Christina L. Costantino

Expression of indoleamine 2,3-dioxygenase in metastatic malignant melanoma recruits regulatory T cells to avoid immune detection and affects survival.

The mechanism by which malignant melanoma (MM) cells survive in lymph nodes is poorly understood. One possible mechanism by which MM cells can escape immune surveillance is through upregulation of immunomodulatory enzymes such as indoleamine 2,3-dioxygenase (IDO). In this study, 25 cases of MM lymph node metastases from patients with long and short survival were evaluated for expression of IDO and the number of Forkhead box p3 (FOXP3)-expressing regulatory T cells. Moderate to strong cytoplasmic IDO expression was present in all (15/15) MM lymph node metastases in patients with poor survival. Eight of 10 patients with metastatic MM and long survival were negative or only weakly positive for IDO. Upregulation of IDO in metastatic MM cells was associated with an increased number of regulatory T cells (Tregs). There was a statistically significant association between shorter survival and both a stronger IDO expression (p=0.0019) and a higher number of FOXP3 expressing Tregs (p

Cytoplasmic accumulation of the RNA binding protein HuR is central to tamoxifen resistance in estrogen receptor positive breast cancer cells

With prolonged exposure, a majority of estrogen receptor positive cancers develop resistance to tamoxifen and subsequent therapies including selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs). While much is known about overexpression of key growth promoting receptors including EGF, erbB2/Her2 and IGF receptors and subsequent activation of MAPK signaling associated with resistance, the underlying mechanism in the development of resistance still remains unknown. We found that inhibition of JNK, a member of the MAPK family, decreases cytoplasmic accumulation of the RNA binding protein HuR. This data combined with previous reports that erbB2/Her2 and IGF-IR signals through JNK, led us to hypothesize that cytoplasmic accumulation of HuR may be a key contributor to development of tamoxifen resistance. Therefore, we tested the effect of HuR expression on tamoxifen responsiveness in both tamoxifen sensitive MCF7 and tamoxifen resistant BT474 cell lines. We found that decreasing the cytoplasmic HuR levels in the cells increases tamoxifen responsiveness in both cell lines. Conversely, the overexpression of HuR establishes tamoxifen resistance in MCF7 cells. Therefore, our data indicate that HuR is central to tamoxifen resistance. Interestingly, we found that acute exposure (24 and 48 h) of MCF7 cells to tamoxifen increased cytoplasmic levels of HuR and concomitantly it’s ligand pp32, suggesting a novel molecular mechanism of resistance and acute response to tamoxifen through increased stability of mRNA transcripts that code for drug-resistant transcripts. Indeed, evaluation of primary breast tumors revealed a correlation between tumor grade, tamoxifen responsiveness and cytoplasmic HuR status. Therefore, inhibition of the cytoplasmic accumulation of HuR concomitantly with the administration of current therapeutics may be a successful treatment strategy. Our data describe a novel mechanism for the development of tamoxifen resistance and is the first study to identify an RNA binding protein as a key mediator of resistance in breast cancer cells

Nanoparticulate delivery of diphtheria toxin DNA effectively kills Mesothelin expressing pancreatic cancer cells

Pancreatic cancer is the fourth leading cause of cancer-related deaths in this country, and there is currently no effective targeted treatment for this deadly disease. A dire need exists to rapidly translate our molecular understanding of this devastating disease into effective, novel therapeutic options. Mesothelin is a candidate target protein shown by a number of laboratories to be specifically overexpressed in pancreatic cancers and not in the adjacent normal tissue. Translational investigations have shown promising results using this molecule as a therapeutic target (e.g., vaccine strategies). In addition, the mesothelin promoter has been cloned and dissected and can therefore be used as a vehicle for regulating expression of DNA sequences. Using a novel, proven, biodegradable nanoparticulate system, we sought to target mesothelin-expressing pancreatic cancer cells with a potent suicide gene, diphtheria toxin-A (DT-A). We first confirmed reports that a majority of pancreatic cancer cell lines and resected pancreatic ductal adenocarcinoma specimens overexpressed mesothelin at the mRNA and protein levels. High mesothelin-expressing pancreatic cancer cell lines produced more luciferase than cell lines with undetectable mesothelin expression when transfected with a luciferase sequence under the regulation of the mesothelin promoter. We achieved dramatic inhibition of protein translation (>95%) in mesothelin-expressing pancreatic cancer cell lines when DT-A DNA, driven by the mesothelin promoter, was delivered to pancreatic cancer cells. We show that this inhibition effectively targets the death of pancreatic cancer cells that overexpress mesothelin. The work presented here provides evidence that this strategy will work in pre-clinical mouse pancreatic cancer models, and suggests that such a strategy will work in the clinical setting against the majority of pancreatic tumors, most of which overexpress mesothelin.

Adenosquamous carcinoma of the pancreas harbors KRAS2, DPC4 and TP53 molecular alterations similar to pancreatic ductal adenocarcinoma

Adenosquamous carcinoma of the pancreas is one of the most aggressive forms of pancreatic cancer. Molecular characterizations of this rare tumor subtype are sparse. Understanding the common molecular and pathologic features of pancreatic adenosquamous carcinomas could provide critical information for identifying therapeutic targets. Herein, we analyzed the pathologic and molecular features of our series of eight pancreatic adenosquamous carcinomas. We found KRAS2 gene mutations at codon 12 in all eight cases. All the cases showed loss of p16 protein. In three of these cases the loss was attributed to an exon 2 homozygous deletion in the p16/CDKN2a gene. The majority of the cases had loss of Dpc4 protein and strong nuclear p53 positivity, similar to the molecular signature found in pancreatic ductal adenocarcinoma. We found that E-cadherin was either lost or reduced in all cases and that epidermal growth factor receptor was overexpressed in all cases. The squamous component was positive for p63 staining and thus p63 labeling was helpful in identifying squamous differentiation in adenosquamous carcinomas with an acantholytic growth pattern. In summary, although pancreatic adenosquamous carcinoma and ductal adenocarcinoma have overlapping pathologic and molecular characteristics, there are distinct differences that may be helpful in diagnostic and therapeutic strategies.

Limits to Thymidylate Synthase and TP53 Genes as Predictive Determinants for Fluoropyrimidine Sensitivity and Further Evidence for RNA-Based Toxicity as a Major Influence

The major determinants of 5-flurouracil (5-FU) response would seem, based on accumulated literature, to be thymidylate synthase (TYMS, TS) expression levels, TS gene modifications, and TP53 status. We tested 5-FU sensitivity in yeast and human cancer cell models in which TS or TP53 alleles and expression were varied. Polymorphic TS tandem repeat status, TS expression levels reported, TS intragenic mutations, and TP53 status in outbred and experimental cancer cell lines did not predict 5-FU sensitivity or resistance. Novel observations included a dose-resistant persistence of unbound TS protein in many cancers and, upon 5-FU treatment of the colon cancer cell line, HCT116, evidence of allelic switching favoring transcripts of the mutant TS allele. The reported alleles having an intragenic mutation could not be causally associated with major degrees of 5-FU sensitivity. In yeast, TS protein was altered upon treatment with FdUMP, but 5-FU toxicity seemed to be largely RNA-based, being rescued by uridine rather than by thymidine. Cancer cell lines were also rescued from 5-FU toxicity with uridine rather than thymidine. Additionally, a TS (CDC21) knockout yeast strain, obviating any potential role for TS protein as a target, was hypersensitive to 5-FU. When denatured proteins from cancer cells treated with radiolabeled 5-FU were labeled, species with alternative molecular weights other than TS were visualized, providing further evidence for alternative 5-FU protein targets. These data emphasize that TS and TP53 status do not consistently explain the variance in responses of fluoropyrimidine-treated cancer cells, in part due to RNA-based toxicity.

Naturally occurring K vitamins inhibit pancreatic cancer cell survival through a caspase-dependent pathway.

Background and Aims:  Available medical therapies against pancreatic cancer are largely ineffective and have many side‐effects. Physiologically, vitamins K1 and K2 (VK) act as co‐factors for γ‐carboxylation of prothrombin and other coagulation factors. In previous studies, VK analogs have been found to have potent negative effects on the survival of various cancer cells. We hypothesized that the well‐tolerated and naturally occurring VK1 and VK2 may be used to inhibit pancreatic cancer cell survival.

Primary Hepatic Gastrinoma.

A 37-year-old female was admitted to our hospital for evaluation and management of a 5.5-cm heterogeneously enhancing mass in segment IV of her liver, which had been disclosed by an abdominopelvic CT scan obtained in the work-up of severe abdominal pain. She reported a 2-year history of chronic, watery diarrhea, which was judged by the admitting hospitalist to be Bparaneoplastic.^ Following percutaneous liver biopsy, the patient was discharged home. The patient presented again 3 days later with diffuse peritonitis, tachycardia, and hypotension. She was taken to the operating room for exploratory laparotomy, at which time three full-thickness perforations were found in the proximal jejunum (Fig. 1). A 30-cm section of the jejunum was resected, and the patient was admitted to the surgical ICU. At re-exploration 36 h later, multiple full-thickness perforations were found in the transverse duodenum. Accordingly, the transverse duodenum was resected, and the GI tract was reconstructed by means of a retrocolic, posterior gastrojejunostomy. Forty-eight hours following the second operation, the patient’s serum gastrin concentration returned elevated at 1331 pg/mL (normal, fasting value <100 pg/mL). Analysis of the percutaneous liver biopsy specimen disclosed a well-differentiated neuroendocrine tumor, consistent with a gastrinoma. No neoplasm was found in either of the two small bowel specimens. Following an uncomplicated post-operative course, the patient was discharged home on high-dose acid suppression therapy. Over the course of the ensuing several months, the patient was evaluated for additional foci of gastrinoma with somatostatin receptor scintigraphy and endoscopic ultrasonography. Neither study identified the presence of extrahepatic disease. Testing for a mutation in the MEN-1 gene was negative. The patient was taken back to the operating room, approximately 4 months from the time of her initial presentation and underwent an extended left hepatectomy, portal lymph node dissection, and exploratory duodenotomy. The primary hepatic gastrinoma lesion was resected with negative margins (Fig. 2); no additional lesion was found in the examined duodenum or pancreas. At the time of discharge on post-operative day no. 6, the patient’s serum gastrin concentration was normal at 35 pg/mL. Six months post-hepatectomy, the patient’s serum gastrin concentration was still normal at 37 pg/mL. Surveillance abdominopelvic CT and liver MR scans, obtained 10 months post-hepatectomy, were negative for any evidence of recurrent or metastatic gastrinoma. At her most recent clinic visit, 10 months post-hepatectomy, the patient was noted to be looking and feeling well. * Richard A. Hodin rhodin@mgh.harvard.edu

Subglottic Stenosis in Granulomatosis With Polyangiitis: The Role of Laryngotracheal Resection

BACKGROUND Granulomatosis with polyangiitis (GPA) is associated with development of subglottic stenosis in about one-fourth of all patients. Although endoscopic management is the primary treatment method for tracheobronchial stenosis, some patients have refractory disease, and tracheostomy is required. It is unclear if laryngotracheal resection and reconstruction (LTRR) can be safely performed in patients with GPA. METHODS A retrospective review was performed of 11 patients with GPA undergoing LTRR. RESULTS Eleven female patients with GPA and a median age of 47 years underwent LTRR. Six patients were diagnosed with GPA after LTRR and had not received any induction immunosuppression regimen. Five patients had received induction immunosuppression regimen and were in clinical remission before LTRR. LTRR was performed with a protective tracheostomy in 3 patients, which was eventually removed in all. There were no major complications and no postoperative deaths. One patient (9%) failed surgical management and had replacement of a permanent tracheostomy 4 months after LTRR. Six patients (55%) required additional tracheal dilations after LTRR. Ten patients (91%) had durable control of symptoms and freedom from tracheostomy with a median follow-up of 9.7 years. Two patients (18%) experienced subsequent lower airway stenoses. CONCLUSIONS Surgical treatment of subglottic stenosis in highly selected patients with GPA is effective and associated with minimal morbidity. Although long-term outcomes are encouraging, additional procedures may be necessary, and patients are at risk of experiencing lower airway disease.

Pancreatic Neuroendocrine Tumor in Tail of Pancreas with Splenic Vein Thrombus and Sinistral Portal Hypertension

Pancreatic neuroendocrine tumors (PNETs) originate from the pancreatic islet cells. They are rare and account for approximately 2–4% of pancreatic neoplasms. Approximately one-quarter of PNETs are nonfunctional, in that they do not cause a syndrome related to hormonal hypersecretion. As a result, they may be diagnosed at a later stage and present with symptoms of mass compression. Tumors in the tail of the pancreas can cause splenic vein thrombosis, which complicates pancreatic neoplasms in 7% to 10% of patients. Gastric varices can form as a result secondary to sinistral portal hypertension. Patients are at risk for gastrointestinal hemorrhage. Patients should undergo distal pancreatectomy and splenectomy for oncologic resection and decompression of the left-sided portal hypertension.

Idiopathic laryngotracheal stenosis

Idiopathic laryngotracheal stenosis (ILTS) is a rare inflammatory disease of unknown etiology. Infectious, traumatic and immunologic processes must first be excluded. The majority of patients affected are female who present with progressive symptoms of upper airway obstruction, which can extend over a number of years. ILTS is characterized by short segment, circumferential stenotic lesions, located particularly at the level of the cricoid. Bronchoscopic evaluation is essential for establishing the diagnosis and operative planning. Various temporizing interventions have historically been utilized, including dilation and laser ablation, for symptomatic management. However these interventions have demonstrated diminishing returns and poor long-term outcomes. Patients with ILTS should be considered early for definitive surgical intervention to minimize complications and optimize outcomes. Laryngotracheal resection and reconstruction is a viable intervention, which has demonstrated good long-term results and low recurrence rates for this patient population.