c-Met inhibitors attenuate tumor growth of small cell hypercalcemic ovarian carcinoma (SCCOHT) populations

A cellular model (SCCOHT-1) of the aggressive small cell hypercalcemic ovarian carcinoma demonstrated constitutive chemokine and growth factor production including HGF. A simultaneous presence of c-Met in 41% SCCOHT-1 cells suggested an autocrine growth mechanism. Expression of c-Met was also observed at low levels in the corresponding BIN-67 cell line (6.5%) and at high levels in ovarian adenocarcinoma cells (NIH:OVCAR-3 (84.4%) and SK-OV-3 (99.3%)). Immunohistochemistry of c-Met expression in SCCOHT tumors revealed a heterogeneous distribution between undetectable levels and 80%. Further characterization of SCCOHT-1 and BIN-67 cells by cell surface markers including CD90 and EpCAM demonstrated similar patterns with differences to the ovarian adenocarcinoma cells. HGF stimulation of SCCOHT-1 cells was associated with c-Met phosphorylation at Tyr¹³⁴⁹ and downstream Thr²⁰²/Tyr²⁰⁴ phosphorylation of p44/42 MAP kinase. This HGF-induced signaling cascade was abolished by the c-Met inhibitor foretinib. Cell cycle analysis after foretinib treatment demonstrated enhanced G2 accumulation and increasing apoptosis within 72 h. Moreover, the IC₅₀ of foretinib revealed 12.4 nM in SCCOHT-1 cells compared to 411 nM and 481 nM in NIH:OVCAR-3 and SK-OV-3 cells, respectively, suggesting potential therapeutic effects. Indeed, SCCOHT-1 and BIN-67 tumor xenografts in NOD^scid mice exhibited an approximately 10-fold and 5-fold reduced tumor size following systemic application of foretinib, respectively. Furthermore, foretinib-treated tumors revealed a significantly reduced vascularization and little if any c-Met-mediated signal transduction. Similar findings of reduced proliferative capacity and declined tumor size were observed after siRNA-mediated c-Met knock-down in SCCOHT-1 cells demonstrating that in vivo inhibition of these pathways contributed to an attenuation of SCCOHT tumor growth.     

单细胞RNA测序技术揭示卵巢高钙血症型小细胞癌复发病灶的细胞分子特征

背景与目的：卵巢高钙血症型小细胞癌（small cell carcinoma of the ovary, hypercalcemic type,SCCOHT）是高度恶性的、极为罕见的原发性未分化卵巢癌,本研究旨在分析SCCOHT肿瘤微环境及细胞分子特征,探究潜在药物靶标。方法：收集1例SCCOHT患者盆腔复发病灶,应用单细胞RNA测序（single-cell RNA sequencing,scRNA-seq）寻找关键细胞亚群及潜在药物靶点,进一步在细胞学层面进行验证。结果：SCCOHT复发病灶中有诱导性多能干细胞（induced pluripotent stem cell,iPSC）、神经施万细胞、神经上皮细胞和巨噬细胞4种细胞亚群和12个细胞亚簇。iPSCs细胞亚群显著上调的基因主要富集于细胞周期调控,其中PLK1基因上调最为显著。PLK1蛋白在SCCOHT组织切片中呈阳性表达,在SCCOHT细胞系中表达水平升高。结论：揭示了SCCOHT的肿瘤微环境由iPSC、神经施万细胞、神经上皮细胞及巨噬细胞组成,鉴定出iPSC关键细胞亚群,寻找到PLK1基因作为潜在的治疗靶点。     

Ovarian small cell carcinoma of the hypercalcemic type in children and adolescents: a prognostically unfavorable but curable disease

BACKGROUND: Ovarian small cell carcinoma of the hypercalcemic type is a rare neoplasm that is associated with a poor prognosis. The objective of the current study was to investigate the clinicopathologic features of this tumor and to develop preliminary diagnostic and therapeutic guidelines. METHODS: Between 1994 and 2005, 11 girls (ages 9-22 years) who were registered on the German Maligne Keimzelltumoren studies and the Kiel Pediatric Tumor Registry were analyzed. Prior to histopathologic review, 8 patients had been misdiagnosed with either germ cell tumor or juvenile granulosa cell tumor. RESULTS: According to the International Federation of Gynecologic Oncology, 4 patients had Stage IA disease, 3 patients had Stage IC disease, and 4 patients had Stage III disease. After resection, 4 patients were followed without additional therapy, and all 4 patients developed recurrent disease after 3 to 11 months. Seven patients received adjuvant chemotherapy during first-line treatment. One patient with Stage III disease received additional regional deep hyperthermia. During first-line treatment, high-dose chemotherapy was received by 4 patients who achieved a complete response (CR) after conventional chemotherapy. All 4 of those patients remained in CR for 7 to 73 months, whereas the other 3 patients developed recurrent disease. Salvage treatment after recurrence or tumor progression consisted of surgery and chemotherapy. One patient received high-dose chemotherapy in 2nd CR and remained in 2nd CR. In total, 5 patients remained alive with no evidence of disease. CONCLUSIONS: Patients with ovarian small cell carcinoma of the hypercalcemic type require multiagent chemotherapy during first-line treatment. High-dose chemotherapy may be used to consolidate the therapeutic success.     

Results of a prospective dose-intensive regimen in 27 patients with small cell carcinoma of the ovary of the hypercalcemic type.

BACKGROUND: The evaluation of first-line intensive combination therapy in small cell carcinoma of the ovary (SCCO). PATIENTS AND METHODS: Debulking surgery; four to six cycles of chemotherapy with cisplatin (P) 80 mg/m(2) day 1, adriamycin (A) 40 mg/m(2) day 1, vepeside (V) 75 mg/m(2)/day days 1-3, cyclophosphamide (EP) 300 mg/m(2)/day days 1-3, every 3 weeks and granulocyte colony-stimulating factor with, in case of a complete remission, high-dose chemotherapy with carboplatin, vepeside, cyclophosphamide and stem-cell support. RESULTS: Twenty-seven patients (median age 25 years); International Federation of Gynecology and Obstetrics stage: five I, four IIC, 17 IIIC-IV and one unknown. Twenty patients underwent complete surgery. Eight patients progressed under chemotherapy. Among 18 patients in complete response (CR), 10 received high-dose chemotherapy (CT) (three stem-cell collection failures, two protocol violations, two disease progression and one refusal). The main grade 3-4 toxic effects were hematologic. There were eight relapses among the 18 CR, four of which were pelvic alone. Among the 27 patients, 13 died and 10 patients are in CR1, three in CR2. The median follow-up is 37 months (8-166) and the median duration of the 18 CR is 30 months (5-111). Overall survival at 1 and 3 years is 58% [confidence interval (CI) 40% to 75%] and 49% (CI 30% to 67%). CONCLUSIONS: Initial dose-intensive therapy achieves interesting overall survival in SCCO.     

11例卵巢高钙血症型小细胞癌临床分析并文献复习

背景与目的:卵巢高钙血症型小细胞癌(small cell carcinoma of the ovary,hypercalcemic type,SCCOHT)是一种罕见的恶性程度极高的妇科肿瘤,好发于年轻女性.分析SCCOHT的临床特征及诊疗情况,并通过文献复习梳理其临床表现、治疗模式及预后因素等特征.方法:对复旦大学附...     

siRNA对卵巢上皮癌细胞HMGA1基因表达抑制的实验研究

目的:探讨小分子干扰RNA对卵巢上皮癌细胞中高迁移率蛋白家族A1 (Hish mobility group A1, HMGA1)基因表达的影响,了解HMGA1基因在卵巢上皮癌发生、发展中的作用.方法:设计并合成HMGA1 siRNA,分为两组,实验组以不同浓度[(1.0,2.5,5.0)μg/L,下同]的HMGA1 siRNA转染HMGA1基因高表达的卵巢上皮癌细胞系OVCAR细胞,对照组以等量阴性对照siRNA转染OVCAR细胞,实时荧光定量RTPCR技术检测转染后OVCAR细胞中HMGA1 mRNA的表达, HMGA1 mRNA的表达以PCR循环数阈值(Ct值)表示;蛋白印迹法测定OVCAR细胞中HMGA1蛋白的表达;显微镜观察并计数转染后存活的OVCAR细胞数.结果: HMGA1 siRNA转染OVCAR细胞后,明显下调OVCAR细胞中HMGA1 mRNA及蛋白的表达水平,实验组不同浓度HMGA1 siRNA转染后, OVCAR细胞的Ct值分别为(19.62±0.02),(20.46±0.02),(20.57±0.03)个循环数,与对照组[分别为(19.30±0.02),(19.45±0.01),(19.58±0.03)个循环数]比较,差异有统计学意义(P<0.05).上述浓度转染后,实验组OVCAR细胞的HMGA1蛋白表达水平(分别为0.75±0.02,0.56±0.02,0.19±0.03)分别与对照组(分别为1.92±0.25,1.80±0.29,1.15±0.18)比较,差异有统计学意义(P<0.01).实验组OCAR细胞生长较对照组明显受到抑制(P<0.05).结论: HMGA1 siRNA可以下调HMGA1 mRNA及其蛋白的表达水平,抑制细胞生长,提示HMGA1基因在卵巢上皮癌的发生、发展中具有重要作用.     

A mouse model for the molecular characterization of brca1-associated ovarian carcinoma

Little is known about the mechanisms that underlie Brca1-associated ovarian tumorigenesis, mainly due to the lack of an appropriate experimental model. We developed genetically defined primary mouse ovarian surface epithelial (OSE) cell lines in which the loss of functional Brca1 and p53 recapitulates the events that are thought to occur in early ovarian cancer development in patients with Brca1 mutations. This system allows for the introduction of additional oncogenes that are thought to cooperate with the loss of Brca1 and p53 to induce tumorigenesis. We showed that Myc is sufficient to induce transformation of ovarian cells that are deficient for both Brca1 and p53 but not sufficient for the transformation of cells that are deficient for either Brca1 or p53. The transformed Brca1-deficient OSE cells display an increased number of centrosomes, acquire complex chromosome aberrations, and lack Rad51 nuclear foci in the presence of DNA-damaging agents, such as mitomycin C and cisplatin. Immunocompetent mice injected with transformed OSE cells develop tumors that resemble human metastatic serous ovarian carcinoma, the most common type of ovarian cancer in women. Consistent with the reported platinum chemosensitivity in patients with Brca1-associated ovarian cancer, the Brca1-deficient OSE cells have increased sensitivity to the DNA-damaging agent cisplatin, whereas sensitivity to the microtubule poison paclitaxel is similar between Brca1 wild-type and Brca1-deficient cells. The Brca1 wild-type and Brca1-deficient mouse ovarian tumors and cell lines provide a new experimental system for the evaluation of therapies that target the Brca1 pathway.     

Cancer-associated galactosyltransferase as a new tumor marker for ovarian clear cell carcinoma

Serum cancer-associated galactosyltransferase antigen (caGT) was assayed in gynecological cancer patients by means of a GT-II-reactive monoclonal antibody (MAb 3872)-based immunoassay. Thirty-six of 47 (75%) ovarian cancer patients showed a significant elevation of caGT in serum above the cutoff level of 200 milliunits/ml (mean +/- 2 SD) determined from normal controls. Particularly, serum caGT levels in eight of nine patients with ovarian clear cell carcinoma were above the cutoff value, and six of them gave more than 200 milliunits/ml. Elevation of caGT in serum from pregnant women was also detected, and the level increased during the course of gestation. Immunohistochemical study revealed that not only various ovarian carcinoma cells in vivo and in vitro, but also syncytiotrophoblast of early gestational placenta, fetal tissues such as mucus-producing cells in the lower alimentary tract, and renal tubules at the 11th week of gestation were stained with MAb 3872, thus indicating its oncofetal character. Compared with CA-125, caGT showed a lower false-positive rate (10%) in benign gynecological diseases, and there was no correlation between caGT and CA-125 values. Therefore, caGT will be a useful tumor marker for ovarian cancers, especially for clear cell carcinoma.     

Transforming growth factor beta 1 (TGF-beta 1) inhibits growth of a human ovarian carcinoma cell line (OVCCR1) and is expressed in human ovarian tumors.

The effects of EGF and TGF-beta 1 on the proliferation of 2 ovarian carcinoma cell lines (IGROV1 and OVCCR1) were evaluated. The cell lines were adapted to grow in a restricted serum (0.5%) medium. EGF was required for proliferation of both ovarian cell lines. Low doses of TGF-beta 1 inhibited clonogenic capacity and attenuated the EGF-mediated stimulation of DNA synthesis in OVCCR1 cells. TGF-beta 1 inhibited OVCCR1 cell proliferation by blocking the cell cycle at the G1/S transition. TGF-beta 1 did not affect either clonal or monolayer growth of IGROV1 cells. Both cell lines express type-I and type-III TGF-beta receptors, suggesting that the unresponsiveness of IGROV1 cells to TGF-beta 1 occurs at a post-receptor level. TGF-beta 1 mRNA was detected in OVCCR1 cells and in 8 out of 11 of the ovarian tumor specimens examined.     

A cellular model reflecting the phenotypic heterogeneity of mutant HRAS driven squamous cell carcinoma

Squamous cell carcinomas have a range of histopathological manifestations. The parameters that determine this clinically observed heterogeneity are not fully understood. Here, we report the generation of a cell culture model that reflects part of this heterogeneity. We have used the catalytic subunit of human telomerase hTERT and large T to immortalize primary UV‐unexposed keratinocytes. Then, mutant HRAS G12V has been introduced to transform these immortal keratinocytes. When injected into immunosuppressed mice, transformed cells grew as xenografts with distinct histopathological characteristics. We observed three major tissue architectures: solid, sarcomatoid and cystic growth types, which were primarily composed of pleomorphic and basaloid cells but in some cases displayed focal apocrine differentiation. We demonstrate that the cells generated represent different stages of skin cancerogenesis and as such can be used to identify novel tumor‐promoting alterations such as the overexpression of the PADI2 oncogene in solid‐type SCC. Importantly, the cultured cells maintain the characteristics from the xenograft they were derived from while being amenable to manipulation and analysis. The availability of cell lines representing different clinical manifestations opens a new tool to study the stochastic and deterministic factors that cause case‐to‐case heterogeneity despite departing from the same set of oncogenes and the same genetic background.     

A re-evaluation of squamous cell carcinoma antigen (SCC) as a serum marker for non-small cell lung cancer

Squamous cell carcinoma antigen (SCC) is still a widely used tumor marker for monitoring non-small cell lung cancer (NSCLC), although recent reports discourage its routine use because of low sensitivity. This is a study evaluating the efficacy of SCC and CYFRA21-1 in diagnosing NSCLC. A chart review was performed in a university hospital in Japan, covering a period of 10 years, up to October 2004. During the study period, 142 (35.5%) among 400 NSCLC patients diagnosed, received serum assays of both SCC and CYFRA21-1. Elevated SCC and CYFRA21-1 levels were found in 29.6% and 59.2% of patients, respectively. SCC sensitivity was only 13.0% but CYFRA21-1 sensitivity rose to 73.9% in metastatic patients. The adjunct of SCC increased the CYFRA21-1 sensitivity by 6.3% in the overall population and by only 2.2% for patients with metastases. SCC determination should be considered an inefficient method as a potential diagnosing tool for NSCLC patients, and it provides no additional value when used in combination with CYFRA21-1.     

Ultrastructural study of small cell carcinoma (author's transl)

Fifty bronchopulmonary tumors were studied by light and electron microscopy: 39 carcinomas (25 small cell carcinomas, 8 undifferentiated carcinomas, 6 poorly differentiated squamous cell carcinomas) and 11 carcinoid tumors (5 central, 4 peripheral, 1 atypical, 1 of the tumorlet type). The results indicate that small cell carcinoma is a precise entity characterized by the presence of neurosecretory granules in the cytoplasm. Ultrastructurally carcinoid tumor resemble small cell carcinomas and there is a continuous spectrum of differentiation between the less undifferentiated neuro-endocrine tumors, i.e. the small cell anaplastic carcinomas, and the higher differentiated types, i.e. the carcinoids. The diagnostic and prognostic value of the ultrastructural findings are discussed.     

Prognosis after nonradical resections for small cell lung carcinoma (SCLC)

Out of 52 consecutive patients resected for small cell lung carcinoma (SCLC) from 1976 to 1986, 19 were selected because they underwent nonradical surgery, 10 of them for locoregional spread and 9 for distant metastases. Of the former subset all received postoperative radiotherapy and 8 chemotherapy also. Three patients are alive and disease-free 37, 56 and 91 months after resection. Four patients had a distant recurrence, and 3 a locoregional failure. Patients of the latter subgroup received chemotherapy in 7 instances. None survived more than 16 months, distant metastases being the cause of death. In these patients N0 status was associated with 13.3 months of mean survival, N1 with 8.5 months, and N2 with 6.7 months. Surgery and adjuvant treatments seem effective in achieving local control of SCLC despite nonradical resections. Tumor burden at locoregional sites does not preclude the possibility of long term survival.     

LABAZ1: A metastatic tumor model for renal cell carcinoma expressing the carbonic anhydrase type 9 tumor antigen

A metastatic renal cell carcinoma (RCC) tumor model xenograft that expresses the targetable, membrane-bound tumor-associated antigen carbonic anhydrase type 9 (CA IX) is described. The xenograft, established from a high-grade type-2 chromophil RCC (cRCC), has been serially transplanted in immune compromised mice, in which it grows orthotopically under the renal capsule, doubling its size every 9 weeks and sending metastases to the lung and liver at approximately 20 weeks. Tumors were capable of being imaged using a micro-PET (micro-positron emission tomograph) with an 18-fluorodeoxyglucose (18-FDG) tracer. Subsequent xenograft generations have conserved immunohistochemical and ultrastructural properties typical for malignant renal epithelium-derived neoplasia (vimentin+, CK-19+, CA IX+ with hypoxia-inducible factor (HIF)-1 alpha constitutive expression) and have demonstrated extensive proliferation, lack of apoptosis, severe genetic alterations, and molecular expression alterations; transforming growth factor beta 1 (TGF-beta 1), hepatocyte growth factor (HGF), proto-oncogene (c-met), matrix metalloproteinase (MMP)-1, and vascular endothelial growth factor (VEGF) C and D were overexpressed, whereas human epidermal growth factor receptor (HER)-2, MMP-2 and MMP-9, VEGF-R3, p53, and p27 were severely down-regulated, suggesting a proangiogenic environment, local invasiveness, and facilitated lymphatic metastasis. Altogether, LABAZ1 provides a relevant and flexible model to study the biology of cRCC, the role of CA IX in RCC tumorigenesis, progression, and metastasis, and a platform for testing new targeted therapeutic strategies.     

Combined radiotherapy-chemotherapy in inoperable bronchial carcinoma (small cell carcinoma excluded)

When treated by irradiation alone, non small cell carcinomas of the lung usually experience a very poor outcome. Consequently, more interest is being focussed on radiotherapy and chemotherapy association. The analysis of a randomized trial comparing irradiation alone to a sequential or an alternated combination of radio and chemotherapy did not reveal any clear advantage of the association of both therapies on each technique used alone. It is only when using concurrent irradiation and chemotherapy that some encouraging results are reported. Several non randomized studies used radio and chemotherapy prior to surgery, aiming at increasing the resectability rate. However, the criteria for initial unresectability were not homogeneous and it is not yet clear if such an association really allowed the state of surgical resection to be improved. In the near future it could be worth considering the use of radiosensitizing drugs, synchroneously given with an irradiation delivered with a multiple daily fractionation. This technique would possibly lead to a therapeutic gain, by increasing the local control rate and reducing the metastasis rate, while keeping it to an acceptable level of toxicity.     

Anti-transferrin receptor antibody linked to Pseudomonas exotoxin as a model immunotoxin in human ovarian carcinoma cell lines

The present in vitro study was performed to evaluate the potential usefulness of immunotoxins in treating human ovarian carcinomas. A monoclonal antibody against the human transferrin receptor was covalently linked to Pseudomonas exotoxin. The activity of this immunotoxin (anti-TFR-PE) was studied in five ovarian carcinoma cell lines, a breast carcinoma cell line (MCF-7), and in KB cells. The ovarian carcinoma cell lines included one previously established cell line (A1847) and four recent isolates obtained from the malignant ascites of patients with metastatic ovarian carcinoma (OVCAR cell lines). While all cell lines showed inhibition of protein synthesis by anti-TFR-PE, there were quantitative differences when the level of protein synthesis was assayed after a 12-hr incubation with the immunotoxin. These differences resulted from different kinetics of anti-TFR-PE activity in the various cell lines. Higher levels of cellular binding and internalization of anti-TFR were shown to contribute to increased toxicity of anti-TFR-PE. Verapamil increased the rate of protein synthesis inhibition and thus enhanced the toxicity of anti-TFR-PE in the OVCAR cell lines.