Metastatic Merkel Cell carcinoma (MCC) is a highly unusual and aggressive skin cancer that presents as a small, pink to violet skin lesion and metastasizes early in its growth. Metastatic MCC is generally treated with small cell lung cancer chemotherapy regimens, because the tumor consists of neuroendocrine cells, but patients generally do not have durable responses. The pathogenesis of MCC has recently been attributed to the Merkel Cell polyoma virus. This virus activates the cellular retinoblastoma oncoprotein and cell cycle machinery, triggering continual cellular proliferation. A 77-year-old man developed extensive MCC metastases, involving more than one fourth of his scalp and numerous cervical lymph nodes. Following failure of initial chemotherapy and radiation, effective palliation was achieved by using a sequence of electron-beam radiotherapy, low dose gemcitabine, and etoposide, resulting in significant periods of tumor regression and prolonged survival. A novel circulating tumor cell (CTC) culture assay was performed on four separate clinic visits during the treatment period. Tumor colonies were cultured from the patient’s peripheral blood and CTC colony counts were correlated with clinical treatment response. Not only did the patient respond to palliative cell cycle directed chemotherapy and electron beam radiation, but we demonstrated that CTC can be cultured from peripheral blood of MCC patients and serve as a predictive marker to monitor treatment response.
10614 Background: Identification of rare (>2-5) circulating tumor cells (CTC) in 7.5 ml blood by immunofluorescence assay (IFA) correlates with a poor prognosis in colon, breast, prostate and lung cancer. Changes in CTC count during treatment also predict the eventual patient progression and survival in these cancers. Existing assays do not detect melanoma CTC, however. In addition, isolation of viable CTC remains problematic. To overcome these limitations we attempted to develop novel melanoma CTC assays, using IFA and cell culture approaches. Methods: Blood samples were obtained from patients and controls following informed consent. The buffy coat (white cells + tumor) was isolated by Ficoll/Hypaque centrifugation, and split into 6 replicate cultures in proprietary TrueCells medium. After 21 days in culture, tumor colonies were counted, and stained for melanoma and leukocyte markers. Buffy coat cells from parallel blood samples were stained with a panel of CSPG4-specific mAb (a pan-melanoma marker) on ultraclean glass slides for analysis by immunofluorescence microscopy. Results: Blood samples were obtained from 16 melanoma patients, ages 28-87. Eight patients were men and 8 were women. CSPG4+ events (>2) were detected in 8/16 patients by IFA (range 0-52). In contrast, tumor cell colonies of >50 cells grew in 12 out of 16 patients with Stage 3 or 4 melanoma (range 0-1054), shown in Table. Cells isolated from CTC colonies produced melanin, stained for CSPG4 and other melanoma markers, but not for leukocyte markers. Control cultures grew no tumor colonies. Conclusions: Our pilot study shows that melanoma CTC can be identified by both IFA and cultured from blood in many patients with stage 3 or 4 melanoma. These CTC exhibited cytologic characteristics diagnostic of melanoma. The culture assay may represent a useful means of enumerating, isolating, and expanding viable melanoma CTC for further molecular study. [Table: see text]
The interactions that occur between leukemic cells and the microenvironment are currently poorly understood. The definition of novel pathways that deliver survival and drug resistance signaling from the milieu to leukemic cells may lead to the discovery of new drugs that are able to target microenvironmental cues perpetuating the leukemic clones. Since the bone marrow (BM) environment is comprised of a complex assortment of different cell types, matrices and soluble and membrane-bound factors, it is crucial to start delineating specific cell-cell interactions and their effects on leukemic cell survival. Mesenchymal stem cells (MSC) are a fundamental component of the BM niche as they support hematopoiesis and are able to differentiate into neighboring cellular types. Therefore, in these studies we investigated the effect of Stro-1+ MSC on the growth and survival of KG1a, a primitive, differentiation-resistant subtype of acute myeloid leukemia. MSC and KG1a cells were co-cultured for 3 days under contact or non-contact conditions, at incremental ratios of 1:1, 1:5, 1:10 and 1:100 respectively, while maintaining a constant initial total number of cells for each dilution (n=3). In transwell non-contact cultures, KG1a cells proliferated at the same rate as KG1a alone with a 3 to 5 fold increase in cell numbers, regardless of the ratio. By contrast, when KG1a were cultured in direct contact with MSC, KG1a growth inhibition was directly proportional to the MSC:KG1a ratio. Specifically, MSC cultured with KG1a cells at ratios of 1:1, 1:5, 1:10, and 1:100 inhibited the growth of KG1a cells by 84%, 76%, 74%, and 45%, respectively while remaining 99-100% viable. Thus, we next performed cell cycle analysis using flow cytometry and found that while the cell cycle distribution of KG1a cultures were: 23% in G0/G1; 19% in G2/M; and 58% in S phase, KG1a co-cultured with MSC had undergone cell cycle arrest in S-phase (98%). Although our results contrast with prior studies showing that MSC inhibited KG1a in G1phase through the production of soluble factors, it is possible that the differences seen are due to the population of MSC used. Within the MSC, Stro-1+ cells constitute a primitive population of cells in intimate relation with the osteoblastic niche, the role of which is to retain stem cells in a quiescent state. Therefore, studies are currently underway to identify molecules responsible cell cycle arrest upon KG1a/MSC interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 538.
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