Enrichment and mutation detection of circulating tumor cells from blood samples

The potential of circulating tumor cells (CTCs) in the diagnosis and prognosis of cancer patients has become increasingly attractive. However, molecular analysis of CTCs is hindered by low sensitivity and a high level of background leukocytes in CTC enrichment technologies. We have developed a novel protocol using a microfluidic device, which enriches and retrieves CTCs from blood samples. The principle of CTC capturing is that tumor cells are larger and less deformable than normal blood cells. To evaluate the potential of utilizing Celsee PREP100 in CTC molecular analysis, we prepared prostate cancer cell lines PC3 and LNCaP, retrieved the captured cells and analyzed them using PCR amplicon sequencing. We were able to recover an average of 79% of 110‑1,100 PC3 and 60‑1,500 LNCaP cells, and detect the p.K139fs*3 deletion of the p53 gene in PC3 cells and p.T877A mutation of the androgen receptor gene in LNCaP cells. Next, we spiked these two types of cells into normal donor blood samples, captured the cells and analyzed them using PCR amplicon sequencing. The PC3 and LNCaP cells were captured and retrieved with the ratio of captured CTCs to the background leukocytes reaching 1:1.5 for PC3 and 1:2.9 for LNCaP cells. We further revealed that the p.K139fs*3 deletion and p.T877A mutation can be detected in the captured PC3 and LNCaP cells, respectively. We successfully validated this approach using clinical blood samples from patients with metastatic prostate cancer. Our results demonstrated a novel approach for CTC enrichment and illustrated the potential of CTC molecular characterization for diagnosis, prognosis and treatment selection of patients with metastatic malignancy

Enrichment and mutation detection of circulating tumor cells from blood samples.

The potential of circulating tumor cells (CTCs) in the diagnosis and prognosis of cancer patients has become increasingly attractive. However, molecular analysis of CTCs is hindered by low sensitivity and a high level of background leukocytes in CTC enrichment technologies. We have developed a novel protocol using a microfluidic device, which enriches and retrieves CTCs from blood samples. The principle of CTC capturing is that tumor cells are larger and less deformable than normal blood cells. To evaluate the potential of utilizing Celsee PREP100 in CTC molecular analysis, we prepared prostate cancer cell lines PC3 and LNCaP, retrieved the captured cells and analyzed them using PCR amplicon sequencing. We were able to recover an average of 79% of 110‑1,100 PC3 and 60‑1,500 LNCaP cells, and detect the p.K139fs*3 deletion of the p53 gene in PC3 cells and p.T877A mutation of the androgen receptor gene in LNCaP cells. Next, we spiked these two types of cells into normal donor blood samples, captured the cells and analyzed them using PCR amplicon sequencing. The PC3 and LNCaP cells were captured and retrieved with the ratio of captured CTCs to the background leukocytes reaching 1:1.5 for PC3 and 1:2.9 for LNCaP cells. We further revealed that the p.K139fs*3 deletion and p.T877A mutation can be detected in the captured PC3 and LNCaP cells, respectively. We successfully validated this approach using clinical blood samples from patients with metastatic prostate cancer. Our results demonstrated a novel approach for CTC enrichment and illustrated the potential of CTC molecular characterization for diagnosis, prognosis and treatment selection of patients with metastatic malignancy

Brief Report: Prognostic Relevance of 3q Amplification in Squamous Cell Carcinoma of the Lung

INTRODUCTION: Amplification of 3q is the most common genetic alteration identified in squamous cell carcinoma of the lung (LUSC), with the most frequent amplified region being 3q26 to 3q28. METHODS: In this analysis, we aim to describe the prognostic relevance of 3q amplification by focusing on a minimal common region (MCR) of amplification constituted of 25 genes. We analyzed 511 cases of LUSC from The Cancer Genome Atlas and included 476 in the final analysis. RESULTS: We identified a 25-gene MCR that was amplified in 221 (44.3%) cases and was associated with better disease-specific survival (not reported [NR] versus 9.25 y, 95% confidence interval [CI]: 5.24-NR, log-rank p = 0.011) and a progression-free interval of 8 years (95% CI: 5.1-NR) versus 4.9 years (95% CI: 3.5-NR, log-rank p = 0.020). Multivariable analysis revealed that MCR amplification was associated with improved disease-specific survival and progression-free interval. CONCLUSIONS: Amplification of the 25-gene MCR within 3q was present in 44% of this cohort, consisting mainly of Caucasian patients with early stage LUSC. This analysis strongly indicates the prognostic relevance of the 25-gene MCR within 3q. We are further evaluating its prognostic and predictive relevance in a racially diverse patient population with advanced LUSC

Transcriptional network involving ERG and AR orchestrates Distal-less homeobox-1 mediated prostate cancer progression

Distal-less homeobox-1 (DLX1) is a well-established non-invasive biomarker for prostate cancer (PCa) diagnosis, however, its mechanistic underpinnings in disease pathobiology are not known. Here, we reveal the oncogenic role of DLX1 and show that abrogating its function leads to reduced tumorigenesis and metastases. We observed that ~60% of advanced-stage and metastatic patients display higher DLX1 levels. Moreover, ~96% of TMPRSS2-ERG fusion-positive and ~70% of androgen receptor (AR)-positive patients show elevated DLX1, associated with aggressive disease and poor survival. Mechanistically, ERG coordinates with enhancer-bound AR and FOXA1 to drive transcriptional upregulation of DLX1 in ERG-positive background. However, in ERG-negative context, AR/AR-V7 and FOXA1 suffice to upregulate DLX1. Notably, inhibiting ERG/AR-mediated DLX1 transcription using BET inhibitor (BETi) or/and anti-androgen drugs reduce its expression and downstream oncogenic effects. Conclusively, this study establishes DLX1 as a direct-target of ERG/AR with an oncogenic role and demonstrates the clinical significance of BETi and anti-androgens for DLX1-positive patients

A pediatric case of pigmented epithelioid melanocytoma with chromosomal copy number alterations in 15q and 17q and a novel NTRK3‐SCAPER gene fusion

Pigmented epithelioid melanocytoma (PEM) represents a group of rare, heavily pigmented melanocytic tumors encompassing lesions previously designated as “animal‐type melanomas” and “epithelioid blue nevi.” Despite the association of multiple such tumors in the setting of Carney complex, most cases of PEM occur spontaneously as solitary neoplasms in otherwise healthy patients. PEM may arise in both children and adults, and has a known propensity to spread to the regional lymph nodes. Despite this latter finding, recurrence at the biopsy site or spread beyond the lymph node basin is exceptionally uncommon. Although the molecular basis for PEM continues to be characterized, findings to date suggest that this category of melanocytic neoplasia has genetic alterations distinct from those seen in common nevi, dysplastic nevi, Spitz nevi, and melanoma. Herein, we present an in‐depth clinical, histopathologic, and molecular analysis of a case of PEM occurring on the scalp of a young African American girl found to have a novel NTRK3‐SCAPER gene fusion.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152480/1/cup13566.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152480/2/cup13566_am.pd

A pediatric case of pigmented epithelioid melanocytoma with chromosomal copy number alterations in 15q and 17q and a novel NTRK3‐SCAPER gene fusion

Pigmented epithelioid melanocytoma (PEM) represents a group of rare, heavily pigmented melanocytic tumors encompassing lesions previously designated as “animal‐type melanomas” and “epithelioid blue nevi.” Despite the association of multiple such tumors in the setting of Carney complex, most cases of PEM occur spontaneously as solitary neoplasms in otherwise healthy patients. PEM may arise in both children and adults, and has a known propensity to spread to the regional lymph nodes. Despite this latter finding, recurrence at the biopsy site or spread beyond the lymph node basin is exceptionally uncommon. Although the molecular basis for PEM continues to be characterized, findings to date suggest that this category of melanocytic neoplasia has genetic alterations distinct from those seen in common nevi, dysplastic nevi, Spitz nevi, and melanoma. Herein, we present an in‐depth clinical, histopathologic, and molecular analysis of a case of PEM occurring on the scalp of a young African American girl found to have a novel NTRK3‐SCAPER gene fusion.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152480/1/cup13566.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152480/2/cup13566_am.pd

Pseudosarcomatous myofibroblastic proliferations of the genitourinary tract are genetically different from nodular fasciitis and lack USP6, ROS1 and ETV6 gene rearrangements

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145266/1/his13526_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145266/2/his13526.pd

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention

Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109645/1/cup12387.pd

Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147795/1/his13758.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147795/2/his13758_am.pd