Project description:Circulating tumor cells (CTCs) are critical in the development of distant organ tumor metastasis, and are associated with advanced cancer stage and poor patient outcome. Here, we present the first genome-wide nucleotide-level characterization of CTCs. Our single-nucleotide polymorphism (SNP) analysis in patients with melanoma involved: 1) global comparative genomic analysis of CTCs and matched regional metastases, 2) identification of key genomic aberrations in CTCs, 3) verification of these target genes in aggressive distant tumor metastases, and 4) evidence of selective expression and functional consequence of CTC-associated genes in melanomas. We report 131 aberrant loci in CTCs that are potentially pro-metastatic, and show that such expression of a 5-marker gene panel (CSMD2, CNTNAP5, FLJ14051, ADAM6, TRPM2) in melanomas confers prognostic utility. Successful treatment of melanoma requires understanding of the metastatic process and identification of patients with tumors most likely to develop aggressive metastatic disease. Melanomas are heterogeneous, and CTCs have long been recognized as vehicles for cancer spread, representing particularly aggressive tumor clones that can evolve into successful clinical metastases. Elucidation of genomic aberrations in CTCs will aid in the development of prognostic biomarkers and therapeutic strategies to target CTCs to prevent or control distant cancer spread. This study provides the first detailed genomic confirmation of the close relation between CTCs and tumor metastases, and illustrates how CTCs can be utilized as a novel approach and rational source for identification of pro-metastatic genes in cancer research. Three individual patient cohorts were utilized in the study. CNV and LOH loci were evaluated initially in metastatic melanoma patients (n=13) in a discovery cohort. SNP loci that harbored CNV/LOH in CTCs were then separately verified for: (a) presence in distant organ metastasis (AJCC Stage IV melanoma) (n=27), and (b) relevance to prognosis in regional melanoma metastasis (AJCC Stage III melanoma) (n=35). The first discovery patient cohort group was utilized for capture of CTCs, and consisted of peripheral blood mononuclear cell (PBMC) and tumor specimens from metastatic melanoma patients (n=13). CTC-related loci were verified in a second cohort of patients with Stage IV distant organ metastases (n=27), including 15 brain, 4 lung, and 8 gastrointestinal (bowel, liver) metastases. The third patient cohort consisted of early passage (<12) established melanoma cell lines derived from 35 regional melanoma metastases (Stage III) for evaluation of the prognostic utility of the CTC-associated aberrant loci.

Project description:Metastatic disease remains one of the most urgent clinical challenges accounting for over 90% of cancer-related deaths. Yet, the identification of novel therapeutic targets to fight or prevent metastatic disease has been hampered by the limited availability of clinically relevant mouse models of metastasis formation. To address this caveat, we developed a novel preclinical mouse model of spontaneous metastatic breast cancer that recapitulates the key biological events of the metastatic cascade and mimics the clinical course of metastatic disease in humans. Exploiting the conditional K14cre;CdhF/F;Trp53F/F mouse model of de novo mammary tumor formation, we orthotopically transplanted K14cre;CdhF/F;Trp53F/F derived mouse invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once recipient mammary tumors were established, we mimicked the clinical setting and performed a mastectomy. Following surgery, recipient mice eventually succumbed to wide-spread clinically overt metastatic disease in lymph nodes, lungs and gastrointestinal tract. Using aCGH analyses, we explored the relationship between the genomic profiles of mammary donor tumors and paired recipient outgrowths and observed a strong correlation, indicating that the genomic profile of the parental K14cre;CdhF/F;Trp53F/F mILC is highly conserved in recipient mammary tumors. To investigate the genomic relationship between recipient mammary tumors and their metastases, we examined the correlation structure of genomic profiles derived from paired sets of primary tumors and metastases. Genomic profiles of clonally-related recipient mammary tumors were highly conserved in local and distant metastases, indicating that few genomic alterations occur during transition from a primary tumor to a distant site. To more thoroughly examine potential site-specific genomic alterations, we constructed so-called ‘delta-profiles’ by calculating the difference between the genomic profile of a recipient mammary tumor and its paired lymph node- and lung metastasis. Site-specific recurrent alterations were not observed in lymph node nor lung metastases. Taken together, these data show that genomic profiles of metastases are highly similar to those of parental recipient tumors and that, if changes occurred, they did not recur in different independent samples. We performed aCGH analyses on DNA isolated from K14cre;Cdh-/-;Trp53-/- derived donor mILCs (n=3) and their recipient mammary tumor outgrowths (n=10). Furthermore, we also analyzed genomic profiles derived from lung (n=10), tumor-draining (n=7) and distant lymph node metastases (n=5) isolated from the same recipient mice. DNA from each of these samples was hybridized against related donor splenic DNA.

Project description:Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice, rapidly declining after B-RAF inhibitor treatment. CTCs were shed early from localized tumors and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled comparison of RNA sequencing profiles with the matched primary tumor. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. In patients with metastatic melanoma, CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF targeted therapy. Together, the capture of CTCs and their molecular characterization provide insight into the hematogenous spread of melanoma. We adapted a microfluidic platform, the HbCTC-Chip (Stott et al., 2010, Pubmed ID: 20930119), to capture melanoma CTCs derived from mouse tumors, using panels of antibodies against melanoma-specific cell surface markers, followed by staining for melanoma antigens and optimized on-chip fluorescent imaging. We used a tamoxifen inducible BRAF(CA/+)/PTEN(flox/flox) melanoma mouse model (Dankort et al., 2009, Pubmed ID: 19282848) derived from a C57BL/6 background. Such mice received focal subcutaneous injection of tamoxifen (Sigma) (50ul at 5mg/ml in 50% EtOH suspension) at the left flank at 6-7 weeks after birth. Blood samples were collected from five mice with high tumor burden following tamoxifen injection. Blood from each mouse was split and processed through the CTC-chips functionalized with anti-CSPG4/MCAM antibody and control IgGs, respectively. Matched primary (from the tamoxifen injection site) and metastatic (from upper or lower back) tumors were harvested from the same mouse and immediately flash-frozen in liquid nitrogen. RNA extraction, single molecular sequencing and determination of Digital Gene Expression was as in Yu et al., 2012 (Pubmed ID: 22763454). One of the five mice yielded no CTC or IgG data. In addition, skin was taken from a sixth mouse, which was a complete wild type C57BL/6 mouse (without the BRAF/PTEN transgenes).The skin was taken after euthanasia of the animal and was processed as the tissue from the matched primary and metastatic tumors.

Project description:The identification of novel tumor-specific markers may improve understanding of melanoma progression and prognostic accuracy. Whole genome expression profiling of 46 primary melanomas, 12 metastases, and 16 normal skin samples using Affymetrix U133 PLUS 2.0 array generated gene lists including both known and new melanoma genes. The molecular genetic alterations contributing to the pathogenesis of melanoma are incompletely defined and few independent prognostic features have been identified beyond the pathologic characteristics of the primary tumor. Early stage melanoma is frequently curable, in contrast to the inferior prognosis of melanomas with regional lymph nodes involvement and the incurability of distant metastatic disease. The identification of novel tumor-specific markers may improve our understanding of melanoma progression and prognostic accuracy. To find differentially expressed genes that can distinguish melanoma from normal tissue, we performed a whole genome expression profiling of 46 primary melanoma samples, 12 regional or distant metastases, and 16 normal skin samples using Affymetrix U133 2.0 Plus array. Our study generated lists of differentially expressed genes in melanoma and identified novel prognostic marker HMGA2. It is a novel, highly overexpressed melanoma gene associated with poor prognosis. The overexpression of HMGA2 is strongly associated with regional and distant metastases and serves as an independent predictor of disease-free survival and overall survival in melanoma. Melanoma samples were obtained through an IRB-approved protocol using informed consent at the University of Michigan Multidisciplinary Melanoma Clinic. A portion of pigmented lesions clinically suspicious for melanoma and known melanoma were sampled by punch biopsy at the time of excision. The punch biopsy was bisected; half was sent to for clinical diagnosis and the other half along with adjacent normal skin when available immediately snap-frozen in liquid nitrogen. Snap-frozen tissue was embedded in OCT (TissueTek) followed by frozen sectioning and H&E slide preparation. The slides were evaluated by dermatopathologist who identified areas with greater than 70% tumor cellularity, which were sampled for RNA extraction. Primary melanomas and melanoma metastases were derived from different patients. Metastatic samples included lymph nodes (n=8), subcutaneous soft tissue (n=2), spleen (n=1), and small intestine (n=1).

Project description:Metastatic disease remains one of the most urgent clinical challenges accounting for over 90% of cancer-related deaths. Yet, the identification of novel therapeutic targets to fight or prevent metastatic disease has been hampered by the limited availability of clinically relevant mouse models of metastasis formation. To address this caveat, we developed a novel preclinical mouse model of spontaneous metastatic breast cancer that recapitulates the key biological events of the metastatic cascade and mimics the clinical course of metastatic disease in humans. Exploiting the conditional K14cre;CdhF/F;Trp53F/F mouse model of de novo mammary tumor formation, we orthotopically transplanted K14cre;CdhF/F;Trp53F/F derived mouse invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once recipient mammary tumors were established, we mimicked the clinical setting and performed a mastectomy. Following surgery, recipient mice eventually succumbed to wide-spread clinically overt metastatic disease in lymph nodes, lungs and gastrointestinal tract. Using aCGH analyses, we explored the relationship between the genomic profiles of mammary donor tumors and paired recipient outgrowths and observed a strong correlation, indicating that the genomic profile of the parental K14cre;CdhF/F;Trp53F/F mILC is highly conserved in recipient mammary tumors. To investigate the genomic relationship between recipient mammary tumors and their metastases, we examined the correlation structure of genomic profiles derived from paired sets of primary tumors and metastases. Genomic profiles of clonally-related recipient mammary tumors were highly conserved in local and distant metastases, indicating that few genomic alterations occur during transition from a primary tumor to a distant site. To more thoroughly examine potential site-specific genomic alterations, we constructed so-called ‘delta-profiles’ by calculating the difference between the genomic profile of a recipient mammary tumor and its paired lymph node- and lung metastasis. Site-specific recurrent alterations were not observed in lymph node nor lung metastases. Taken together, these data show that genomic profiles of metastases are highly similar to those of parental recipient tumors and that, if changes occurred, they did not recur in different independent samples.

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Whole Genome Characterization of Circulating Tumor Cells Identifies Novel Prognostic Genomic Alterations in Melanoma Metastasis

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