Receptor Tyrosine Kinase Expression Predicts Response to Sunitinib in Breast Cancer.

BACKGROUND: Preliminary data indicate that tyrosine kinase inhibitors (TKIs) function through rearranged during transfection (RET) in breast cancer. However, TKIs are not specific and can block several receptor tyrosine kinases (RTKs). This study used cell lines and primary breast cancer specimens to determine factors associated with TKI response. METHODS: Proliferation was assessed after short interfering RNA knockdown with or without sunitinib in breast cancer cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Breast cancer tissue and matched normal breast was obtained from 30 women with invasive breast carcinoma. Gene expression was assessed by reverse transcriptase-polymerase chain reaction. Fresh tissue was treated in vitro with sunitinib or control media for 30 min, and response was assessed by phosphorylation-specific western blot. RESULTS: The RTKs including epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR1-3), platelet-derived growth factor receptor (PDGFRa/b), and Kit were overexpressed in triple-negative breast tumors relative to HER2- and estrogen receptor-alpha (ERα)-positive tumors and normal breast tissue. Knockdown of EGFR reduced in vitro proliferation in MCF-7 and MDA-MB-231 but not in SKBR-3 or ZR-75-1 breast cancer cells. With the exception of RET, response to sunitinib was independent of RTK expression in all four cell lines. Both ERα-positive and low-EGFR-expressing tumors had an increased in vitro sunitinib response, as determined by alteration of Erk activation. Expression of other RTKs and additional clinical factors were not associated with response. CONCLUSION: Triple-negative breast cancers overexpress RTKs but have decreased in vitro response to the TKI sunitinib. In addition to RET, TKIs that block EGFR may increase the therapeutic efficacy of TKIs in breast cancer.

GIPR expression in gastric and duodenal neuroendocrine tumors.

BACKGROUND: Compounds targeting somatostatin-receptor-type-2 (SSTR2) are useful for small bowel neuroendocrine tumor (SBNET) and pancreatic neuroendocrine tumor (PNET) imaging and treatment. We recently characterized expression of 13 cell surface receptor genes in SBNETs and PNETs, identifying three drug targets (GIPR, OXTR, and OPRK1). This study set out to characterize expression of this gene panel in the less common neuroendocrine tumors of the stomach and duodenum (gastric and duodenal neuroendocrine tumors [GDNETs]). METHODS: Primary tumors and adjacent normal tissue were collected at surgery, RNA was extracted, and expression of 13 target genes was determined by quantitative polymerase chain reaction. Expression was normalized to GAPDH and POLR2A internal control genes. Expression relative to normal tissue (ddCT) and absolute expression (dCT) were calculated. Wilcoxon tests compared median expression with false discovery rate correction for multiple comparisons. RESULTS: Gene expression was similar in two gastric and seven duodenal tumors, and these were analyzed together. Like SBNETs (n = 63) and PNETs (n = 51), GDNETs showed significant overexpression compared with normal tissue of BRS3, GIPR, GRM1, GPR113, OPRK1, and SSTR2 (P < 0.05 for all). Of these, SSTR2 had the highest absolute expression in GDNETs (median dCT 4.0). Absolute expression of BRS3, GRM1, GPR113, and OPRK1 was significantly lower than SSTR2 in GDNETs (P < 0.05 for all), whereas expression of GIPR was similar to SSTR2 (median 4.3, P = 0.4). CONCLUSIONS: As in SBNETs and PNETs, GIPR shows absolute expression close to SSTR2 but has greater overexpression relative to normal tissue (21.1 versus 3.5-fold overexpression). We conclude that GIPR could provide an improved signal-to-noise ratio for imaging versus SSTR2 and represents a promising novel therapeutic target in GDNETs.

The value of preoperative imaging in small bowel neuroendocrine tumors.

BACKGROUND: Neuroendocrine tumors of the small bowel (SBNETs) are a rare but important subgroup of malignancies. Since 30 % of SBNETs present with metastatic disease, often with an occult primary, preoperative imaging is critical for determining who will benefit most from abdominal exploration. We set out to evaluate the usefulness of the two most commonly performed imaging modalities in predicting the extent of disease found at exploration in patients with SBNETs. METHODS: A retrospective chart review was performed on patients with SBNETs resected at 1 institution. Data from preoperative computed tomography (CT) scans were reviewed to determine whether the primary tumor, nodal, or liver metastases were seen, then compared with intraoperative findings. Results of preoperative somatostatin receptor scintigraphy (SRS) were similarly examined. RESULTS: A total of 62 patients with SBNETs were included. Of these patients, 42 of 62 (68 %) had distant metastases and 48 of 62 (77 %) had nodal metastases at exploration. A total of 56 patients had preoperative CT scans and 47 had SRS. Using CT, a primary tumor was localized to the small bowel in 27 of 56 (48 %) and nodal metastases seen in 33 of 56 (79 %) of cases. SRS found intra-abdominal uptake in 35 of 47 cases (74 %). CONCLUSIONS: CT and SRS are complementary in making the diagnosis of SBNET, with CT giving more precise anatomical detail, while SRS helps to confirm that lesions are NETs and is useful for identifying occult extrahepatic sites of metastatic disease. However, 10-15 % of SBNETs were not identified by either test preoperatively, and therefore surgical exploration still plays an important role in making the diagnosis in these patients.

Discriminating pheochromocytomas from other adrenal lesions: the dilemma of elevated catecholamines.

BACKGROUND: Screening tests for pheochromocytoma involve measuring levels of catecholamines in the urine or plasma, which have significant false-positive rates. We reviewed patients with adrenal masses and elevated levels of catecholamines to determine the value of different preoperative tests in diagnosing pheochromocytomas. METHODS: A retrospective chart review identified patients who underwent adrenalectomy between 1997 and 2011 with elevation of urine or serum catecholamines. A database of clinicopathologic factors was created including preoperative urine and plasma metanephrines, normetanephrines, vanillylmandelic acid, and fractionated catecholamines, and tumor dimensions on imaging and pathology. RESULTS: A total of 70 patients underwent adrenalectomy because of presence of an adrenal mass and elevation of catecholamines or normetanephrines or metanephrines. Of these, 46 had pathologically confirmed pheochromocytomas. To improve our ability to discriminate between pheochromocytoma and other pathology, we examined different combinations of clinicopathologic factors and catecholamine levels and found the best test was a scoring system. Points are awarded for a hierarchy of elevated normetanephrine, norepinephrine, metanephrines, with additional points received for age <50 and size on imaging >3.3 cm. A score of 2 is suggestive of pheochromocytoma, with a positive predictive value of 86-87%, while a score of 4 is diagnostic with positive predictive value of 100%. CONCLUSION: We found that urine/serum normetanephrine levels were the most valuable screening tool; however, a score examining the size of adrenal mass on preoperative CT, age, and either plasma or urine norepinephrine, metanephrine, and normetanephrine values leads to a higher positive predictive value, making this scoring system superior to individual lab tests.

Overexpression of membrane proteins in primary and metastatic gastrointestinal neuroendocrine tumors.

BACKGROUND: Small 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. METHODS: Primary 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. RESULTS: Gene 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. CONCLUSIONS: OXTR 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.

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.

Comparison of clinicopathologic factors in 122 patients with resected pancreatic and ileal neuroendocrine tumors from a single institution.

BACKGROUND: Recent population-based studies have demonstrated significant differences in outcome between patients with pancreatic and ileal neuroendocrine tumors. The objective of this study was to examine the clinicopathologic differences between ileal and pancreatic neuroendocrine tumors following resection. METHODS: A retrospective chart review was performed and data on clinicopathologic variables, biochemical markers, and follow-up of patients with resected ileal (INETs) and pancreatic (PNETs) neuroendocrine tumors were collected. The t test or analysis of variance (ANOVA) was used to compare means. Survival analysis was performed using the Kaplan-Meier method. RESULTS: Between 1998 and 2010, 122 patients with PNETs and INETs were explored (70 PNETs and 52 INETs). Several variables were found to be significantly different between patients in both groups. INETs were more often associated with flushing (44 vs. 14%; P < 0.001) and diarrhea (63 vs. 16%; P < 0.001) and were more often associated with elevation in preoperative serum levels of pancreastatin (88 vs. 42%; P < 0.001), chromogranin A (78 vs. 54%; P = 0.036), and serotonin (90 vs. 43%; P < 0.001). INETs more frequently had vascular invasion on pathology (96 vs. 60%; P < 0.001), and presented more often with nodal and/or distant metastases (77 vs. 37%; P < 0.001). There was no significant difference in overall survival between patients in both groups. CONCLUSION: In this series, patients with INETs presented with a more advanced stage of disease compared with PNETs, had higher preoperative levels of 3 markers, and were more often symptomatic. Despite these factors, there was no significant difference in overall survival between patients with these 2 tumor types.

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-ß 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 Student's 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.

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.

Gene expression accurately distinguishes liver metastases of small bowel and pancreas neuroendocrine tumors.

Small bowel (SBNETs) and pancreatic neuroendocrine tumors (PNETs) often present with liver metastases. Although liver biopsy establishes a neuroendocrine diagnosis, the primary tumor site is frequently unknown without exploratory surgery. Gene expression differences in metastases may distinguish primary SBNETs and PNETs. This study sought to determine expression differences of four genes in neuroendocrine metastases and to create a gene expression algorithm to distinguish the primary site. Nodal and liver metastases from SBNETs and PNETs (n = 136) were collected at surgery under an Institutional Review Board-approved protocol. Quantitative PCR measured expression of bombesin-like receptor-3, opioid receptor kappa-1, oxytocin receptor, and secretin receptor in metastases. Logistic regression models defined an algorithm predicting the primary tumor site. Models were developed on a training set of 21 nodal metastases and performance was validated on an independent set of nodal and liver metastases. Expression of all four genes was significantly different in SBNET compared to PNET metastases. The optimal model employed expression of bombesin-like receptor-3 and opioid receptor kappa-1. When these genes did not amplify, the algorithm used oxytocin receptor and secretin receptor expression, which allowed classification of all 136 metastases with 94.1 % accuracy. In the independent liver metastasis validation set, 52/56 (92.9 %) were correctly classified. Positive predictive values were 92.5 % for SBNETs and 93.8 % for PNETs. This validated algorithm accurately distinguishes SBNET and PNET metastases based on their expression of four genes. High accuracy in liver metastases demonstrates applicability to the clinical setting. Studies assessing this algorithm's utility in prospective clinical decision-making are warranted.

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.

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.

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.