Jennifer C. Carr

The response to neoadjuvant chemotherapy predicts clinical outcome and increases breast conservation in advanced breast cancer

BACKGROUND The aim of this study was to determine outcomes in patients with breast cancer treated with neoadjuvant chemotherapy. METHODS Seventy-two consecutive patients receiving neoadjuvant chemotherapy for breast cancer were enrolled. RESULTS Mastectomy was avoided in 46% of patients, and 42% converted to negative nodes after neoadjuvant chemotherapy. Thirteen patients (18%) achieved a pathologic complete response, which was associated with the estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (Her2)-negative subtype (58%) and was significantly less likely to occur in the ER+/Her2- subtype (2%) (P < .01). Patients with the ER+/Her2+ subtype were most likely to have no response or progression during chemotherapy, compared with those with the ER-/Her2- subtype (50% vs 0%, P = .01). Five-year survival for patients achieving a pathologic complete response was 100%, compared with 74% in the group with partial response and 48% in the group with no response or progression (P = .01). CONCLUSIONS Neoadjuvant chemotherapy for patients with advanced breast cancer provided prognostic information, allowed evaluation of response to chemotherapy, decreased the mastectomy rate, and potentially reduced the need for axillary lymph node dissection.

Juvenile polyposis and other intestinal polyposis syndromes with microdeletions of chromosome 10q22–23

Dahdaleh FS, Carr JC, Calva D, Howe JR. Juvenile polyposis and other intestinal polyposis syndromes with microdeletions of chromosome 10q22–23.

Differentiation of small bowel and pancreatic neuroendocrine tumors by gene-expression profiling.

BACKGROUND Between 10% and 20% of patients with neuroendocrine tumors (NETs) present with metastases of unknown primary site. Because knowledge of the primary site has important implications for treatment, we set out to define gene-expression profiles to differentiate between small-bowel NETs (SBNETs) and pancreatic NETs (PNETs). METHODS RNA was extracted from tumor and normal tissues in 11 patients with SBNETs and 15 patients with PNETs, and qPCR was performed for 367 GPCR genes. Differentially expressed genes were identified using the RT2 Profiler. Whole genome expression analysis was performed on 11 SBNETs, 5 PNETS, and corresponding normal tissues. Statistical significance was evaluated by the Student t test and ANOVA. RESULTS Whole-genome analysis revealed 173 significantly differentially expressed genes in SBNETs and normal tissues and in 52 in PNETs. GPCR arrays identified 28 genes in SBNETs and 18 in PNETs, with significant expression differences from normal tissues. In all SBNETs, 2 genes were significantly upregulated by more than fivefold: OXTR and GPR113. No PNETs shared this profile, whereas 73% had a greater than fivefold downregulation of ADORA1 and SCTR. These genes also allowed for determination of the primary site in 8 of 10 liver metastases. CONCLUSION Differential expression patterns using as few as 2 to 4 GPCR genes successfully discriminated primary sites in small bowel and pancreatic NETs.

Gastric inhibitory polypeptide receptor (GIPR) is a promising target for imaging and therapy in neuroendocrine tumors

BACKGROUND Ligands binding the somatostatin receptor type 2 (SSTR2) are useful for imaging and treatment of neuroendocrine tumors (NETs), but not all tumors express high levels of these receptors. The aim of this study was to evaluate gene expression of new therapeutic targets in NETs relative to SSTR2. METHODS RNA was extracted from 103 primary small bowel and pancreatic NETs, matched normal tissue, and 123 metastases. Expression of 12 candidate genes was measured by quantitative polymerase chain reaction normalized to internal controls; candidate gene expression was compared with SSTR2. RESULTS Relative to normal tissue, primary NET expression of SSTR2, GPR98, BRS3, GIPR, GRM1, and OPRK1 were increased by 3, 8, 13, 13, 17, and 20-fold, respectively. Similar changes were found in metastases. Although most candidate genes showed lesser absolute expressions than SSTR2, absolute GIPR expression was closest to SSTR2 (mean dCT 3.6 vs. 2.7, P = .01). Absolute OPRK1 and OXTR expression varied greatly by primary tumor type and was close to SSTR2 in small bowel NETs but not pancreatic NETs. CONCLUSION Compared with the current treatment standard SSTR2, GIPR has only somewhat lesser absolute gene expression in tumor tissue but much lesser expression in normal tissue, making it a promising new target for NET imaging and therapy.

Discovery of SMAD4 promoters, transcription factor binding sites and deletions in juvenile polyposis patients.

Inactivation of SMAD4 has been linked to several cancers and germline mutations cause juvenile polyposis (JP). We set out to identify the promoter(s) of SMAD4, evaluate their activity in cell lines and define possible transcription factor binding sites (TFBS). 5′-rapid amplification of cDNA ends (5′-RACE) and computational analyses were used to identify candidate promoters and corresponding TFBS and the activity of each was assessed by luciferase vectors in different cell lines. TFBS were disrupted by site-directed mutagenesis (SDM) to evaluate the effect on promoter activity. Four promoters were identified, two of which had significant activity in several cell lines, while two others had minimal activity. In silico analysis revealed multiple potentially important TFBS for each promoter. One promoter was deleted in the germline of two JP patients and SDM of several sites led to significant reduction in promoter activity. No mutations were found by sequencing this promoter in 65 JP probands. The predicted TFBS profiles for each of the four promoters shared few transcription factors in common, but were conserved across several species. The elucidation of these promoters and identification of TFBS has important implications for future studies in sporadic tumors from multiple sites, and in JP patients.

Germline mutations in SMAD4 disrupt bone morphogenetic protein signaling.

INTRODUCTION Juvenile polyposis (JP) is an autosomal dominant disease that predisposes to GI malignancies. Germline mutations in the tumor suppressor gene SMAD4 account for approximately 20% of JP cases. SMAD4 is the common intracellular mediator of the TGF-β and bone morphogenetic protein (BMP) pathways. Since mutations in BMP receptor 1A also cause JP, we hypothesize that altered BMP signaling is the underlying defect in JP. We therefore set out to investigate the effect of SMAD4 mutations on BMP signaling. METHODS SMAD4 mutations identified in JP patients were selected for analysis. These were created in SMAD4 pCMV expression vectors (EV) using a PCR-based, site-directed mutagenesis (SDM) approach. SDM clones were confirmed by direct sequencing, then co-transfected with an IdI-BMP Luciferase Responsive Element (BRE-Luc) vector and Renilla control vector into HEK-293T cells. Lysates were then collected after 48 hours, and luciferase activity was quantified using a luminometer. A pCMV empty vector was used as a negative control, and its luciferase activity was considered the baseline for cellular BMP signaling. Results obtained for each SDM clone were compared to those with the wild type (WT) vector. Statistical analysis was performed with the Students t-test. RESULTS Eleven distinct mutations from 16 JP patients were analyzed; seven mutations were nonsense, and four were missense. Both type of mutations resulted in reduction of BMP signaling; missense mutations produced an 8-30% reduction in luciferase activity, whereas nonsense mutations led to 30-60% reduction in luciferase activity when compared to the WT clone (Figure 1). All nonsense mutations led to significantly reduced activity relative to WT (P < 0.05), while the reduction in signaling seen in missense mutations was not statistically significant. CONCLUSION SMAD4 germline mutations as seen in the JP patients appear to negatively impact downstream BMP signaling. Nonsense mutations resulted in significantly reduced luciferase activity when compared to missense mutations. These results support the hypothesis that disruption of the BMP signaling pathway is the likely etiology of JP in patients with SMAD4 mutations.

Overexpression of Membrane Proteins in Primary and Metastatic Gastrointestinal Neuroendocrine Tumors

BackgroundSmall bowel and pancreatic neuroendocrine tumors (SBNETs and PNETs) are rare tumors whose incidence is increasing. Drugs targeting the somatostatin receptor are beneficial in these tumors. To identify additional cell-surface targets, we recently found receptors and membrane proteins with gene expression significantly different from adjacent normal tissues in a small number of primary SBNETs and PNETs. We set out to validate these expression differences in a large group of primary neuroendocrine tumors and to determine whether they are present in corresponding liver and lymph node metastases.MethodsPrimary SBNETs and PNETs, normal tissue, nodal, and liver metastases were collected and mRNA expression of six target genes was determined by quantitative PCR. Expression was normalized to GAPDH and POLR2A internal controls, and differences as compared to normal tissue were assessed by Welch’s t test.ResultsGene expression was determined in 45 primary PNETs with 20 nodal and 17 liver metastases, and 51 SBNETs with 50 nodal and 29 liver metastases. Compared to normal tissue, the oxytocin receptor (OXTR) showed significant overexpression in both primary and metastatic SBNETs and PNETs. Significant overexpression was observed for MUC13 and MEP1B in PNET primary tumors, and for GPR113 in primary SBNETs and their metastases. SCTR and ADORA1 were significantly underexpressed in PNETs and their metastases. OXTR protein expression was confirmed by immunohistochemistry.ConclusionsOXTR is significantly overexpressed relative to normal tissue in primary SBNETs and PNETs, and this overexpression is present in their liver and lymph node metastases, making OXTR a promising target for imaging and therapeutic interventions.

BMPR1A mutations in juvenile polyposis affect cellular localization

BACKGROUND Juvenile polyposis (JP) is characterized by the development of hamartomatous polyps of the gastrointestinal tract that collectively carry a significant risk of malignant transformation. Mutations in the bone morphogenetic protein receptor type 1A (BMPR1A) are known to predispose to JP. We set out to study the effect of such missense mutations on BMPR1A cellular localization. METHODS We chose eight distinct mutations for analysis. We tagged a BMPR1A wild-type (WT) expression plasmid with green fluorescent protein on its C-terminus. Site-directed mutagenesis was used to recreate JP patient mutations from the WT-green fluorescent protein BMPR1A plasmid. We verified mutant expression vector sequences by direct sequencing. First, we transfected BMPR1A expression vectors into HEK-293T cells; then, we performed confocal microscopy to determine cellular localization. Four independent observers used a scoring system from 1 to 3 to categorize the degree of membrane versus cellular localization. RESULTS Of the eight selected mutations, one was within the signaling peptide, four were within the extracellular domain, and three were within the intracellular domain. The WT BMPR1A vector had strong membrane staining, whereas all eight mutations had much less membrane and much more intracellular localization. Enzyme-linked immunosorbent assays for BMPR1A demonstrated no significant differences in protein quantities between constructs, except for one affecting the start codon. CONCLUSIONS Bone morphogenetic protein receptor type 1A missense mutations occurring in patients with JP affected cellular localization in an in vitro model. These findings suggest a mechanism by which such mutations can lead to disease by altering downstream signaling through the bone morphogenetic protein pathway.

SP1 Regulates the Transcription of BMPR1A

BACKGROUND BMPR1A is a cell surface receptor in the bone morphogenetic protein (BMP) pathway. Mutations in BMPR1A predispose to juvenile polyposis (JP). Sp1 and related proteins are widely expressed regulators of gene transcription, including members of the BMP pathway. We set out to identify important transcription factor binding sites (TFBS) in the recently identified BMPR1A promoter and to assess for the role of Sp1 and associated proteins in its regulation. MATERIALS AND METHODS The BMPR1A promoter was cloned into a luciferase reporter vector. Deletion fragments of this promoter insert were then constructed, of varying lengths and opposing directions, and were used to transfect HEK-293 and CRL-1459 cells. In silico analysis was performed to screen for relevant TFBS. Site-directed mutagenesis (SDM) was then employed to individually disrupt these TFBS in the wild-type (WT) vector. SDM constructs were then assessed for activity. RESULTS Light activity from the deletion constructs ranged between 3% and 129% of the WT promoter. ModelInspector identified eight potential binding sites for Sp1- and Sp1-associated proteins that mapped to areas of marked loss or gain of activity from the deletion constructs. SDM of these TFBS led to a drop in activity in five mutants, which included 3 Sp1 sites, an ETSF site, and NFκB site. CONCLUSIONS By combining in silico analysis and experimental data, Sp1 was found to be a candidate factor that likely plays a role in the transcriptional regulation of BMPR1A. This study potentially provides further insight toward the molecular basis of JP, and suggests that Sp1 plays a role in BMP signaling.