecologic cancer-related death. We have previously shown that CTCFL (also known as BORIS, Brother of the Regulator of Imprinted Sites) is expressed in most ovarian cancers, and is associated with global and promoter-specific DNA hypomethylation, advanced tumor stage, and poor prognosis. To explore its role in HGSC, we expressed BORIS in human fallopian tube secretory epithelial cells (FTSEC), the presumptive cells of origin for HGSC. BORIS-expressing cells exhibited increased motility and invasion, and BORIS expression was associated with alterations in several cancer-associated gene expression networks, including fatty acid metabolism, TNF signaling, cell migration, and ECM-receptor interactions. Importantly, GALNT14, a glycosyltransferase gene implicated in cancer cell migration and invasion, was highly induced by BORIS, and GALNT14 knockdown significantly abrogated BORIS-induced cell motility and invasion. In addition, in silico analyses provided evidence for BORIS and GALNT14 coexpression in several cancers. Finally, ChIP-seq demonstrated that expression of BORIS was associated with de novo and enhanced binding of CTCF at hundreds of loci, many of which correlated with activation of transcription at target genes, including GALNT14. Taken together, our data indicate that BORIS may promote cell motility and invasion in HGSC via upregulation of GALNT14, and suggests BORIS as a potential therapeutic target in this malignancy. IMPLICATIONS: These studies provide evidence that aberrant expression of BORIS may play a role in the progression to HGSC by enhancing the migratory and invasive properties of FTSEC.

TGF-ß signaling promotes tumor vasculature by enhancing the pericyte-endothelium association.

BACKGROUND: The breast cancer microenvironment promotes tumor vascularization through the complex interactions involving tumor-associated fibroblasts (TAFs). Emerging data indicate that TAFs increase production and signaling by TGF-ß cytokines, while the role of TGF-ß signaling in the regulation of tumor blood vessels is not fully understood. The current study presents evidence that TAFs enhance the organization of tumor blood capillaries, and TGF-ß signaling plays an important role in this response. METHODS: Tumor vascularization was studied in xenograft models of breast carcinoma cells, alone and in combination with fibroblasts. TGF-ß signaling in breast cancer cells was modulated by expression of kinase-inactive TGFBR1-K232R (dnTGFBR1) or constitutive-active TGFBR1-T204D (caTGFBR1) receptor mutants. The architecture of tumor blood capillaries was assessed by immune-histochemical analysis of endothelium and pericytes. The role of TGF-ß-Smad signaling in fibronectin expression was examined using adenoviral transduction of signaling components. RESULTS: Our studies revealed that TAFs significantly increase the lumen size of blood microvessels. Inactivation of TGF-ß signaling in tumor cells by dnTGFBR1 reduced the microvessel density and lumen sizes, decreasing tumor growth. In contrast, caTGFBR1-tumors exhibited greater vessel density and lumen sizes. Tumors with inactive dnTGFBR1 showed lower amounts of TAFs, while caTGFBR1 increased amounts of TAFs compared to the control. Inspection of pericytes and endothelial cells in tumor vasculature revealed that TAFs enhanced vessel coverage by pericytes, vascular cells supporting capillaries. This effect was impaired in dnTGFBR1-tumors, whereas active caTGFBR1 enhanced the association of pericytes with endothelium. Accordingly, dnTGFBR1-tumors exhibited the presence of hemorrhages, a sign of fragile blood vessels. Biochemical analysis showed that TGFBR1-SMAD signaling up-regulates fibronectin, a prominent regulator of endothelium-pericyte interactions. CONCLUSIONS: The current study indicates that tumor-fibroblast crosstalk enhances tumor vascularization by increasing the pericyte-endothelium association via a mechanism involving the TGFß-fibronectin axis. The tumor-fibroblast model represents a useful system for dissecting the complex interactions governing tumor angiogenesis and developing new approaches to therapeutic targeting tumor vasculature.

Tumor p38MAPK signaling enhances breast carcinoma vascularization and growth by promoting expression and deposition of pro-tumorigenic factors.

The breast carcinoma microenvironment strikingly influences cancer progression and response to therapy. Various cell types in the carcinoma microenvironment show significant activity of p38 mitogen-activated protein kinase (MAPK), although the role of p38MAPK in breast cancer progression is still poorly understood. The present study examined the contribution of tumor p38MAPK to breast carcinoma microenvironment and metastatic capacity. Inactivation of p38MAPK signaling in metastatic breast carcinoma cells was achieved by forced expression of the kinase-inactive mutant of p38/MAPK14 (a dominant-negative p38, dn-p38). Disruption of tumor p38MAPK signaling reduced growth and metastases of breast carcinoma xenografts. Importantly, dn-p38 markedly decreased tumor blood-vessel density and lumen sizes. Mechanistic studies revealed that p38 controls expression of pro-angiogenic extracellular factors such as matrix protein Fibronectin and cytokines VEGFA, IL8, and HBEGF. Tumor-associated fibroblasts enhanced tumor growth and vasculature as well as increased expression of the pro-angiogenic factors. These effects were blunted by dn-p38. Metadata analysis showed elevated expression of p38 target genes in breast cancers and this was an unfavorable marker of disease recurrence and poor-outcome. Thus, our study demonstrates that tumor p38MAPK signaling promotes breast carcinoma growth, invasive and metastatic capacities. Importantly, p38 enhances carcinoma vascularization by facilitating expression and deposition of pro-angiogenic factors. These results argue that p38MAPK is a valuable target for anticancer therapy affecting tumor vasculature. Anti-p38 drugs may provide new therapeutic strategies against breast cancer, including metastatic disease.

Tumor-fibroblast interactions stimulate tumor vascularization by enhancing cytokine-driven production of MMP9 by tumor cells.

Advance-stage breast carcinomas include significant amounts of fibroblasts and infiltrating immune cells which have been implicated in tumor growth, recurrence, and response to therapy. The present study investigated the contribution of fibroblasts to tumor growth using direct tumor-fibroblast co-cultures and tumor xenograft models. Our findings revealed that fibroblasts enhance breast carcinoma growth by promoting the tumor vasculature via the MMP9-dependent mechanism. In tumor-fibroblast co-cultures, fibroblasts increased expression of TGF-ß, TNF, and IL-1ß cytokines in tumor cells. These cytokines cooperatively induced expression of matrix metalloproteinase MMP9 in tumor cells. Knockdown of MMP9 by shRNA significantly reduced tumor vascularization induced by fibroblasts. Mechanistically, our findings argue that expression of MMP9 in tumor cellsis regulated by crosstalk of TGF-ß with TNF and/or IL-1ß cytokines. The mechanism of this cooperative response did not involve cross-activation of the canonical signaling pathways as TGF-ß did not activate RELA/p65 signaling, while TNF did not affect SMAD signaling. Instead, TGF-ß and TNF cytokines co-stimulated MAP kinases and expression of JUN and JUNB, AP1 transcription factor subunits, which together with RELA/p65 were essential for the regulation of MMP9. Depletion of JUN and JUNB or RELA in tumor cells blocked the cooperative induction of MMP9 by the cytokines. Thus, our studies uncovered a previously unappreciated role of tumor-fibroblast interactions in the stimulation of tumor angiogenesis, and an essential role of the MAPK-AP1 axis in the cooperative up-regulation of the angiogenic driver MMP9 by cytokine crosstalk.

Metaplastic ossification of the temporal artery with osteoclast-like giant cells: a mimicker of giant cell (temporal) arteritis.

PURPOSE: To describe a patient presenting with suspected giant cell (temporal) arteritis (GCA) in whom subsequent temporal artery biopsy showed luminal narrowing by medial calcification, metaplastic ossification, and fibrointimal proliferation, consistent with calciphylaxis. METHODS: A 55-year-old man with end-stage renal disease presented with unilateral loss of vision and elevated erythrocyte sedimentation rate and was initially treated as though he had GCA; however, a subsequent temporal artery biopsy showed marked luminal narrowing by medial calcification, metaplastic ossification, and fibrointimal proliferation, consistent with calciphylaxis. In addition, the tunica media of the affected artery contained multinucleate giant cells, but these represented osteoclasts and foreign body giant cells reacting to calcium, rather than a part of GCA. RESULTS: This is a rare report of metaplastic ossification and the finding of non-GCA-related giant cells in the tunica media of the temporal artery, thus representing a clinical and histopathologic mimicker of GCA. CONCLUSIONS: The clinical differential diagnosis of GCA includes other etiologies that can present similarly; however, temporal artery biopsy can discern the underlying pathology. Importantly, the identification of giant cells is not required for the diagnosis of GCA, and likewise, as our case shows, the finding of giant cells in the wall of a temporal artery does not always imply a diagnosis of GCA.

Hyperplasia and carcinoma in secretory endometrium: a diagnostic challenge.

The diagnosis of endometrial hyperplasia or carcinoma in a background of secretory endometrium can be difficult. We attempt to establish the diagnostic criteria to be used in such cases. We examined 80 cases of endometrial hyperplasia, carcinoma, and other conditions with glandular crowding arising in secretory endometrium, analyzed their morphologic features, assessed the volume percentage stroma in each case and performed Ki67 immunostaining on 27 cases. Thirteen cases each of secretory and gestational endometrium served as controls. The mean age of the patients was 45 yr. The non-neoplastic diseases included simple hyperplasia without atypia (56%), endometrial polyps (12.5%), and chronic endometritis with glandular crowding (3%). The proportion of cases with complex hyperplasia without atypia was 10%. Neoplastic diseases included atypical complex hyperplasia (12.5%) and endometrioid carcinoma (6%). The secretory changes were usually less advanced in the hyperplastic glands than in the background endometrium. The morphologic features that best distinguished hyperplasia or carcinoma from secretory endometrium included glandular crowding that stood out from the background; architectural disorder (the long axes of the glands pointing in different directions or parallel to the endometrial surface); dilated, irregularly shaped glands, including budding or branching glands and staghorn-shaped glands; stroma of a polyp; cribriform or confluent glands in cases of carcinoma; nuclear atypia in cases of atypical hyperplasia and carcinoma; and crowded nonsecretory glands. The volume percentage stroma of neoplastic lesions was less than that of non-neoplastic ones (34% vs. 61%, P=0.000001) and that of secretory endometrium (34% vs. 68%, P=0.000038). Non-neoplastic lesions did not have significantly more crowded glands than secretory endometrium (61% vs. 68%, P=0.11). Gestational endometrium had more crowded glands than non-neoplastic lesions (39% vs. 61%, P=0.000004), an approximately equal volume percentage stroma with complex hyperplasia without atypia (39% vs. 43%, P=0.51), and less crowded glands than neoplastic lesions (39% vs. 34%, P=0.03). The Ki67 index of the neoplastic lesions was higher than that of the controls, including secretory and gestational endometria (positive nuclei per 100 epithelial cells, 44.8 vs. 4.6, P=0.0004), of the non-neoplastic lesions (44.8 vs. 5.4, P=0.002) and of complex hyperplasia without atypia (44.8 vs. 9.3, P=0.007). Hyperplasia and carcinoma in secretory endometrium can be diagnosed on the basis of increased glandular crowding, architectural irregularity, nuclear atypia, and increased Ki67 index.

Variability of pathologists' utilization of p16 and ki-67 immunostaining in the diagnosis of cervical biopsies in routine pathology practice and its impact on the frequencies of cervical intraepithelial neoplasia diagnoses and cytohistologic correlations.

CONTEXT: The use of p16 in cervical biopsies improves the accuracy of cervical intraepithelial neoplasia (CIN) diagnosis and grading and decreases its interpathologist variability. OBJECTIVE: To determine the impact of the frequency of use of p16 immunostains in cervical biopsies on pathologists' diagnoses of CIN grade 1 and grade 2 or above (CIN1 and CIN2+) and on cytohistologic correlations. DESIGN: We identified all cervical biopsy specimens with cytologic correlations subjected or not to p16 staining from January 1, 2005, to September 30, 2010; calculated each pathologist's percentage of p16 use; and correlated it with their major cytohistologic discrepancy rates, CIN2+ diagnoses, and CIN1/CIN2+ ratios. RESULTS: During the study period, each of the 23 pathologists interpreted 59 to 1811 (mean, 518) of 11 850 cervical biopsy specimens, used p16 for 0% to 21.31% (mean, 10.14%) of these, had CIN2+ detection rates of 9.5% to 24.1% (mean, 18.9%), and CIN1/CIN2+ ratios of 0.7 to 4.5 (mean, 1.5). Compared to the 12 "low users" of p16, who used p16 fewer times than the institution's mean for p16 use, the 11 "high users" of p16 diagnosed more biopsies (8391 versus 3459), had a lower rate of major cytohistologic discrepancies (12.62% versus 14.92%, P < .001), a higher rate of CIN2+ diagnoses (19.9% versus 16.4%, P < .001), a lower range of CIN2+ rates (15.0%-23.1% versus 9.5%-24.1%), and lower CIN1/CIN2+ ratios (1.2 versus 2.3). CONCLUSIONS: We found a high intrainstitutional variability of p16 use in cervical biopsies, CIN2+ rates, and CIN1/CIN2+ ratios. Use of p16 for greater than 10% of cervical biopsies was associated with improved cytohistologic correlation rates and with lower variability in the frequencies of histologic diagnoses.

Large-cell neuroendocrine carcinoma of the cervix.

Large cell neuroendocrine carcinoma (LCNEC) of the uterine cervix is a rare and aggressive malignancy with poor prognosis even in its early stage, despite multimodality treatment strategy. Here, we report a case of a woman with clinical polypoid stage IB LCNEC of the cervix, which was detected in her 6-week postpartum checkup. A literature review was also conducted to evaluate current therapeutic approaches and potential new strategies.

Metanephric adenoma of the kidney: an unusual diagnostic challenge.

Although metanephric adenoma (MA) is a rare, benign neoplasm of epithelial cells, it is often difficult to distinguish this entity from other malignant neoplasms preoperatively. We report a case of a large renal mass for which preoperative diagnosis was indeterminate, with the differential diagnosis including Wilm's tumor, MA, and papillary renal cell carcinoma (PRCC). Accurate postoperative differentiation of MA from PRCC is critical because adjuvant therapy is considered after surgical resection of PRCC tumors.

Small-cell carcinoma with an epidermal growth factor receptor mutation in a never-smoker with gefitinib-responsive adenocarcinoma of the lung.

Activating mutations in the epidermal growth factor receptor (EGFR) gene are extremely rare in small-cell lung cancer (SCLC). Here, we present a case of an EGFR-mutant gefitinib-responsive non-small-cell lung cancer (NSCLC) of adenocarcinoma histology occurring in a never-smoker followed by subsequent diagnosis of metastatic SCLC carrying an EGFR mutation. Although gefitinib therapy of the primary NSCLC resulted in disease control for over 3 years, the patient subsequently developed metastatic SCLC to the liver. Epidermal growth factor receptor mutation analysis revealed that the exon 21 L858R activating mutation was present in both the original lung adenocarcinoma and the metastatic SCLC. We hypothesize that SCLC either evolved from the previously diagnosed NSCLC or that both arose from a common precursor. Further comparative molecular analysis of these histologically distinct tumors would be of value to better understand the potential role of EGFR in the pathogenesis of SCLC in never-smokers, and the role of selection for an EGFR-mutant SCLC subclone as an unusual mechanism of acquired resistance to EGFR inhibitors in NSCLC.

Collision tumor versus multiphenotypic differentiation: a case of carcinoma with features of colonic and lung primary tumors.

BACKGROUND: Collision tumors are rare tumors composed of two histologically distinct neoplasms coinciding at the same location. Collision tumors need to be distinguished from tumors originating from a progenitor cell with potential for multiphenotypic differentiation. CASE REPORT: A clinically puzzling case of an intricate malignant pulmonary nodule in a patient with previous history of colorectal carcinoma is reported. A brief review of the clinical literature on collision tumor and tumor stem cells is presented. CONCLUSION: This case report emphasizes the importance of detailed histopathologic and immunohistochemical analyses, and clinical history in diagnosing a tumor composed of multiple malignant morphologies.

Rare case of hemangiopericytoma responds to sunitinib.

Hemangiopericytoma (HP) is a rare mesenchymal tumor associated with the capillary wall. We describe a 62-year-old man with a 20-year history of recurrent metastatic HP, in which a favorable clinical benefit was observed with sunitinib, an oral VEGFR, and PDGFR kinase inhibitor. Disease progression was evident during the 3 months before starting sunitinib therapy. Radiologic evaluation indicated that metastatic lesions in the lungs and pelvis remained stable during the 6 months on sunitinib therapy. Histologic analysis of the tumor 3 weeks after active sunitinib therapy did not reveal tumor necrosis, but recanalized thrombi within tumor blood vessels indicated previous tumor insult. Additional studies are warranted to confirm the possible effects of sunitinib in controlling widespread metastatic HP.

Thymic carcinoid responds to neoadjuvant therapy with sunitinib and octreotide: a case report.

Carcinoids are malignant neuroendocrine tumors consisting of a spectrum of neoplasms from low-grade typical carcinoid to high-grade small cell carcinoma. We report a case of atypical thymic carcinoid that responded to neoadjuvant therapy with octreotide and sunitinib, an oral multikinase inhibitor. After 3 weeks of treatment, tumor size significantly decreased to allow for a safe surgical resection with clear margins. We believe that further study of sunitinib and octreotide with the neoadjuvant intent of preparing tumors for resection is warranted as a strategy to improve curative management of neuroendocrine tumors.

Endometrial microcalcifications detected by ultrasonography: clinical associations, histopathology, and potential etiology.

Endometrial microcalcifications are uncommon, with alleged clinical implications ranging from innocuous to ominous. We reviewed the histopathologic slides from 29 patients who had endometrial echogenic foci on pelvic ultrasound and found many endometrial microcalcifications. The extent of microcalcifications in each specimen was graded on a semiquantitative scale from 0 to 3. The mean patient age was 54 years (range, 34-81 years). The specimens included endometrial biopsies, curettages, and hysterectomies. Most of the patients had presented with abnormal vaginal bleeding. Fifteen patients (51.7%) were postmenopausal, 10 (34.5%) were premenopausal, and the rest were perimenopausal. The most frequent endometrial types were atrophic (39.5%), inactive (23.3%), and proliferative (14%). Six specimens (14%) showed benign endometrial polyps. One patient had well-differentiated endometrioid carcinoma of the endometrium without myometrial invasion. Specimens from 16 patients (55.2%) had microcalcifications. The patients with calcifications were older than those without calcifications (mean age, 60 vs. 47 years, respectively; P = 0.017). The extent of microcalcifications positively correlated with the presence of endometrial polyps (P = 0.00076), postmenopausal state (P = 0.004), atrophic endometrium (P = 0.002), and hormone replacement therapy (P = 0.013). The microcalcifications were concentric or amorphous, intraglandular or stromal. They were focally associated with minute papillary epithelial projections or with degenerated endometrial glands. Follow-up was available on 26 patients (89.7%). Except for the patient with endometrioid carcinoma, none has developed uterine, adnexal, or peritoneal malignancy. In summary, endometrial microcalcifications are histologically heterogeneous and are associated with older patient age, postmenopausal state, atrophic endometrium, and endometrial polyps. Those found incidentally by means of pelvic ultrasonography, in our experience, did not portend malignancy.