Miriam D. Post

Restoration of miR-200c to Ovarian Cancer Reduces Tumor Burden and Increases Sensitivity to Paclitaxel

A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. miRNAs have emerged as potential therapeutic candidates due to their ability to downregulate multiple targets involved in tumor progression and chemoresistance. miRNA-200c (miR-200c) is downregulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. miR-200c increases sensitivity to taxanes in vitro by targeting class III β-tubulin gene (TUBB3), a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors having low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) as compared with those having high TUBB3 (average survival 42.56 ± 3.19 months). miR-200c also targets TrkB, a mediator of resistance to anoikis. We show that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biologic substrates in vitro. Because both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately before cytotoxic chemotherapy may allow for a better response or lower effective dose. Mol Cancer Ther; 11(12); 2556–65. ©2012 AACR.

Tissue-Specific Pathways for Estrogen Regulation of Ovarian Cancer Growth and Metastasis

Menopausal estrogen (E2) replacement therapy increases the risk of estrogen receptor (ER)-positive epithelial ovarian cancers (EOC). Whether E2 is tumorigenic or promotes expansion of undiagnosed preexisting disease is unknown. To determine E2 effects on tumor promotion, we developed an intraperitoneal mouse xenograft model using ZsGreen fluorescent ER(-) 2008 and ER(+) PEO4 human EOC cells. Tumor growth was quantified by in vivo fluorescent imaging. In ER(+) tumors, E2 significantly increased size, induced progesterone receptors, and promoted lymph node metastasis, confirming that ERs are functional and foster aggressiveness. Laser-captured human EOC cells from ER(-) and ER(+) xenografted tumors were profiled for expression of E2-regulated genes. Three classes of E2-regulated EOC genes were defined, but <10% were shared with E2-regulated breast cancer genes. Because breast cancer selective ER modulators (SERM) are therapeutically ineffective in EOC, we suggest that our EOC-specific E2-regulated genes can assist pharmacologic discovery of ovarian-targeted SERM.

Large cell neuroendocrine carcinoma of the cervix: Prognostic factors and survival advantage with platinum chemotherapy

OBJECTIVE Large cell neuroendocrine carcinoma of the cervix (LCNEC) is a rare cervical neoplasm associated with poor survival. Our objective was to identify treatments associated with improved survival. METHODS Relevant data were abstracted from an English literature MEDLINE search, SEER database, and a patient treated at our institution. Multivariate analysis was performed by generating Cox proportional hazard ratios. RESULTS We identified 62 patients with LCNEC: 49 cases from the English literature, 12 patients in the SEER database and our patient. Out of the 62 women, median age was 37 (range, 21-75). FIGO stage was as follows: 58% had stage I disease, 16% had stage II, 2% had stage III, 8% had stage IV disease and 16% had no stage documented. Of all patients, 73% underwent primary surgery, 4.7% underwent primary radiation, 4.7% underwent chemotherapy, 8% had chemoradiation, and 9.6% had no primary treatment. Of all patients, 58% died of disease, 26% had no evidence of disease, 3% were alive with disease, and 13% had no survival data. The overall median survival was 16.5 months (0.5-151 months). Median overall survival for stage I, II, III, and IV cancers was 19, 17, 3, and 1.5 months, respectively. In a multivariate analysis, earlier stage (p<0.00001) and the addition of chemotherapy (p=0.04) were associated with improved survival. Both platinum agents (p=0.034) and platinum and etoposide together (p=0.027) were associated with improved survival. CONCLUSIONS Perioperative chemotherapy, in particular platinum with or without etoposide, improves survival in the rare LCNEC.

DUF1220 protein domains drive proliferation in human neural stem cells and are associated with increased cortical volume in anthropoid primates

Genome sequences encoding DUF1220 protein domains show a burst in copy number among anthropoid species and especially humans, where they have undergone the greatest human lineage-specific copy number expansion of any protein coding sequence in the genome. While DUF1220 copy number shows a dosage-related association with brain size in both normal populations and in 1q21.1-associated microcephaly and macrocephaly, a function for these domains has not yet been described. Here we provide multiple lines of evidence supporting the view that DUF1220 domains function as drivers of neural stem cell proliferation among anthropoid species including humans. First, we show that brain MRI data from 131 individuals across 7 anthropoid species shows a strong correlation between DUF1220 copy number and multiple brain size-related measures. Using in situ hybridization analyses of human fetal brain, we also show that DUF1220 domains are expressed in the ventricular zone and primarily during human cortical neurogenesis, and are therefore expressed at the right time and place to be affecting cortical brain development. Finally, we demonstrate that in vitro expression of DUF1220 sequences in neural stem cells strongly promotes proliferation. Taken together, these data provide the strongest evidence so far reported implicating DUF1220 dosage in anthropoid and human brain expansion through mechanisms involving increasing neural stem cell proliferation.

Significance of histologic pattern of carcinoma and sarcoma components on survival outcomes of uterine carcinosarcoma

BACKGROUND To examine the effect of the histology of carcinoma and sarcoma components on survival outcome of uterine carcinosarcoma. PATIENTS AND METHODS A multicenter retrospective study was conducted to examine uterine carcinosarcoma cases that underwent primary surgical staging. Archived slides were examined and histologic patterns were grouped based on carcinoma (low-grade versus high-grade) and sarcoma (homologous versus heterologous) components, correlating to clinico-pathological demographics and outcomes. RESULTS Among 1192 cases identified, 906 cases were evaluated for histologic patterns (carcinoma/sarcoma) with high-grade/homologous (40.8%) being the most common type followed by high-grade/heterologous (30.9%), low-grade/homologous (18.0%), and low-grade/heterologous (10.3%). On multivariate analysis, high-grade/heterologous (5-year rate, 34.0%, P = 0.024) and high-grade/homologous (45.8%, P = 0.017) but not low-grade/heterologous (50.6%, P = 0.089) were independently associated with decreased progression-free survival (PFS) compared with low-grade/homologous (60.3%). In addition, older age, residual disease at surgery, large tumor, sarcoma dominance, deep myometrial invasion, lymphovascular space invasion, and advanced-stage disease were independently associated with decreased PFS (all, P < 0.01). Both postoperative chemotherapy (5-year rates, 48.6% versus 39.0%, P < 0.001) and radiotherapy (50.1% versus 44.1%, P = 0.007) were significantly associated with improved PFS in univariate analysis. However, on multivariate analysis, only postoperative chemotherapy remained an independent predictor for improved PFS [hazard ratio (HR) 0.34, 95% confidence interval (CI) 0.27-0.43, P < 0.001]. On univariate analysis, significant treatment benefits for PFS were seen with ifosfamide for low-grade carcinoma (82.0% versus 49.8%, P = 0.001), platinum for high-grade carcinoma (46.9% versus 32.4%, P = 0.034) and homologous sarcoma (53.1% versus 38.2%, P = 0.017), and anthracycline for heterologous sarcoma (66.2% versus 39.3%, P = 0.005). Conversely, platinum, taxane, and anthracycline for low-grade carcinoma, and anthracycline for homologous sarcoma had no effect on PFS compared with non-chemotherapy group (all, P > 0.05). On multivariate analysis, ifosfamide for low-grade/homologous (HR 0.21, 95% CI 0.07-0.63, P = 0.005), platinum for high-grade/homologous (HR 0.36, 95% CI 0.22-0.60, P < 0.001), and anthracycline for high-grade/heterologous (HR 0.30, 95% CI 0.14-0.62, P = 0.001) remained independent predictors for improved PFS. Analyses of 1096 metastatic sites showed that carcinoma components tended to spread lymphatically, while sarcoma components tended to spread loco-regionally (P < 0.001). CONCLUSION Characterization of histologic pattern provides valuable information in the management of uterine carcinosarcoma.

Estrogen switches pure mucinous breast cancer to invasive lobular carcinoma with mucinous features

Mucinous breast cancer (MBC) is mainly a disease of postmenopausal women. Pure MBC is rare and augurs a good prognosis. In contrast, MBC mixed with other histological subtypes of invasive disease loses the more favorable prognosis. Because of the relative rarity of pure MBC, little is known about its cell and tumor biology and relationship to invasive disease of other subtypes. We have now developed a human breast cancer cell line called BCK4, in which we can control the behavior of MBC. BCK4 cells were derived from a patient whose poorly differentiated primary tumor was treated with chemotherapy, radiation and tamoxifen. Malignant cells from a recurrent pleural effusion were xenografted in mammary glands of a nude mouse. Cells from the solid tumor xenograft were propagated in culture to generate the BCK4 cell line. Multiple marker and chromosome analyses demonstrate that BCK4 cells are human, near diploid and luminal, expressing functional estrogen, androgen, and progesterone receptors. When xenografted back into immunocompromised cycling mice, BCK4 cells grow into small pure MBC. However, if mice are supplemented with continuous estradiol, tumors switch to invasive lobular carcinoma (ILC) with mucinous features (mixed MBC), and growth is markedly accelerated. Tamoxifen prevents the expansion of this more invasive component. The unexpected ability of estrogens to convert pure MBC into mixed MBC with ILC may explain the rarity of the pure disease in premenopausal women. These studies show that MBC can be derived from lobular precursors and that BCK4 cells are new, unique models to study the phenotypic plasticity, hormonal regulation, optimal therapeutic interventions, and metastatic patterns of MBC.

Pulmonary Capillary Hemangiomatosis A Rare Cause of Pulmonary Hypertension

Pulmonary capillary hemangiomatosis is a rare cause of pulmonary hypertension characterized by extensive proliferation of pulmonary capillaries within alveolar septae. Clinical presentation is nonspecific and includes dyspnea, cough, chest pain, and fatigue. Radiology shows diffuse centrilobular ground-glass opacities. Pulmonary capillary hemangiomatosis is clinically and radiographically indistinguishable from peripheral venoocclusive disease, making microscopic diagnosis essential. Histologically, pulmonary capillary hemangiomatosis shows an abnormal proliferation of small, thin-walled capillaries that expand the alveolar septae. The endothelial cells that comprise these lesions are cytologically bland and show no mitotic activity. Pulmonary capillary hemangiomatosis is important to recognize because prostacyclin therapy, a mainstay in the treatment of pulmonary hypertension, has been reported to cause sudden respiratory distress and death in these patients. Prognosis of this disease remains poor, and the only definitive treatment is lung transplantation.

Specificity and prognostic validation of a polyclonal antibody to detect Six1 homeoprotein in ovarian cancer

OBJECTIVE The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer. METHODS A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1-Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival. RESULTS The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann-Whitney U test) and by protein (presence vs. absence, p=0.05 Fischers Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001). CONCLUSIONS The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.

p16 expression in squamous and trophoblastic lesions of the upper female genital tract.

p16, a surrogate marker for human papillomavirus (HPV) infection, is uniformly present in HPV-related carcinomas. This study aims to further characterize p16 expression in trophoblastic lesions and squamous lesions of the upper female genital tract, as little data exists. p16 immunostaining was performed on sections from ichthyosis uteri (1), primary uterine corpus squamous cell carcinoma (UCSCC) (2), primary ovarian SCC (OSCC) (5; 2 associated with a dermoid cyst), endometrial endometrioid adenocarcinoma with extensive squamous differentiation (EC-SD) (5), ovarian endometrioid adenocarcinoma with extensive squamous differentiation (OC-SD) (4), placental site nodule (5), and placental site trophoblastic tumor (PSTT) (6). We evaluated the percentage of positive cytoplasmic and nuclear staining (focal=<10%, multifocal=10% to 50%, and diffuse=>50%) and staining intensity (weak, moderate, and strong). HPV-DNA analysis by polymerase chain reaction was performed on 5 OSCC. Ichthyosis uteri, all UCSCC and 1 OSCC (arising in a dermoid) were negative; the other dermoid-associated OSCC showed focal moderate staining, the remaining OSCC displayed strong (100%), diffuse (2), or multifocal (1) p16 positivity. Three of the 5 EC-SD cases showed strong diffuse staining of the squamous component. The glandular component focally showed strong p16 positivity (2), with variably intense focal staining in 3 cases. The squamous component of all OC-SD showed focal moderate staining, with variable staining of the glandular component. Overall, 3 EC-SD had 80% to 90% p16 positivity. Five of the 5 placental site nodules and 4 of the 6 PSTT showed focal weak staining, whereas 2 PSTT were p16 negative. HPV-DNA analysis was negative in 3 of the 5 OSCC, the other 2 cases being technical failures. p16 is expressed in OSCC and in the squamous and glandular components of EC-SD and OC-SD. As p16 is negative in UCSCC, it may help to identify the origin of SCC diffusely involving the corpus and cervix, and suggests different pathogeneses for SCC of the upper female genital tract, likely to be unrelated to HPV infection. In contrast to earlier data, we found weak and focal p16 expression in trophoblastic lesions. Thus, when considering the differential diagnosis of cervical SCC and trophoblastic lesions, only strong diffuse p16 staining should be considered helpful.

Patterns of placental pathology in preterm premature rupture of membranes

Inflammation is associated with preterm premature rupture of membranes (PPROM) and adverse neonatal outcomes. Subchorionic thrombi, with or without inflammation, may also be a significant pathological finding in PPROM. Patterns of inflammation and thrombosis may give insight into mechanisms of adverse neonatal outcomes associated with PPROM. To characterize histologic findings of placentas from pregnancies complicated by PPROM at altitude, 44 placentas were evaluated for gross and histological indicators of inflammation and thrombosis. Students t-test (or Mann-Whitney U-test), χ 2 analysis (or Fishers exact test), mean square contingency and logistic regression were used when appropriate. The prevalence of histologic acute chorioamnionitis (HCA) was 59%. Fetal-derived inflammation (funisitis and chorionic plate vasculitis) was seen at lower frequency (30% and 45%, respectively) and not always in association with HCA. There was a trend for Hispanic women to have higher odds of funisitis (OR = 5.9; P = 0.05). Subchorionic thrombi were seen in 34% of all placentas. The odds of subchorionic thrombi without HCA was 6.3 times greater that the odds of subchorionic thrombi with HCA (P = 0.02). There was no difference in gestational age or rupture-to-delivery interval, with the presence or absence of inflammatory or thrombotic lesions. These findings suggest that PPROM is caused by or can result in fetal inflammation, placental malperfusion, or both, independent of gestational age or rupture-to-delivery interval; maternal ethnicity and altitude may contribute to these findings. Future studies focused on this constellation of PPROM placental findings, genetic polymorphisms and neonatal outcomes are needed.