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Supplementary Figure 1 from Human Endogenous Retrovirus K Triggers an Antigen-Specific Immune Response in Breast Cancer Patients

Recent evidence indicates that human cancer cells reactivate the expression of latent human endogenous retroviral (HERV) proteins. However, the extent to which cancer patients mount de novo immune responses against expressed HERV elements is unclear. In this study, we determined the extent of HERV-K env expression in human breast cancer (BC) and whether both humoral and cell-mediated immunity against HERV-K can be found in BC patients. We found HERV-K env protein expression in 88% of BC (n = 119) but not in normal breast (n = 76) tissues. ELISA screening assays detected significant titers of anti–HERV-K env IgG in a large proportion of BC patients. T-cell responses against HERV-K were also detected in peripheral blood mononuclear cells (PBMC) from BC patients stimulated with autologous dendritic cells pulsed with HERV-K env SU antigens. These responses included induction of T-cell proliferation (P = 0.0043), IFN-γ production measured by enzyme-linked immunospot (P < 0.0001), and multiplex cytokine secretion (P = 0.0033). Multiplex cytokine analysis found a T-helper 1 cytokine response, including interleukin (IL)-2 (P = 0.0109), IL-6 (P = 0.0396), IL-8 (P = 0.0169), and IP-10 (P = 0.0045) secretion during in vitro stimulation of BC PBMC with HERV-K antigen. We also found HERV-K–specific CTLs that were capable of lysing target cells expressing HERV-K env protein in BC patients but not in normal female controls without cancer. These findings suggest that retroviral gene products are capable of acting as tumor-associated antigens activating both T-cell and B-cell responses in BC patients. [Cancer Res 2008;68(14):5869–77]

Objective Self-sampling for human papillomavirus (HPV) testing is an effective option to increase the cervical screening coverage. How to best triage HPV-positive self-samples remains controversial. Here, we evaluated the performance of a novel p16INK4a immunocytology approach (p16) and HPV genotyping in triaging HPV-positive self-samples. Methods A cohort of 73699 women were screened via SeqHPV assay on self-samples. HPV-positive women who met any sequential positive result of HPV16/18 or VIA or p16 were referred for colposcopy. A triage strategy was considered favorable if the NPV for CIN3+ was ≥98%, combined with an improvement of sensitivity and specificity in comparison to the comparator, being the ‘ASC-US+’ triage and the guideline strategy (HPV16/18+ or ASC-US+). Results A total of 3510 HPV-positive women were included, 422 (12.0%) CIN2+ and 247 (7.0%) CIN3+ were identified. The positivity of p16 and ASC-US+ were 36.3% and 22.2%, respectively. p16 was more sensitive and less specific than ASC-US+ (P Conclusions Our findings indicate the promising utility of p16 immunocytology via adjusting the staining score or serving as an ancillary tool to liquid-based cytology or combining with genotyping for the triage of HPV-positive self-samples, which promotes the precise screening of cervical cancer.

Background For cervical cancer screening, routine cytology has a high specificity but a lower sensitivity. In older women, atrophy, which may mimic HSIL, presents a diagnostic challenge. p16 is a widely used biomarker for histological diagnosis of HSIL. Our objective was to evaluate PathCIN® p16INK4a immunocytology in identification of high grade dysplasia vs. benign atrophy. Methods As part of a multi-center screening program, 3351 women were co-tested by p16 immunocytology. Among women referred for colposcopy on basis of cytology and high-risk HPV status, those with atrophy were older than the population screened (52 vs. 43 years). Cases from older women with atrophy (n ​= ​116) and controls without atrophy (n ​= ​47) were identified by re-examination of Pap smears. The detection of CIN2+ was compared for p16, cytology and HR-HPV results. Results The sensitivity of routine cytology (≥LSIL) was much lower for cases with atrophy (17%) than non-atrophic cases (75%). The sensitivity of p16 immunocytology and of HR-HPV testing was high (88%–100%) both with and without atrophy. The specificity of routine cytology (≥LSIL) was higher for cases with atrophy (79%) than non-atrophic cases (38%). The specificity of p16 immunocytology was high (88–95%) and the specificity of HR-HPV testing was low (31%–33%) both with and without atrophy. Combining p16 with HPV testing and/or routine cytology had no benefit, as compared to p16 staining alone. Conclusions p16 immunocytology compares favorably with routine cytology and HPV testing in the differential diagnosis of HSIL and benign atrophy. It is more sensitive than cytology for atrophic specimens, and is more specific than HPV testing. p16 immunocytology may decrease the need for colposcopy referrals and could be a useful tool for early detection of cervical cancer in peri- and post-menopausal women, who are more likely to have HSIL coexisting with atrophy.

Purpose When used for cervical cancer primary screening, liquid-based cytology (LBC) has a high specificity but a low sensitivity. For histological diagnosis of high-grade lesions, p16INK4a immunostaining has proven to be useful. Therefore, our objective was to evaluate the use of p16INK4a immuno-cytology as a primary screen and a secondary screen after primary high-risk human papillomavirus (hrHPV) screening or LBC screening. Methods A total of 1197 cytology slides were immuno-stained using automatic p16INK4a staining system (PathCIN®p16INK4a) in two studies from cervical screening programs. In the primary screening study, 875 slides were randomly selected and analyzed for p16INK4a. In the secondary screening study, 322 of the remaining slides were chosen by virtue of being HPV 16/18+, other hrHPV+/LBC≥ASC-US, or HPV-negative/LBC ≥LSIL. The sensitivity and specificity for detection of cervical intraepithelial neoplasia 2/3 or worse (CIN2+/CIN3+) were compared based on p16INK4a, LBC and HPV test results. Results In combining two studies, there were 431 cases with biopsy pathology. They included 83 cases with CIN2+ and 41 cases with CIN3+. The p16 positivity rate increased with pathologic and cytologic severity (P

Background Self-sampling for human papillomavirus (HPV) testing is a feasible option to improve the cervical screening coverage. However, an ideal triage method for HPV-positive self-samples does not yet exist. The aim of this study was to explore the utility of HPV genotyping and p16INK4a immunostaining (p16) in triaging HPV-positive self-samples, focusing on HPV-positive, cytology-negative (HPCN) women. Methods A total of 73,699 women were screened in a cervical screening project in China via SeqHPV assay on self-samples. HPV-positive women were called-back and collected cervical sample for p16 immunostaining and liquid-based cytology, those who met any result of HPV16/18+ or visual inspection with acetic acid (VIA) + or p16+ were referred for colposcopy, and HPCN women with adequate data on p16 and pathology were analyzed. A triage strategy was considered acceptable if the negative predictive value (NPV) for cervical intraepithelial neoplasia 3 or worse (CIN3+) was 98% or more, combined with an improvement of sensitivity and specificity for CIN2+/CIN3+ in reference to the comparator, being HPV16/18 + . Results A total of 2731 HPCN women aged 30–64 years were enrolled, 136 (5.0%) CIN2+ and 53 (1.9%) CIN3+ were detected. Five triage strategies met the criteria: p16+; HPV16/33+; ‘HPV16+ or HPV33/58/31/35+&p16+’; ‘HPV16/33+ or HPV58/31/35+&p16+’; HPV16/18/31/33/45/52/58 + & p16+. These strategies required less or similar colposcopy referrals, and less colposcopies to detected one case of CIN2+/CIN3+, achieving favorable false positive (negative) rates to the comparator. Among them, p16 staining detected 83.1% (79.2%) of underlying CIN2 + (CIN3+) in HPCN women. Moreover, three triage strategies were favorable in sensitivity and/or specificity to the ‘HPV16/33+’ strategy: p16+; ‘HPV16+ or HPV33/58/31/35 + &p16+’; HPV16/18/31/33/45/52/58 + &p16 + . Conclusions Genotyping for HPV16/33 could be utilized to optimize the management of HPCN women. Moreover, p16 immunostaining, either alone or combined with extended genotypes, is more effective than HPV genotypes alone in the triage of HPCN women.

Self-sampling for human papillomavirus (HPV) testing is an effective option to increase the cervical screening coverage. How to best triage HPV-positive self-samples remains controversial. Here, we evaluated the performance of a novel p16A cohort of 73699 women were screened via SeqHPV assay on self-samples. HPV-positive women who met any sequential positive result of HPV16/18 or VIA or p16 were referred for colposcopy. A triage strategy was considered favorable if the NPV for CIN3+ was ≥98%, combined with an improvement of sensitivity and specificity in comparison to the comparator, being the 'ASC-US+' triage and the guideline strategy (HPV16/18+ or ASC-US+).A total of 3510 HPV-positive women were included, 422 (12.0%) CIN2+ and 247 (7.0%) CIN3+ were identified. The positivity of p16 and ASC-US+ were 36.3% and 22.2%, respectively. p16 was more sensitive and less specific than ASC-US+ (P0.0001). However, when combined p16 with cytology or genotypes, two triage strategies were superior to the 'ASC-US+' strategy: p16 scored 3+; HPV16/33/58/31+p16+. Moreover, four strategies were favorable to the guideline strategy: ASC-US+ or p16+; LSIL+ or p16+; HPV16+ or p16+; HSIL+ or p16+ or HPV16+. These strategies achieved better balance between diseases detection and colposcopy referral.Our findings indicate the promising utility of p16 immunocytology via adjusting the staining score or serving as an ancillary tool to liquid-based cytology or combining with genotyping for the triage of HPV-positive self-samples, which promotes the precise screening of cervical cancer.

Additional file 1: Table S1. Positive Predictive Value (PPV) for CIN2+ and CIN3+. Table S2. p16 positivity stratified by HPV genotypes and histologic grades (n, %). Fig. S1. Management protocol of HPV-positive women. VIA, visual inspection under acetic acid; LBC, liquid-based cytology. Genotyping for HPV16/18, VIA, and p16 immunostaining were used for triage sequentially. Fig. S2. p16 immunostaining, (A) positive, one or more cervical epithelial cells with nuclear and/or cytoplasmic stained brownish or yellow were defined as positive regardless of their morphology. (B) negative.

Purpose When used for cervical cancer primary screening, liquid-based cytology (LBC) has a high specificity but a low sensitivity. For histological diagnosis of high-grade lesions, p16INK4a immunostaining has proven to be useful. Therefore, our objective was to evaluate the use of p16INK4a immuno-cytology as a primary screen and a secondary screen after primary high-risk human papillomavirus (hrHPV) screening or LBC screening. Methods A total of 1197 cytology slides were immuno-stained using automatic p16INK4a staining system (PathCIN®p16INK4a) in two studies from cervical screening programs. In the primary screening study, 875 slides were randomly selected and analyzed for p16INK4a. In the secondary screening study, 322 of the remaining slides were chosen by virtue of being HPV 16/18+, other hrHPV+/LBC≥ASC-US, or HPV-negative/LBC ≥LSIL. The sensitivity and specificity for detection of cervical intraepithelial neoplasia 2/3 or worse (CIN2+/CIN3+) were compared based on p16INK4a, LBC and HPV test results. Results In combining two studies, there were 431 cases with biopsy pathology. They included 83 cases with CIN2+ and 41 cases with CIN3+. The p16 positivity rate increased with pathologic and cytologic severity (P

Epithelioid angiosarcoma of bone is a rare entity. Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a secreted glycoprotein that has been implicated in tumorigenesis. We report a case of epithelioid angiosarcoma involving the long bones of the lower extremity showing diffuse and strong expression for SPARC immunohistochemistry in tumor cells. Ki-67 was positive in ~50% of tumor cell nuclei and the accompanying mitotic index was 19 mitotic figures/10 high power fields.Expression of SPARC in tumors has been correlated with sensitivity to nanoparticle albumin-bound paclitaxel (Nab-paclitaxel), particularly in the context of robust cell cycle progression into the mitotic phase. This finding could suggest new therapeutic options for further consideration.

Aberrant expression of subgroup k human endogenous retroviruses (HERV-K) has been observed in prostate cancer. This subgroup is unique because it encodes sequences in the human genome containing open reading frames for near intact retroviruses. We hypothesized that HERV-K reactivation could serve as a non-invasive early disease detection marker for prostate cancer. We evaluated HERV-K gag messenger RNA (mRNA) expression in blood samples of African-American and European-American men using a case-control design via quantitative real-time PCR. Additionally, we examined HERV-K envelope protein expression in prostate tumors by immunohistochemistry. HERV-K envelope protein was commonly upregulated in prostate tumors, but more so in tumors of African-American than European-American patients (61% versus 40%, P < 0.01). Examining HERV-K gag expression in peripheral blood mononuclear cells (PBMC) from 294 cases and 135 healthy men, we found that the abundance of HERV-K gag message was significantly higher in cases than controls and was associated with increased plasma interferon-gamma. Men with gag expression in the highest quartile had > 12-fold increased odds {odds ratio = 12.87 [95% confidence interval 6.3-26.25]} of being diagnosed with prostate cancer than those in the lowest quartile. Moreover, our results showed that HERV-K expression may perform better as a disease biomarker in older than younger men (whereas the sensitivity of prostate-specific antigen (PSA) testing decreases with age) and in men with a smoking history compared with never smokers. Combining non-invasive HERV-K testing with PSA testing may improve the efficacy of prostate cancer detection specifically among older men and smokers who tend to develop a more aggressive disease.

Lesions arising from anogenital mammary-like glands (AGMLG) are rare and could be benign or malignant, and epithelial or stromal (Kazakov et al. 2010). Fibroepithelial lesions of AGMLG, including f...

BACKGROUND & AIMS: Helicobacter pylori is found within the gastric mucous gel layer and in association with the epithelial surface. The aim of this study was to determine the effect of H. pylori infection on mucin gene expression (MUC6, MUC5, and MUC1) in the gastric epithelium. METHODS: Gastric biopsy specimens were examined by immunohistochemistry and in situ hybridization for mucin gene expression. RESULTS: MUC6 was limited to mucous glands of H. pylori-negative patients, but 72% of H. pylori-positive patients also expressed MUC6 in surface mucous cells. In contrast, MUC5 mucin was found in significantly fewer surface mucous cells of H. pylori-positive specimens. Carbohydrates recognized by Lewis (Le) X and paradoxical concanavalin A (con A) staining were aberrantly expressed in the surface mucous cells of 16 of 27 and 17 of 23 H. pylori-positive tissues, respectively. Surface Le(b) was present in 26 of 27 H. pylori-positive and 19 of 30 H. pylori-negative tissues. Eradication of H. pylori resulted in reversal of surface MUC5 and MUC6 expression toward normal patterns. Purified gastric mucins of H. pylori- positive patients bound more anti-MUC6 and anti-Le(b) than mucins from H. pylori-negative patients. CONCLUSIONS: The type of mucin that is normally limited to mucous glands is aberrantly expressed in surface mucous cells of H. pylori-positive patients, whereas less of the surface epithelium expresses normal surface-type gastric mucin. (Gastroenterology 1997 Aug;113(2):455-64)

✓ The poor prognosis of patients with malignant gliomas is at least partially due to the invasive nature of these tumors. In this study, the authors investigated the possibility that the cysteine protease cathepsin B (CB) is a participant in the process of glial tumor cell invasion. To accomplish this, an immunohistochemical analysis was made of the localization of antibodies to CB in biopsies of five specimens of normal brain, 16 astrocytomas, 33 anaplastic astrocytomas, and 33 glioblastomas multiforme. Staining was scored according to the percentage of positive cells and the intensity of the stain, graded from 0 to 3+. Staining for CB was not seen in any of five samples of normal brain except for occasional neuronal cell bodies and microglia. Only five (31%) of 16 astrocytomas showed a small percentage of positive cells (0.01%–3%) that were stained in a light, diffuse cytoplasmic pattern (1+). Twenty-nine (87.8%) of 33 anaplastic astrocytomas showed positive light, granular staining in 2% to 40% of cells. In anaplastic astrocytoma, the staining within a tumor was heterogeneous with intensities of 1+ (17%), 1+ to 2+ (29%), or 2+ (55%). In contrast, all 33 (100%) glioblastomas were positive in 10% to 90% of cells. The staining was present in a coarse, granular pattern with an intensity of 2+ (12%) or 3+ (88%). Tumor cells infiltrating into brain adjacent to malignant gliomas stained positively in 26 cases that could be evaluated for glioblastoma multiforme; these invading cells frequently followed penetrating blood vessels as typical “secondary structures of Scherer.” Moderate to intense CB staining associated with endothelial proliferation in high-grade tumors was also observed, especially in regions of tumor infiltration into adjacent normal brain. These results provide evidence consistent with the hypothesis that CB is functionally significant in the process of tumor invasion and angiogenesis in the clinical progression of the malignant phenotype in astrocytomas.

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (3H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon.

Small cell carcinomas are among the most aggressive, poorly differentiated, and highly malignant of the neuroendocrine tumors (NETs). Of which, small cell gastric carcinoma is a rare small cell neuroendocrine tumor. The purpose of our study was to present this case and perform a comprehensive literature review.We review a case of small cell gastric carcinoma that is particularly unusual in that it occurred in a woman from the US when the majority of cases of small cell gastric carcinoma have been reported in men from East Asia, and more specifically, from Japan. The diagnosis was made after endoscopy revealed a large ulcerated mass in the gastric cardia of Borrmann type 3. Biopsies revealed multiple small basophilic cells underlying the squamous epithelium of the esophagus and cardiac mucosa, indicating the presence of a tumor at the gastroesophageal junction. Immunostaining established the diagnosis with positive stains for chromogranin, synaptophysin, and CD56. Our patient is being treated with chemotherapy, but many different treatment regimens have been tried for small cell gastric carcinoma with variable success.Overall prognosis for small cell gastric carcinoma is dismal. Neuroendocrine tumors in general have variable clinical behaviors and prognosis is dependent on the neuroendocrine tumor type. The adoption of a standardized classification system for neuroendocrine tumors could improve the recognition of infrequently encountered neuroendocrine tumors like small cell gastric carcinoma and will enhance strategies for treatment and thus improve prognosis for patients with these rare and aggressive tumors.

Recent evidence indicates that human cancer cells reactivate the expression of latent human endogenous retroviral (HERV) proteins. However, the extent to which cancer patients mount de novo immune responses against expressed HERV elements is unclear. In this study, we determined the extent of HERV-K env expression in human breast cancer (BC) and whether both humoral and cell-mediated immunity against HERV-K can be found in BC patients. We found HERV-K env protein expression in 88% of BC (n = 119) but not in normal breast (n = 76) tissues. ELISA screening assays detected significant titers of anti–HERV-K env IgG in a large proportion of BC patients. T-cell responses against HERV-K were also detected in peripheral blood mononuclear cells (PBMC) from BC patients stimulated with autologous dendritic cells pulsed with HERV-K env SU antigens. These responses included induction of T-cell proliferation (P = 0.0043), IFN-γ production measured by enzyme-linked immunospot (P < 0.0001), and multiplex cytokine secretion (P = 0.0033). Multiplex cytokine analysis found a T-helper 1 cytokine response, including interleukin (IL)-2 (P = 0.0109), IL-6 (P = 0.0396), IL-8 (P = 0.0169), and IP-10 (P = 0.0045) secretion during in vitro stimulation of BC PBMC with HERV-K antigen. We also found HERV-K–specific CTLs that were capable of lysing target cells expressing HERV-K env protein in BC patients but not in normal female controls without cancer. These findings suggest that retroviral gene products are capable of acting as tumor-associated antigens activating both T-cell and B-cell responses in BC patients. [Cancer Res 2008;68(14):5869–77]

Embedded in the genomes of nearly all vertebrates, are sequences of retroviral origin. These endogenous retroviruses (ERVs) are the result of ancient retroviral infections of the germline. 8% of the human genome consists of such sequences (HERVs).To date, no active, infectious HERV has been observed in humans, but high levels of retroviral mRNA and protein from a group known as HERV-K (HML-2) have been observed in some human cancers. Benign prostatic hyperplasia (BPH) is a non-malignant enlargement of the prostate commonly diagnosed in older men. Its prevalence negatively impacts on the sensitivity of PSA scores. We previously reported an association between high levels of HERV-K (HML-2) gag mRNA expression and a prostate cancer diagnosis (Wallace et al., 2014). The strength of this association increased with age. We hypothesised that measuring HERV-K (HML-2) expression could improve the discriminatory ability of PSA in the diagnosis of prostate cancer versus BPH. We set out to analyse differences in HERV-K (HML-2) protein expression between prostate adenocarcinoma and BPH. To this end, we conducted immunohistochemistry on prostate tissue microarrays (LS-SPRCA26, LS-SCA19; LifeSpan Biosciences) containing cores from prostate adenocarcinoma and BPH cases. TMAs were stained with an anti-HERV-K gag antibody (LS-C65287, LifeSpan Biosciences) and scored by an independent pathologist. We observed significantly greater epithelial HERV-K gag protein expression in BPH cores versus prostate adenocarcinoma cores (Fisher exact test, p = 0.001). Similar differences were also observed in stromal tissue (Fisher exact text, p = 0.02). Significant differences were also seen after positive expression levels were dichotomized into high versus low subgroups for both epithelial tissue (Fisher exact test, p = 0.001) and for stromal tissue (Fisher exact test, p = 0.034). Finally, we considered an association between HERV-K gag protein expression levels and tumor stage, but we did not observe any significant differences. Our data indicate that measuring HERV-K (HML-2) expression may improve the discriminatory ability of PSA in the diagnosis of prostate cancer versus BPH and that its potential utility as an adjunct biomarker warrants further investigation. Furthermore, increased sample size in relation to tumor stage will enable its prognostic potential to be evaluated. Citation Format: Ronan Downey, Laura Murillo, Teresa McHale, Tiffany Wallace, Caleb Seufert, Aaron Schetter, Tiffany Dorsey, Carol Johnson, Radoslav Goldman, Christopher Loffredo, Peisha Yan, Francis Sullivan, Francis Giles, Feng Wang-Johanning, Stefan Ambs, Sharon Glynn. Human endogenous retrovirus K expression as a possible adjunct to PSA in the diagnosis of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 536. doi:10.1158/1538-7445.AM2015-536

Background Distinguishing endocervical adenocarcinoma (ECA) from endometrial mucinous adenocarcinoma (EMMA) is clinically significant in view of the differences in their management and prognosis. In this study, we used a panel of tumor markers to determine their ability to distinguish between primary endocervical adenocarcinoma and primary endometrial mucinous adenocarcinoma. Methods Immunohistochemistry using monoclonal antibodies to MUC1 (Ma695), p16, estrogen receptor (ER), progesterone receptor (PR), and vimentin, was performed to examine 32 cases, including 18 EMMAs and 14 ECAs. For MUC1, cases were scored based on the percentage of staining pattern, apical, apical and cytoplasmic (A/C), or negative. For p16, cases were scored based on the percentage of cells stained. For the rest of the antibodies, semiquantitative scoring system was carried out. Results For MUC1, majority of EMMA (14 of 18 cases, 78%) showed A/C staining, whereas only few ECA (2 of 14, 14%) were positive. The difference of MUC1 expression in the two groups of malignancy was statistically significant (p < 0.001). Staining for p16 was positive in 10 of 14 (71%) ECA and 4 of 18 (22%) EMMA. Estrogen receptor was positive in 3 of 14 (21%) ECA and 17 of 18 (94%) EMMA. Progesterone receptor was positive in 3 of 14 (21%) ECA and 16 of 18 (89%) EMMA. Vimentin was positive in 1 of 14 (7%) ECA, and 9 of 18 (50%) EMA, with median and range of 0 (0–6), and 1.5 (0–9) respectively. Conclusion A panel of immunohistochemical markers including MUC1, p16, ER, PR, and vimentin is recommended, when there is morphological and clinical doubt as to the primary site of endocervical or endometrial origin.

5049 Background: Aberrant expression of human endogenous retrovirus-k (HERV-K) has been observed in prostate cancer (PCa). HERV-K is unique as it encodes sequences in the human genome containing op...

Calcitonin gene-related peptide (CGRP) is a neurotransmitter present in the peripheral ends of sensory neurons of the gut and may modulate reflexes of the enteric nervous system. We studied the release of CGRP in normal human gallbladders and in those containing gallstones to test the hypothesis that abnormalities of regulation of CGRP release participate in gallstone formation. Human gallbladder strips were obtained from histologically normal organs removed during liver surgery (n = 8) or from patients operated upon for symptomatic cholelithiasis (n = 14). After removal of the mucosa, muscle strips were superfused with oxygenated Kreb's buffer in an organ bath at 37°C. Pharmacologic agents were added to the superfusate and samples were collected at 2-min intervals for analysis. CGRP release was measured by a sensitive and specific radioimmunoassay and adjusted for tissue weight. In normal gallbladders, CGRP release was stimulated sixfold over basal by capsaicin (10 -5 M) to 363 ± 75 pg per gram of muscle per 2 min. This release was abolished by addition of somatostatin (SS) or the neural blocker tetrodotoxin (TTX). Lesser degrees of CGRP release were observed after nonspecific stimulation with K + or phosphodiesterase inhibition with caffeine. In gallbladders with gallstones, capsaicin-induced CGRP release was 74 ± 16 pg per gram of muscle per 2 min (20% of normal, P < 0.001). Release induced by caffeine and K + was also inhibited compared to normal gallbladder strips. Release of CGRP from diseased strips was abolished by TTX and inhibited by SS to degrees similar to normal tissue. CGRP release in normal human gallbladder muscle strips is increased by the sensory neuron stimulant capsaicin, the nonspecific stimulant K + , and phosphodiesterase inhibition by caffeine. This release is abolished by blockade of axonal transmission with TTX and by SS. CGRP content and release from gallbladders containing gallstones are inhibited. Whether these observations have significance in gallstone formation or are secondary processes remains to be seen. © 1995 Academic Press, Inc.

The amino acid and sugar composition of mucins from various organs is similar but not identical. This could arise by one or more of the following: organ-specific processing of a single core protein, organ-specific splicing of a single mucin mRNA, or organ-specific expression of various mucin genes. To begin to investigate the source of this variability, we examined (a) immunological cross-reactivity and (b) cDNA cross-hybridization, among several mucin-secreting organs of the human body. Peptide-directed antibodies raised against both nondeglycosylated (LS) and deglycosylated (HFB) intestinal mucin strongly stained mucous cells in the bronchial epithelium and submucosal glands, indicating homology between mucins of the bronchus and intestine at the peptide level. By screening a bronchus cDNA library with an intestinal mucin cDNA, SMUC-41, we isolated a bronchus mucin cDNA, HAM-1. This cDNA is 96% homologous to the first repeat of SMUC-41. HAM-1 hybridized to restriction fragments of human genomic DNA identical to those hybridizing to SMUC-41 on Southern blots. SMUC-41 also hybridized to polydisperse transcripts in the bronchus, cervix, gall bladder, and mammary gland, indicating mucin homology among all these organs at the RNA level. We conclude that the bronchus and intestine express a common mucin gene, which is likely co-expressed by at least several other mucin-secreting organs.