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Supplementary Figures S1-S2; Supplementary Tables S1-S2.

Supplementary Data from CXCR2 Promotes Ovarian Cancer Growth through Dysregulated Cell Cycle, Diminished Apoptosis, and Enhanced Angiogenesis

Purpose: Chemokine receptor CXCR2 is associated with malignancy in several cancer models; however, the mechanisms involved in CXCR2-mediated tumor growth remain elusive. Here, we investigated the role of CXCR2 in human ovarian cancer.Experimental Design: CXCR2 expression was silenced by stable small hairpin RNA in ovarian cancer cell lines T29Gro-1, T29H, and SKOV3. Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, flow cytometry, electrophoretic mobility shift assay, and mouse assay were used to detect CXCR2, interleukin-8, Gro-1, cell cycle, apoptosis, DNA binding of NF-κB, and tumor growth. Immunohistochemical staining of CXCR2 was done in 240 high-grade serous ovarian carcinoma samples.Results: Knockdown of CXCR2 expression by small hairpin RNA reduced tumorigenesis of ovarian cancer cells in nude mice. CXCR2 promoted cell cycle progression by modulating cell cycle regulatory proteins, including p21 (waf1/cip1), cyclin D1, CDK6, CDK4, cyclin A, and cyclin B1. CXCR2 inhibited cellular apoptosis by suppressing phosphorylated p53, Puma, and Bcl-xS; suppressing poly(ADP-ribose) polymerase cleavage; and activating Bcl-xL and Bcl-2. CXCR2 stimulated angiogenesis by increasing levels of vascular endothelial growth factor and decreasing levels of thrombospondin-1, a process likely involving mitogen-activated protein kinase, and NF-κB. Overexpression of CXCR2 in high-grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival (P < 0.001) and of early relapse (P = 0.003) in the univariate analysis.Conclusions: Our data provide strong evidence that CXCR2 regulates the cell cycle, apoptosis, and angiogenesis through multiple signaling pathways, including mitogen-activated protein kinase and NF-κB, in ovarian cancer. CXCR2 thus has potential as a therapeutic target and for use in ovarian cancer diagnosis and prognosis. Clin Cancer Res; 16(15); 3875–86. ©2010 AACR.

Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. Significance: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception. This article is highlighted in the In This Issue feature, p. 2355

Duodenal eosinophilia may play a role in functional dyspepsia (FD), but existing study results are conflicted. We investigated the association between duodenal eosinophils (count and degranulation) and FD symptoms, accounting for atopic conditions, medications, and seasonal variations. In a cross-sectional study conducted in the Michael E. DeBakey VA Medical Center in Houston, Texas, we analyzed duodenal histopathology of 436 patient samples from a prospective cohort with a validated symptom survey data and chart reviews. FD was defined using Rome II symptom criteria. Eosinophil count was number per 5 high-power fields (HPF), and eosinophil degranulation was eosinophilic granules in the stroma both determined by two independent investigators. The study cohort was predominantly male (87.4%) with a mean age of 59.3 (standard deviation (SD) ± 9.8). Mean and median eosinophil counts were 75.5 (± 47.8) and 63 (IQR: 43, 101) per five HPF, respectively. Duodenal eosinophilia (defined as ≥ 63 per 5 HPF) and eosinophil degranulation were present in 50.5% and 23.1% of patient samples, respectively. FD was observed in 178 patients (41.7%), but neither the mean eosinophil count nor duodenal eosinophilia was associated with FD. Eosinophil degranulation was independently associated with FD overall (OR 1.74; 95% CI 1.08, 2.78; p = 0.02) and early satiety (OR 2.04; 95% CI 1.26, 3.30; p = 0.004). In this large, ethnically diverse cohort of adult patients, we found no significant association between duodenal eosinophilia and FD. However, the presence of duodenal eosinophilic degranulation, an activated eosinophil marker, was significantly associated with FD, especially early satiety.

BACKGROUND AND AIMS: Risk of esophageal adenocarcinoma (EA) in those with Barrett’s esophagus (BE) is 11-fold greater than the general population. It remains unclear which BE patients are at highest risk of progression to EA. We aimed to validate a predictive model risk stratifying BE patients. METHODS: We conducted a retrospective cohort study at the Houston VA of consecutive patients with a new BE diagnosis from November 1990 to January 2019. Study follow-up was through 2/2020. Patients were excluded if they had no follow-up esophagogastroduodenoscopy (EGD) with esophageal biopsy after the initial BE-diagnosing EGD or evidence of high-grade dysplasia (HGD) or EA on initial EGD. We performed an external validation study of a risk model containing sex, smoking, BE length, and low-grade dysplasia (LGD) status and assessed discriminatory ability using area under the receiver operating characteristic curve (AUROC). RESULTS: Among 608 BE patients, 24 progressed to HGD/EA. The points-based model discriminated well with an AUROC of 0.72 (95% confidence interval [CI], 0.63–0.82). When categorized into low/intermediate/high-risk groups according to published cut-offs, the AUROC was poor at 0.57. Restructured into low-risk versus high-risk groups, the AUROC was 0.72 (95% CI, 0.64–0.80). Excluding baseline LGD did not reduce discriminatory ability (AUROC 0.73; 95% CI, 0.64–0.82). CONCLUSION: This external validation provides further evidence that the model including sex, LGD status, smoking status, and BE length may help to risk stratify BE patients. A simplified version excluding LGD status and/or reducing the number of risk groups has increased utility in clinical practice without loss of discriminatory ability.

Background & Aims Nonalcoholic steatohepatitis (NASH) is associated with an increase in healthcare resource use and poor health-related quality of life (HRQoL). We assessed the humanistic and economic burden of NASH, disease management, and patient journey. Methods We performed a cross-sectional analysis of data, collected from July through November 2017, from the Growth from Knowledge Disease Atlas Real-World Evidence program, reported by physicians in United States, France, and Germany. We extracted demographic and medical data from medical records. Some patients voluntarily completed a survey that provided information on disease history, treatment satisfaction, and patient-reported outcomes. Results We analyzed data from 1216 patients (mean age, 54.9±12.3 years; 57.5% male; mean body mass index, 31.7±6.9); 64.6% had biopsy-confirmed NASH and comorbidities were recorded for 41.3%. Treatments included lifestyle modification (64.6%) or use of statins (25.0%), vitamin E (23.5%), or metformin (20.2%). Patients with biopsy-confirmed NASH reported more physician (4.5 vs 3.7) and outpatient visits (1.8 vs.1.4) than patients with suspected NASH not confirmed by biopsy. Among the 299 patients who completed the survey, 47.8% reported various symptoms associated to their NASH. Symptomatic patients reported significantly lower HRQoL than patients without symptoms. Conclusions In an analysis of data from 3 countries, we found NASH to be associated with regular use of medical resources; patients with symptoms of NASH had reduced HRQoL. The burden of NASH appears to be underestimated. Studies are needed to determine the burden of NASH by fibrosis stage and disease severity.

Background & Aims Limitations of endoscopic sampling may result in missed dysplasia at the diagnosis of Barrett’s esophagus (BE). However, the role of close follow-up endoscopy is unclear. The aim was to evaluate the proportion of patients diagnosed with “missed” dysplasia within 18 months of their index nondysplastic BE (NDBE) diagnosis. Methods This was a retrospective analysis of a cohort of BE patients diagnosed during 1990–2019 at the Houston VA. Patients with BE on index esophagogastroduodenoscopy (EGD) were classified as NDBE, indefinite dysplasia, or dysplastic (low- or high-grade dysplasia) based on initial biopsies. We identified NDBE patients who had follow-up EGD within 3–18 months after index EGD. We used logistic regression models to estimate odds ratios and 95% confidence intervals for risk factors of dysplasia on follow-up EGD. Results We identified 614 patients who had BE on index EGD. Among those with NDBE and follow-up EGD within 3–18 months (n = 271), 4.1% had definite dysplasia on follow-up, and an additional 14.0% had indefinite dysplasia. Proportions of definite or indefinite dysplasia at follow-up within 3–18 months significantly decreased from 32.6% among patients with index EGD before 2009 to 11.7% among patients with index EGD after 2013 (P for trend = .068). Those with any indefinite or definite dysplastic BE at follow-up within 3–18 months after index EGD (n = 49) were more likely to have BE length ≥3 cm on index EGD (odds ratio, 3.39; 95% confidence interval, 1.63–7.08) than those with persistent NDBE or no BE on follow-up. Conclusions The occurrence of missed dysplasia on an index EGD has decreased over time. However, those with long segment BE were more than 3 times as likely to have missed dysplasia, and this group could benefit from dysplasia surveillance within 18 months of BE diagnosis.

Duodenal eosinophilia may play a role in functional dyspepsia (FD), but existing study results are conflicted. We investigated the association between duodenal eosinophils (count and degranulation) and FD symptoms, accounting for atopic conditions, medications, and seasonal variations.In a cross-sectional study conducted in the Michael E. DeBakey VA Medical Center in Houston, Texas, we analyzed duodenal histopathology of 436 patient samples from a prospective cohort with a validated symptom survey data and chart reviews. FD was defined using Rome II symptom criteria. Eosinophil count was number per 5 high-power fields (HPF), and eosinophil degranulation was eosinophilic granules in the stroma both determined by two independent investigators.The study cohort was predominantly male (87.4%) with a mean age of 59.3 (standard deviation (SD) ± 9.8). Mean and median eosinophil counts were 75.5 (± 47.8) and 63 (IQR: 43, 101) per five HPF, respectively. Duodenal eosinophilia (defined as ≥ 63 per 5 HPF) and eosinophil degranulation were present in 50.5% and 23.1% of patient samples, respectively. FD was observed in 178 patients (41.7%), but neither the mean eosinophil count nor duodenal eosinophilia was associated with FD. Eosinophil degranulation was independently associated with FD overall (OR 1.74; 95% CI 1.08, 2.78; p = 0.02) and early satiety (OR 2.04; 95% CI 1.26, 3.30; p = 0.004).In this large, ethnically diverse cohort of adult patients, we found no significant association between duodenal eosinophilia and FD. However, the presence of duodenal eosinophilic degranulation, an activated eosinophil marker, was significantly associated with FD, especially early satiety.

Microscopic colitis (MC), a subtype of inflammatory bowel disease, is a chronic condition of unknown etiology. Recent evidence has linked MC with intriguing changes in the stool microbiota, which may be linked to disease pathogenesis. The composition of the mucosal microbiome in patients with MC remains unclear. We performed a cross-sectional study comparing colonic tissue samples from patients with MC to those of healthy controls at the Michael E. DeBakey VA Medical Center. We included adults older than 18 who underwent a colonoscopy with biopsies to evaluate chronic diarrhea. Cases were defined by histology consistent with MC and controls by the absence of histologic disease. We conducted structured chart review to exclude other gastrointestinal diseases and obtain demographic (age, sex, race) and clinical (duration of symptoms and concurrent medications) information for cases and controls. We extracted bacterial DNA from formalin-fixed paraffin-embedded tissue samples and sequenced the v4 region of the 16S rRNA gene. Operational taxonomic unit (OTU) clustering was performed using UPARSE, and OTUs were assigned using the SILVA database. Statistical analysis was performed in QIIME and LEfSe. Comparisons with FDR-adjusted p values of less than 0.05 were considered statistically significant. We included 20 MC patients and 20 controls with mean ages of 62 and 54, respectively. Most cases were White (95%), 60% had symptoms for greater than 12 months, and 50% were taking PPIs and NSAIDs at the time of their diagnosis. Compared to controls, MC patients had a significant increase in the proinflammatory sulfur-reducing bacterial family Desulfovibrionales. The Coriobacteriaceae family, abundant in the healthy gut, was significantly decreased in MC cases. There was also an increase in the genus Actinomyces in MC patients on PPI and an increase in the class Bacilli among those taking NSAIDs. Patients with MC have an increase in the proinflammatory family Desulfovibrionales. Actinomyces and Bacilli were associated with medications (PPI and NSAID) known to increase the risk of MC. Our findings may have important implications for understanding the pathogenesis of MC.

Somatic mutations can promote malignant transformation of airway epithelial cells and induce inflammatory responses directed against resultant tumors. Tumor-infiltrating T lymphocytes (TIL) in early-stage non–small cell lung cancer (NSCLC) secrete distinct proinflammatory cytokines, but the contribution of these TILs to tumor development and metastasis remains unknown. We show here that TILs in early-stage NSCLC are biased toward IL17A expression (Th17) when compared with adjacent tumor-free tissue, whereas Th17 cells are decreased in tumor infiltrating locoregional lymph nodes in advanced NSCLC. Mice in which Pten and Smad4 (Pts4d/d) are deleted from airway epithelial cells develop spontaneous tumors, that share genetic signatures with squamous- (SQ.2b), and adeno- (AD.1) subtypes of human NSCLC. Pts4d/d mice globally lacking in IL17a (Pts4d/dIl17a–/–) showed decreased tumor latency and increased metastasis. Th17 cells were required for recruitment of CD103+ dendritic cells, and adoptive transfer of IL17a-sufficient CD4+ T cells reversed early tumor development and metastasis in Pts4d/dIl17a–/– mice. Together, these findings support a key role for Th17 cells in TILs associated with the Pts4d/d model of NSCLC and suggest therapeutic and biomarker strategies for human SQ2b and AD1 lung cancer. Cancer Immunol Res; 6(6); 645–57. ©2018 AACR.

Lung adenocarcinoma (LUAD) is the most commonly diagnosed histological subtype of lung cancer. While earlier work has underscored genomic and immune alterations in LUAD, the roles of individual cell populations in early-stage human LUAD evolution in space remain unknown. Here, we provide a detailed cellular atlas of early-stage LUAD and its spatial ecosystem along the peripheral lung. We performed single-cell RNA sequencing of 186,916 cells including enriched epithelial fractions from five early-stage LUADs with fourteen multi-region normal lung tissues of defined spatial proximities from the primary LUADs. We show that major epithelial and immune cellular lineages, states, and transcriptomic features geospatially and progressively evolve across normal regions and with increasing LUAD proximity. Analysis of 70,030 lung epithelial cells unraveled diverse lineage trajectories, transcriptional lineage plasticity programs underlying KRAS-mutant cells, and intratumoral heterogeneity within single sites. T regulatory cell programs including multiple immune checkpoints increased in tissues with closer proximity to LUADs, in sharp contrast to signatures of CD8+ cytotoxic T cells, antigen presentation by macrophages, and inflammatory dendritic cells. We found that some spatial signatures (e.g. a B cell signature score) were increased along the pathologic spectrum of normal lung, preneoplastic lesions, and matched invasive LUADs. LUAD cell-cell communication networks were enriched with ligand-receptor interactions involving CD24, LGALS9 and TIM3 immune checkpoints, including crosstalk between CD24 antigen in LUAD epithelial cells and SIGLEC10 in myeloid subsets. CD24 was markedly increased in preneoplasias relative to normal lung and further in LUAD, and its expression was highly positively correlated with immunosuppressive phenotypes. These data provide an atlas of cellular states and phenotypes underlying early-stage LUAD evolution in space, and a scalable resource for identification of targets for early treatment. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Kieko Hara, Patrick Brennan, Minghao Dang, Dapeng Hao, Ruiping Wang, Enyu Dai, Hitoshi Dejima, Jiexin Zhang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Kyle Chang, Danielle R. Little, Samer Bazzi, Linh Tran, Kostyantyn Krysan, Carmen Behrens, Dzifa Duose, Edwin R. Parra, Maria Gabriela Raso, Luisa M. Solis, Junya Fukuoka, Jianjun Zhang, Boris Sepesi, Tina Cascone, Lauren A. Byers, Don L. Gibbons, Jichao Chen, Seyed Javad Moghaddam, Edwin J. Ostrin, Daniel G. Rosen, John V. Heymach, Paul Scheet, Steven Dubinett, Ignacio I. Wistuba, Junya Fujimoto, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Resolving the spatial and cellular architecture of lung adenocarcinoma by multi-region single-cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 130.

Tumor response to neoadjuvant therapy is a significant predictive indicator of recurrence-free survival. We measured tumor response using residual tumor thickness at the tumor-normal tissue interface (TNI) and evaluated its association with outcome in patients with liver metastasis of breast cancer. We included 48 patients who underwent neoadjuvant therapy followed by partial liver resection at MD Anderson Cancer Center between 1997 and 2010. The hematoxylin-eosin–stained tumor sections were evaluated for both pathologic response and the residual tumor thickness at the TNI by 3 pathologists who were blinded to the clinical information, treatment regimen, and patient outcome. The residual tumor thickness at the TNI was correlated with recurrence-free survival using Kaplan-Meier method and log-rank test. Cox proportional hazard model was used to identify predictors of recurrence-free survival. All patients were women with a median age of 43 years. The median duration of follow-up was 52.1 months. Residual tumor thickness less than or equal to 3 mm at the TNI correlated with major pathologic response and was associated with longer recurrence-free survival in both univariate and multivariate analyses. Residual tumor thickness at the TNI predicts recurrence-free survival and provides an objective outcome end point in patients who underwent neoadjuvant therapy and liver resection of metastatic breast cancer. We suggest using both the pathologic response and the residual tumor thickness at the TNI to measure tumor response to therapy to improve accuracy.

The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3′-untranslated region (3′-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3′-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression., Epithelial-to-mesenchymal transition is a key process in tumorigenesis but little is known about the molecular mechanism regulating such process at the translational level. Here, the authors identify a subset of mRNAs important for this process that are specifically modulated by the RNA-binding protein CELF1.

Research into the biological role of the Ca(2+)-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca(2+) increases in mouse pancreatic beta cells.

As a putative marker for cancer stem cells in human malignant tumors, including ovarian cancer, CD133 expression may define a tumor-initiating subpopulation of cells and is associated with the clinical outcome of patients. However, at this time its clinical significance in ovarian cancer remains uncertain. The aim of this study was to clarify the clinical role of CD133 expression in human ovarian cancer. Immunohistochemical staining of CD133 expression was performed in 400 ovarian carcinoma samples using tissue microarray. The associations among CD133 expression and clinical factors (diagnosis, tumor grade, cancer stage, and clinical response to chemotherapy), overall survival and disease-free survival time were analyzed. CD133 expression was found in 31% of ovarian carcinoma samples. Fisher’s exact test and one-way analysis of variance suggested that CD133 expression was associated with high-grade serous carcinoma (P = 0.035), late-stage disease (P < 0.001), ascites level (P = 0.010), and non-response to chemotherapy (P = 0.023). CD133 expression was also associated with shorter overall survival time (P = 0.007) and shorter disease-free survival time (P < 0.001) by log-rank test. Moreover, CD133 expression was an independent predictor of shorter disease-free survival time in an unconditional logistic regression analysis with multiple covariates (P = 0.024). Our results thus show that CD133 expression is a predictor of poor clinical outcome for patients with ovarian cancer, supporting the proposed link between CD133 and cancer stem cells.

Perry K D, Reynolds C, Rosen D G, Edgerton M E, Albarracin C T, Gilcrease M Z, Sahin A A, Abraham S C & Wu Y (2011) Histopathology59, 619–630 Metastatic neuroendocrine tumour in the breast: a potential mimic of in-situ and invasive mammary carcinoma Aims: The aim of this study was to review the clinicopathological characteristics of neuroendocrine tumours (NETs) metastasizing to the breast, in order to identify features that could be useful in distinguishing these metastatic lesions from primary breast neoplasms. Methods and results: Eighteen metastatic NETs in the breast were identified from two large hospitals over a 15-year period. Eleven (62%) tumours originated in the gastrointestinal tract, 5 (28%) originated in the lung, and the other two were of indeterminate origin. Eight (44%) cases were initially misdiagnosed as primary mammary carcinomas. In retrospect, all metastatic tumours exhibited architectural and cytological features that would suggest neuroendocrine differentiation. Immunohistochemistry can further aid in the distinction between metastatic neuroendocrine and primary mammary carcinoma. All 11 tumours from the gastrointestinal tract expressed CDX-2, 3 (60%) of five tumours from the lung expressed thyroid transcription factor-1, and only 2 (11%) of 18 showed weak oestrogen receptor positivity. Additionally, unlike primary carcinomas, the majority (82%) of metastatic NETs were negative for cytokeratin 7, and all were negative for gross cystic disease fluid protein 15 and mammoglobin. Conclusions: There is a high propensity for metastatic NETs to mimic primary breast carcinomas. Careful attention to cytological and architectural features can help to identify cases that require further immunophenotypic workup with a panel of tissue-specific antibodies. However, clinical history is paramount for optimal diagnosis.

In March and early April 2009, cases of a new swine-origin influenza A (H1N1) virus were diagnosed in Mexico and the United States. Influenza virus presents as a respiratory infection with high morbidity and mortality. We describe the postmortem findings of eight confirmed cases of influenza A/H1N1 in a medical examiner setting. The eight cases falling under the jurisdiction of the Harris County Medical Examiner (Houston, TX, USA) with confirmed influenza A/H1N1 infection between June and September 2009 were included in this study. All cases were males between 6 months and 54 years of age. All adult patients had a body mass index from 31 to 49.8 kg/m(2). Five cases had comorbid conditions including one case with sleep apnea and mental retardation, three cases with chronic ethanolism, and one case with thymoma, sarcoidosis, and myasthenia gravis. The remaining three cases had no pre-existing medical conditions. All patients presented with severe flu-like symptoms; yet, only five were febrile. Rapid influenza diagnostic tests were performed in three cases by primary-care physicians, two of which were negative. None of the patients received antiviral medication. The average disease duration time was 8.2 days (3-14 days). A wide range of histopathological findings including tracheitis, necrotizing bronchiolitis, alveolitis, intra-alveolar hemorrhage, and hyaline membranes, both in a focal and in a diffuse distribution, were identified. Influenza A/H1N1 viral infection presents with a wide range of histological findings in a diffuse or focal distribution; most consistently with tracheitis, necrotizing bronchiolitis, and alveolitis with extensive alveolar hemorrhage. These histopathological findings at autopsy along with a clinical history of flu-like symptoms should raise suspicion for influenza A/H1N1 infection, and postmortem analysis by the reverse transcription-polymerase chain reaction (RT-PCR) is recommended for an accurate diagnosis.

Papillary differentiation is one of the most common histological features of ovarian cancer, although the underlying mechanism that leads to such differentiation is not known. We hypothesized that human ovarian surface epithelial cells can be transformed into carcinoma with papillary differentiation by overexpressing HER2/neu in these cells. Mice were injected either subcutaneously or intraperitoneally with two immortalized human ovarian surface epithelial cell lines after enforced expression of HER-2/neu. Mice subcutaneously injected with tumor cells from either the T29Nt or T80Nt developed undifferentiated carcinomas. In contrast, mice injected intraperitoneally with T29Nt cells developed papillary carcinoma, and those injected intraperitoneally with T80Nt cells developed undifferentiated carcinoma. Our results demonstrate that ovarian surface epithelial cells can develop into papillary carcinoma in mice, and that the induction of papillary differentiation depends not only on specific genetic modifications but also on the tumor microenvironment and epithelial cell type from ovary from different patients.

Mutations in the beta-catenin gene are common in ovarian endometrioid carcinoma. Few studies have addressed the association of beta-catenin expression with tumor characteristics and patient outcome, yielding controversial results. The purpose of this study was to retrospectively assess the expression of beta-catenin in ovarian endometrioid carcinoma and correlate its expression with the Gynecologic Oncology Group's (GOG) grading system, clinicopathological characteristics, and patient survival. A total of 49 patients with primary ovarian endometrioid carcinoma were included in this study. A four-tier score grading system was used for the membranous staining (negative, weak, moderate, and strong) and the percentage of positive cells for the nuclear staining of beta-catenin. The status of five morphometric parameters, nuclear morphology (uniform or pleomorphic), mitotic count, glandular pattern, degree of squamous differentiation, and status of papillary pattern, was assessed. We found that a low membranous expression of beta-catenin and a high mitotic count (15 per 10 high-power fields) were significantly associated with poor prognosis and early recurrence of ovarian endometrioid carcinoma. In addition, cases with nuclear expression of beta-catenin showed an intermediate overall survival risk and late disease recurrence. Young age at the time of diagnosis, advanced disease stage, and suboptimal debulking were among the clinical factors predicting poor survival and early disease recurrence. The presence of squamous differentiation, a papillary pattern or nuclear pleomorphism did not show any correlation with overall survival or disease-free survival. Low membranous expression of beta-catenin and high mitotic count are poor prognostic indicators in patients with ovarian endometrioid carcinoma, whereas the GOG grading system showed no prognostic value. Our data suggest that there is a need to define a better grading system for ovarian endometrioid carcinoma. Molecular markers such as beta-catenin and mitotic count could aid in defining this grading system.

Aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme responsible for the oxidation of intracellular aldehydes, was shown to have a function in the early differentiation of stem cells, through its function in oxidizing retinol to retinoic acid. It has been shown that ALDH1 is a predictor of poor clinical outcome in breast cancer. The authors hypothesized that the level of ALDH1 expression may be correlated with the clinical outcome of patients with ovarian cancer. Immunohistochemical staining of ALDH1 expression was analyzed in 442 primary ovarian carcinomas using tissue microarray. The associations between the expression of the ALDH1 and clinical factors (diagnosis, tumor grade, stage, and clinical response to chemotherapy), as well as overall and disease-free survival, were analyzed. Expression of ALDH1 was found in 48.9% of the samples. Fisher's exact test suggested that high expression of ALDH1 was significantly associated with endometrioid adenocarcinoma (P

OBJECTIVE To describe a biinstitutional experience with fine needle aspiration (FNA) of breast lesions occurring in men over a period of over 10 years and to assess the role of FNA in detecting male breast cancer. STUDY DESIGN Computer patient records were searched from January 1990 to September 2007. Male patients who had a diagnosis of carcinoma on breast biopsy or resection were identified, and detailed chart review was done to assess demographics and correlate biopsy and FNA results. RESULTS A total of 346, 792 patients' charts were searched, and 217 male patients were found to have had a breast mass and subsequent FNA. Combined in both institutions, FNA results were positive for carcinoma in 12 (5.5%) cases, negative in 181 (83.4%) cases, suspicious in 5 (2.3%) cases, indeterminate/inconclusive in 3 (1.4%) cases and unsatisfactory in 16 (7.4%) cases. Matching biopsies to confirm FNA results were available in 26 (12%) of the 217 cases. The calculated sensitivity and specificity for the malignant cases reached 100%. When suspicious cases were calculated for diagnostic accuracy, the sensitivity was 67% and the specificity 100%. CONCLUSION This biinstitutional study demonstrates that FNA is a reliable but underutilized procedure for the management of male breast lesions.

Background Annexin A1 (ANXA1) is a potential marker of development of breast cancer. However, previous studies of ANXA1 expression in primary breast carcinoma and lymph node metastasis have yielded conflicting results. Therefore, to accurately characterize the ANXA1 expression pattern, we used microarray analysis and matched patient samples to evaluate progressive alterations in ANXA1 protein expression during malignant transformation and metastasis. Design We constructed a tissue microarray using 82 pairs of primary breast cancers and lymph node metastases from archival materials. We also identified 21 cases of breast carcinoma for which a single slide contained the entire progression from benign breast tissue, carcinoma in situ, to invasive carcinoma. Immunohistochemical staining for ANXA1 and various prognostic markers was performed. Result Microarray analysis revealed that ANXA1 expression was lost in 79% of breast carcinomas, and there was no difference in ANXA1 expression between primary breast carcinoma and lymph node metastasis. Most ANXA1-negative tumors were positive for estrogen and progesterone receptors but negative for HER2/neu and epidermal growth factor receptor. In contrast, most ANXA1-positive tumors were negative for estrogen, progesterone, and HER2/neu. In the whole tissue sections, ANXA1 is heterogeneously expressed in benign epithelium and is lost in both in situ carcinoma and invasive carcinoma. Conclusions The lack of ANXA1 expression in the majority of breast carcinomas and the early loss of ANXA1 expression in in situ carcinoma, which is maintained in both invasive and metastatic tumors, suggests a possible role for ANXA1 in the early events of malignant transformation.

The ovarian epithelial cells carrying heterozygous BRCA1 or BRCA2 mutation (we called BRCA1/2 mutation) are known to predispose to the development of ovarian cancer; however, the molecular basis of such predisposition is largely unknown. We hypothesize that BTAK may be a potential target for heterozygous BRCA1/2 mutation. We sought to determine the relationship between the status of BRCA1/2 heterozygous mutation and BTAK expression in prophylactically removed ovaries as compared with normal ovaries and ovarian cancer controls. Western blot analysis of BTAK was performed in a primary cell culture carrying heterozygous BRCA1 mutation and three normal ovarian surface epithelial cell cultures. Immunohistochemical analysis of BTAK expression was also performed by image analysis in ovaries of 21 patients with known BRCA1/2 mutation or very strong family history of breast/ovarian cancer that underwent prophylactic oophorectomy, 38 normal ovaries from patients without any known mutation, and 194 ovarian carcinomas. The BTAK expression was significantly increased in primary culture carrying a heterozygous BRCA1 mutation as compared to those with no known BRCA1/2 mutation. Immunohistochemical staining of BTAK showed increased expression in ovarian epithelial cells carrying BRCA1/2 mutation or strong breast/ovarian family history compared with normal ovaries (P

Purpose: The function of tumor susceptibility gene 101 (TSG101) in ovarian carcinogenesis is largely unexplored. The aim of this study is to investigate the role of TSG101 in human ovarian cancer development, to examine the expression levels of TSG101 in ovarian carcinomas, and to correlate the results with clinicopathologic variables and survival. Experimental Design: Human ovarian cancer tissue arrays that contain duplicates of 422 cases of primary ovarian carcinoma were used to probe the expression levels of TSG101 and p21 in epithelial ovarian cancer. In vitro studies in ovarian cancer cells using TSG101-specific small interfering RNA (siRNA) were done to further elucidate the mechanism of TSG101-mediated p21 regulation. Results: We show that TSG101 is increasingly overexpressed in borderline tumors and low-grade and high-grade carcinomas. Patients with low expression of TSG101 survive longer than those with high expression. Suppressing TSG101 by siRNA in ovarian cancer cells led to growth inhibition, cell cycle arrest, and apoptosis with concurrent increases in p21 mRNA and protein. Consistent with this negative association between TSG101 and p21, expression levels of these two markers are inversely correlated in ovarian cancer. Conclusions: TSG101 negatively regulates p21 levels, and up-regulation of TSG101 is associated with poor prognosis in ovarian cancer.

Homeobox genes control developmental patterning and are increasingly being found to be deregulated in tumors. The DLX4 homeobox gene maps to the 17q21.3-q22 region that is amplified in some epithelial ovarian cancers. Because amplification of this region correlates with poor prognosis, we investigated whether DLX4 overexpression contributes to aggressive behavior of this disease. DLX4 was not detected in normal ovary and cystadenomas, whereas its expression in ovarian carcinomas was strongly associated with high tumor grade and advanced disease stage. Overexpression of DLX4 in ovarian cancer cells promoted growth in low serum and colony formation. Imaging of mice bearing intraperitoneal tumors revealed that DLX4 overexpression substantially increased tumor burden. Tumors that overexpressed DLX4 were more vascularized than vector-control tumors. Conditioned medium of DLX4-overexpressing tumor cells was more effective than medium conditioned by vector-control cells in stimulating endothelial cell growth. These observations were associated with the ability of DLX4 to induce expression of vascular endothelial growth factor as well as intracellular and secreted isoforms of fibroblast growth factor-2. Moreover, increased levels of these fibroblast growth factor-2 isoforms induced vascular endothelial growth factor expression in tumor cells. This study reveals a novel role for a homeobox gene in ovarian tumorigenicity by its induction of a proangiogenic, growth-stimulatory molecular program.

Small GTPase RAS plays a critical role in cellular signaling and oncogenic transformation. Proteomics analysis of genetically defined human ovarian cancer models identified the tumor susceptibility gene 101 (TSG101) as a downstream target of RAS oncogene. Mechanistic studies revealed a novel post-translational regulation of TSG101 through the RAS/RAF/MEK/MAPK signaling pathway and downstream molecules p14(ARF)/HDM2. Immunoanalysis using ovarian cancer samples and microtissue array revealed elevated TSG101 levels in human ovarian carcinomas. Silencing of TSG101 by short interfering RNA in ovarian cancer cells led to growth inhibition and cell death. Concurrent with the apparent growth-inhibitory effect, the levels of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1alpha (HIF-1alpha), as well as its cellular activity, were markedly reduced after TSG101 knockdown. These results demonstrate that TSG101 is important for CITED2- and HIF-1alpha-mediated cellular regulation in ovarian carcinomas.

Ovarian cancer is developed from a single layer of thin epithelial cells covering the surface of ovary, named human ovarian surface epithelial cells. Like all primary human cells, human ovarian surface epithelial cells have a finite life span and will go into senescence and eventually die when cultured in vitro. Immortalized human ovarian surface epithelial cells will provide an important model system with which to study ovarian cancer initiation and progression. Here, we show that silencing p53 expression with retrovirus-mediated small interfering RNA can delay the senescence and extend cell passage number, but is not sufficient to immortalize normal ovarian surface epithelial cells. Introduction of the catalytic subunit of telomerase is similarly insufficient to achieve immortalization. However, concurrent disruption of p53 expression with small interfering RNA retroviral constructs and ectopic expression of the catalytic subunit of telomerase was sufficient to induce cellular immortalization in 3 of 3 human ovarian surface epithelial cell cultures tested. The immortalization is associated with increased telomerase activity and telomere length, and attenuated response of cell-cycle regulatory proteins to irradiation. The resultant immortal cells continued to express the same specific cytokeratins 8 and 18 as parental cells did, indicating that the epithelial characters are still maintained in the immortal cells. In addition, the immortalized cells are non-tumorigenic and nearly diploid, which is in constrast with one immortalized by SV40 T/t antigens and hTERT. As both p53 pathway dysfunction and activation of telomerase are commonly present in human ovarian cancer, these immortal cells provide an authetic cell model system for the study of the human ovarian cancer initiation, progression, differentiation and chemoprevention.

Extraskeletal myxoid chondrosarcoma is a rare tumor with an indolent course, high propensity for local recurrence, metastases, and propensity for the proximal extremities of middle-aged males.We present the case of a 44-year-old man with an extraskeletal myxoid chondrosarcoma in the plantar fascia of the medial arch initially thought to be a plantar fibroma. Magnetic resonance imaging of the lesion demonstrated a lobulated subcutaneous mass plantar to the tarsal bones and inseparable from the fascia. Microscopic examination revealed a lobulated lesion composed of cords and nests of round to spindled malignant cells in a blue-gray myxoid matrix surrounded by fibrous septae. The malignant cells displayed variable positivity for S-100.Plantar extraskeletal myxoid chondrosarcoma is a rare occurrence. It should always be considered in the differential diagnosis of masses arising in the plantar fascia of the foot.

Fluorescent contrast agents were topically applied in vivo to colon tissue in a mouse model of colorectal cancer. Excised tissue was imaged with confocal microscopy ex vivo to differentiate dysplasia from normal tissue and benign inflammatory lesions.

Epithelial–stromal interactions play a critical role in tumor initiation and progression; cancer-associated stroma, but not normal stroma, is known to be tumor-promoting. However, the molecular signal used by epithelial cancer cells to reprogram normal stroma to a tumorigenic stroma is not known. Here, we present evidence to suggest that the chemokine growth-regulated oncogene 1 (Gro-1) may be one such signaling molecule. We showed that the expression of Gro-1 is activated by RAS and is vital for cell survival and the malignant transformation of ovarian epithelial cells. Surprisingly, we found that Gro-1 is a potent inducer of senescence in stromal fibroblasts and that this effect depends on functional p53. Senescent fibroblasts induced by Gro-1 can promote tumor growth whereas abrogation of senescence through immortalization results in loss of such tumor promoting activity. We also demonstrated that stromal fibroblasts adjacent to epithelial cancer cells are senescent in human ovarian cancer specimens and in heterografts from RAS-transformed human ovarian epithelial cells and ovarian cancer cells. Moreover, Gro-1 was expressed at significantly higher amounts in ovarian cancer than in normal tissues and was higher in serum samples from women with ovarian cancer than in serum from women without ovarian cancer. These findings provide strong evidence that RAS-induced Gro-1 can reprogram the stromal microenvironment through the induction of senescence of fibroblasts and thus can promote tumorigenesis. Therefore, Gro-1 may be a therapeutic target as well as a diagnostic marker in ovarian cancer.

The risk of developing ovarian cancer is about 1% over a lifetime, but it is the most deadly gynecologic cancer, in part due to lack of diagnostic markers for early-stage disease and cell model system for studying early neoplastic changes. Most existing immortal human ovarian surface epithelial cells were achieved by using viral protein such as SV40 T/t antigen or E6/E7, which inactivate multiple cellular pathways. In the current study, we used a small interfering RNA (siRNA) against the retinoblastoma gene (pRb) and ectopic expression of human telomerase reverse transcriptase (hTERT) to immortalize the primary ovarian epithelial cell line OSE137 and two additional human ovarian surface epithelial cells. The immortalized OSE137 showed increased telomerase activity, lengthened telomeres, increased G2/M phase, altered cell-cycle regulatory proteins but nontumorigenic. As both Rb and hTERT pathways are commonly altered in human ovarian cancer and these genetic changes are faithfully modeled in these cells without using viral protein, these immortal cells represent an authentic in vitro model system with which to study the initiation and progression of human ovarian cancer.

Purpose: ARHI expression is lost or markedly down-regulated in the majority of ovarian cancers. The mechanism by which ARHI is down-regulated in ovarian cancers is still not clear. Our previous reports indicated that ARHI promoter activity was reduced in ovarian cancer cells, due in part to the effects of negative regulatory transcription factor(s). Experimental Design and Results: We now show that E2F1 and E2F4, but not E2F2, E2F3, or E2F5, bind to the ARHI promoter and repress its activity in ovarian cancer cells. Consistent with this observation, immunochemical staining of cell lines and of 364 samples of ovarian cancer tissue show that the expression of E2F1 and E2F4 proteins is much higher in ovarian cancer cells than in normal ovarian epithelial cells, and that increased expression of E2Fs was negatively correlated with ARHI expression (P < 0.05). Mutation of the putative E2F binding site in the ARHI promoter reversed this inhibitory effect and significantly increased ARHI promoter activity. In addition to the effects of transcriptional regulation, ARHI mRNA also exhibited a significantly reduced half-life in ovarian cancer cells when compared with that in normal ovarian epithelial cells (P < 0.01), suggesting posttranscriptional regulation of ARHI expression. ARHI mRNA contains AU-rich elements (ARE) in the 3′-untranslated region. We have found that these AREs interact with HuR, an ARE-binding protein that stabilizes bound mRNAs, possibly contributing to the rapid turnover of ARHI mRNA. Finally, reduced HuR ARE binding activity was observed in ovarian cancer cells when compared with normal ovarian surface epithelium. Conclusions: Taken together, our data suggest that ARHI expression is regulated at both the transcriptional and the posttranscriptional levels, contributing to the dramatic decrease in ARHI expression in ovarian cancers.

The family of insulin-like growth factor-binding proteins (IGFBPs) comprises six members, which bind and regulate the functions of insulin-like growth factors. Overexpression of IGFBP2 and IGFBP5 contributes to the invasiveness and progression of several human cancers, but their role and clinical significance in ovarian cancer has not been investigated in detail. We examined IGFBP2 and IGFBP5 expression levels using two tissue microarrays, one containing six normal surface epithelium, six benign serous cysts, 10 serous borderline tumors, eight low-grade, and 20 high-grade serous carcinomas. The other comprising 441 ovarian cancers of different histologic types linked to a clinicopathologic database. Each tumor was sampled in duplicate with a 1.0-mm punch core needle. Immunohistochemical staining was performed using antibodies against IGFBP2 or IGFBP5. The staining intensity was scored semiquantitatively as negative (0), weak (1-10%), moderate (10-50%), or strong (50-100%) using computerized image analysis. Statistical analyses used Fisher's exact test and Kaplan-Meier method. IGFBP2 and IGFBP5 were overexpressed in high-grade serous carcinomas compared to normal surface epithelium, benign serous cysts, serous borderline tumors, or low-grade serous carcinoma. They were differentially expressed in different types of ovarian carcinomas, being more often expressed at high levels in high-grade serous carcinoma, malignant mixed mullerian tumors and undifferentiated carcinoma, and more often expressed at low levels or not at all in clear cell and mucinous carcinomas. We concluded that IGFBP2 and IGFBP5 might play a role in the development of high-grade ovarian serous carcinoma, but not in mucinous or clear cell ovarian carcinomas.

Individual classes of human endogenous retrovirus (HERV) genes and proteins are expressed in cancer, but expression of more than one type of HERV is rare. We report here the expression of multiple HERV genes and proteins in ovarian cell lines and tissues. Expression of HERV-K env mRNA was greater in ovarian epithelial tumors than in normal ovarian tissues (N = 254). The expression of this protein on the surface and in the cytoplasm of ovarian cancer cells was confirmed using anti-HERV-K specific antibody by flow cytometric analysis. The frequency of expression of HERV-K env protein in multitissue microarrays (N = 641) was determined by immunohistochemistry and a significant correlation with tumor histotype was found. A significantly increased expression of HERV-K was observed in tumors with low malignant potential and low grade, relative to expression in normal ovarian tissues. The increase in expression of HERV-K env protein took place in a stepwise fashion in serous papillary adenocarcinoma. Interestingly, we found that other classes of HERV env mRNAs, including ERV3 and HERV-E, are expressed in the same ovarian cancer tissues that expressed HERV-K. Furthermore, anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (55%) were detected in patients with ovarian cancer, but not in normal female controls. HERV env proteins are frequently transcribed and translated in ovarian epithelial tumors, and multiple HERV families are detectable in ovarian cancer. HERV env proteins, and especially those expressed on the cell surface, may serve as novel tumor targets for detection, diagnosis and immunotherapy of ovarian cancer.

Microsatellite instability is due to defects in the family of DNA repair genes, primarily hMLH1 and hMSH2, which can be detected by immunohistochemical staining. However, it is unclear whether immunohistochemical staining can accurately predict microsatellite instability status. We sought here to evaluate the sensitivity, specificity, and predictive values of immunostaining for the expression of the DNA mismatch-repair genes hMLH1 or hMSH2 in predicting microsatellite instability in ovarian carcinoma. Tissue microarrays with specimens from 322 women with primary ovarian carcinoma were stained with antibodies to hMLH1 and hMSH2; cases in which either hMLH1 or hMSH2 were negative were analyzed for microsatellite instability with the five-marker panel recommended by the National Cancer Institute (BAT26, BAT25, D5S346, D2S123, and D17S250). Microsatellite instability was also analyzed in another 19 cases selected at random in which both hMLH1 and hMSH2 were positive. Tumors with instability at two or more of the five NCI markers were considered to have a high level of microsatellite instability; tumors showing instability at only one marker were considered microsatellite instability-low; and tumors in which no markers exhibited microsatellite instability were considered microsatellite stable. We found that negative staining for hMLH1 protein (five cases) or hMSH2 protein (two cases) was associated with high level of microsatellite instability. The sensitivity and specificity of immunohistochemical staining for hMLH1 were 62 and 100% and those of hMSH2 alone were 25 and 100%. Combining loss of expression of both hMLH1 and hMSH2 led to sensitivity, specificity, and positive and negative predictive values of 87, 100, 100, and 95%. These results suggest that use of a two-molecule panel (hMLH1 and hMSH2) can accurately determine the microsatellite instability status of patients with ovarian cancer.

BACKGROUND. Signal transducer and activator of transcription 3 (Stat3), which is a latent transcription factor that participates in the transcriptional activation of apoptosis and cell cycle progression, has been implicated as an oncogene in several neoplastic diseases. However, the specific role of Stat3 in ovarian carcinogenesis remains poorly understood. The objectives of the current study were to examine the effect of Stat3 activation on the phenotypic transformation of an immortalized, nontumorigenic ovarian epithelial cell line and to evaluate the expression of tyrosine-activated Stat3 (pStat3) in tissue microarrays from 303 ovarian carcinomas to determine its prognostic relevance and to correlate its expression with several upstream oncogenes of Stat3 and with the oncogenes involved in apoptosis and proliferation. METHODS. Overexpression of pStat3 was weakly tumorigenic and produced measurable tumors in mice in 1 of 3 clones. Using tissue microarrays from a large group of patients with primary ovarian carcinoma, the expression of pStat3 was correlated with the expression of growth factor receptors (HER-2/neu and epidermal growth factor receptor [EGFR]), interleukin 6, and the proliferation and apoptosis markers Ki-67, Bcl-2, and Bcl-xL and with clinicopathologic variables and patient survival. RESULTS. High pStat3 expression in the tumor tissue microarray was associated with high levels of HER-2/neu, EGFR, and Ki-67. No correlation was observed between overall pStat3 levels and any other clinicopathologic variables tested. High nuclear expression of pStat3 (>10% of positive-stained cells) was linked with poor overall survival. CONCLUSIONS. The activation and translocation of pStat3 to the nucleus are frequent events in ovarian carcinoma that are associated with a poor prognosis. Further studies are needed to elucidate the mechanism of activation of Stat3, its effects on downstream targets, and its role in the neoplastic transformation of epithelial ovarian cells. Cancer 2006. © 2006 American Cancer Society.

Background. When ovarian carcinoma is diagnosed in stage I, up to 90% of patients can be cured with surgery and currently available chemotherapy. At present, less than 25% of cases are diagnosed at this stage. To increase the fraction of ovarian cancers detected at an early stage, screening strategies have been devised that utilize a rising serum CA125 level to trigger the performance of transvaginal sonography. One limitation of CA125 as an initial step in such a screening strategy is that up to 20% of ovarian cancers lack expression of the antigen. Serum tumor markers that can be detected in ovarian cancers that lack CA125 expression might improve the sensitivity for early detection. Methods. From 296 ovarian cancers, 65 (22%) were found to have weak or absent CA125 expression on immunoperoxidase staining. Tissue expression of CA125 was compared to serum CA125 levels. Using immunoperoxidase staining of tissue arrays, we have assessed expression of 10 potential serum tumor markers in the 65 epithelial ovarian cancers with little or no CA125 expression and in ovarian cystadenomas, tumors of low malignant potential, normal ovaries, and 16 other normal tissues. Results. Low or absent expression of CA125 in surgical specimens of epithelial ovarian cancer was associated with low levels of serum CA125 in pre-operative serum specimens. In ovarian cancers that lacked CA125, all specimens (100%) expressed human kallikrein 10 (HK10), human kallikrein 6 (HK6), osteopontin (OPN), and claudin 3. A smaller fraction of CA125-deficient ovarian cancers expressed DF3 (95%), vascular endothelial growth factor (VEGF) (81%), MUC1 (62%), mesothelin (MES) (34%), HE4 (32%), and CA19-9 (29%). When reactivity with normal tissues was considered, however, MES and HE4 showed the greatest specificity. Differential expression was also found for HK10, OPN, DF3, and MUC1. Conclusions. At the level of tissue expression, each of 10 potential serum markers could be detected in 29–100% of ovarian cancers that had low or absent expression of CA125. Several markers exhibited more intense expression in cancers than in normal organs. Further investigation is needed to demonstrate complementary expression of markers in serum.

A variety of biomarkers have been developed to monitor growth of ovarian cancer and to detect disease at an early interval. CA125 (MUC16) has provided a useful serum tumor marker for monitoring response to chemotherapy, detecting disease recurrence, distinguishing malignant from benign pelvic masses, and potentially improving clinical trial design. A rapid fall in CA125 during chemotherapy predicts a favorable prognosis and could be used to redistribute patients on multiarmed randomized clinical trials. Several studies now document that CA125 can serve as a surrogate marker for response in phase II trials. Serial measurement of CA125 might also provide a useful marker for monitoring stabilization of disease with cytostatic targeted therapeutic agents. The greatest potential for serum markers may be in detecting ovarian cancer at an early stage. A rising CA125 can be used to trigger transvaginal sonography (TVS) in a small fraction of patients. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risk for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the UK that will test critically the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, as 20% of ovarian cancers have little or no expression of CA125, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Two proteomic approaches have been used: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. Both approaches are promising and require further development. Several groups are placing markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.