Christian N. Arnold

Placebo-Controlled, Double-Blind, Prospective, Randomized Study on the Effect of Octreotide LAR in the Control of Tumor Growth in Patients With Metastatic Neuroendocrine Midgut Tumors: A Report From the PROMID Study Group

4508 Background: Octreotide is currently used for the control of symptoms in patients with gastroenteropancreatic neuroendocrine tumors (NETs). However, the ability of long-acting somatostatin analogues to control the growth of well-differentiated metastatic NETs is a matter of debate. The analysis of the first randomized, double-blind, placebo-controlled, multicenter, phase IIIb study of octreotide LAR in patients with metastatic NETs of the midgut is presented. METHODS Treatment-naïve patients with histologically confirmed locally inoperable or metastasized well-differentiated NETs and a Karnofsky index >60 were randomized to receive either octreotide LAR 30 mg/month (mo) or placebo for 18 mos, or until tumor progression or death. The primary endpoint was median time to tumor progression. Secondary endpoints included objective tumor response rate (WHO criteria), measured every 3 mos, as well as symptom control and overall survival. This was a planned interim analysis using the Lan-DeMets error spending approach. RESULTS Eighty-five patients (n=43 octreotide LAR; n=42 placebo) have been enrolled to date and data from 67 patients with tumor progressions and 16 deaths (n=7 octreotide LAR; n=9 placebo) are included here. Median time to tumor progression in the octreotide LAR and placebo groups were 14.3 mos and 6 mos, respectively (HR: 0.34; 95% CI: 0.20-0.59; P=0.000072). After 6 mos of treatment, stable disease was seen in 67% and 37.2% of patients treated with octreotide LAR and placebo, respectively. Due to the low number of observed deaths, median survival time could not be estimated. CONCLUSIONS Octreotide LAR significantly lengthens median time to tumor progression compared with placebo in patients with metastatic NETs of the midgut. Patients treated with octreotide LAR had a 66% risk reduction of tumor progression compared with patients receiving placebo. Octreotide LAR demonstrates substantial tumor control and shows a more favorable antiproliferative response than placebo as two-thirds of patients treated with octreotide LAR achieved stable disease at 6 mos. [Table: see text].PURPOSE Somatostatin analogs are indicated for symptom control in patients with gastroenteropancreatic neuroendocrine tumors (NETs). The ability of somatostatin analogs to control the growth of well-differentiated metastatic NETs is a matter of debate. We performed a placebo-controlled, double-blind, phase IIIB study in patients with well-differentiated metastatic midgut NETs. The hypothesis was that octreotide LAR prolongs time to tumor progression and survival. PATIENTS AND METHODS Treatment-naive patients were randomly assigned to either placebo or octreotide LAR 30 mg intramuscularly in monthly intervals until tumor progression or death. The primary efficacy end point was time to tumor progression. Secondary end points were survival time and tumor response. This report is based on 67 tumor progressions and 16 observed deaths in 85 patients at the time of the planned interim analysis. RESULTS Median time to tumor progression in the octreotide LAR and placebo groups was 14.3 and 6 months, respectively (hazard ratio [HR] = 0.34; 95% CI, 0.20 to 0.59; P = .000072). After 6 months of treatment, stable disease was observed in 66.7% of patients in the octreotide LAR group and 37.2% of patients in the placebo group. Functionally active and inactive tumors responded similarly. The most favorable effect was observed in patients with low hepatic tumor load and resected primary tumor. Seven and nine deaths were observed in the octreotide LAR and placebo groups, respectively. The HR for overall survival was 0.81 (95% CI, 0.30 to 2.18). CONCLUSION Octreotide LAR significantly lengthens time to tumor progression compared with placebo in patients with functionally active and inactive metastatic midgut NETs. Because of the low number of observed deaths, survival analysis was not confirmatory.

Octreotide Versus Octreotide Plus Interferon-Alpha in Endocrine Gastroenteropancreatic Tumors: A Randomized Trial

BACKGROUND & AIMS The effect of octreotide plus interferon-alpha versus octreotide monotherapy on the primary study end points of time to treatment failure (progression, death, stop of study treatment) and long-term survival was investigated in patients with progressive metastatic neuroendocrine foregut (mainly pancreatic) and midgut tumors. METHODS One hundred nine of 125 registered patients were randomized starting in January 1995, and 105 patients (51 monotherapy, 54 combination treatment) were finally analyzed in March 2000. Tumor growth was assessed at 3-month intervals by computed tomography or magnetic resonance imaging. Long-term survival was studied up to April 2004 in all analyzed patients and in 9 patients not randomized because of stable disease. RESULTS Partial tumor regression occurred in 2.9%, 1.9%, and 5.7% and stabilization of tumor growth in 44.8%, 27.6%, and 15.2% at 3, 6, and 12 months, respectively, with no significant differences between both treatment arms. In March 2000, 9.5% of patients were in treatment. Time to treatment failure and long-term survival did not differ significantly between the 2 groups, with a median survival of 32 and 54 months for the octreotide and the combination groups, respectively. Survival was longer in patients not randomized because of stable disease (median, 68 months) and in those with low nuclear Ki-67. A trend toward longer survival was shown for patients with slow spontaneous tumor growth before randomization. Patients responding to treatment lived longer than unresponsive patients. CONCLUSIONS Combination treatment was not superior to monotherapy concerning progression-free and long-term survival. Patients responding to treatment and those with slow spontaneous tumor growth had a survival advantage.

Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines.

Loss of DNA mismatch repair (MMR) occurs in 10–15% of sporadic colorectal cancer, is usually caused by hMLH1 hypermethylation, and has been shown to confer resistance to various chemotherapeutic reagents, including 5‐fluorouracil (5‐FU). We tested the hypothesis that demethylation of the hMLH1 promoter in hypermethylated colorectal cancer cells would restore MMR proficiency and drug sensitivity to 5‐FU. We used the MMR‐deficient cell lines SW48, HCT116, HCT116+chr2 and the ‐proficient cell line HCT116+chr3. After treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5 aza‐dC), hMLH1 mRNA and protein expression were determined by RT‐PCR and immunoblots. The methylation status for hMLH1 was investigated by methylation‐specific PCR. Cells were subsequently treated with 5‐FU and the growth characteristics ascertained by clonogenic assays. hMLH1 hypermethylation was reverted in SW48 cells 24 hr after treatment with 5 aza‐dC and was accompanied by hMLH1 mRNA and protein reexpression. While 5 aza‐dC alone did not affect the growth of SW48 cells, all other cell lines responded with a pronounced growth inhibition. 5‐FU treatment strongly reduced the colony formation of HCT116+chr3 cells. These effects were significantly less in the MMR‐deficient cells. Combined treatment of SW48 cells resulted in a similar growth pattern as seen in 5‐FU only treated HCT116+chr3 cells. We demonstrate that in vitro resistance to 5‐FU can be overcome by reexpression of hMLH1 protein through 5 aza‐dC‐induced demethylation in hypermethylated cell lines. Induction of the expression of methylated tumor suppressor or MMR genes could have a significant impact on the development of future chemotherapy strategies.

EPIGENETIC INACTIVATION OF RUNX3 IN MICROSATELLITE UNSTABLE SPORADIC COLON CANCERS

Runt domain transcription factors are important targets of TGF‐β superfamily proteins and play a crucial role in mammalian development. Three mammalian runt‐related genes, RUNX1, RUNX2 and RUNX3, have been described. RUNX3 has been shown to be a putative tumor suppressor gene localized to chromosome 1p36, a region showing frequent loss of heterozygosity events in colon, gastric, breast and ovarian cancers. Because of the important role of TGF‐β signaling in the human colon, we hypothesized that RUNX3 may serve as a key tumor suppressor in human colon cancers and colon cancer‐derived cell lines. We examined RUNX3 expression and the frequency of RUNX3 promoter hypermethylation in 17 colon cancer cell lines and 91 sporadic colorectal cancers. Semiquantitative analysis of RUNX3 transcripts was performed by RT‐PCR and de novo methylation of the RUNX3 promoter was studied by a methylation‐specific PCR (MSP) assay. Nineteen of 91 informative tumors (21%) and 11 of 17 (65%) colon cancer cell lines exhibited hypermethylation of the RUNX3 promoter. Interestingly, RUNX3 promoter hypermethylation was more common in tumors exhibiting high frequency of microsatellite instability (MSI‐H) (33% of MSI‐H vs. 12% of MSI‐L/MSS tumors; p = 0.012). Hypermethylation of the RUNX3 promoter correlated with loss of mRNA transcripts in all cell lines. RUNX3 promoter methylation was reversed and its expression restored in SW48 and HCT15 colon cancer cells after treatment with the demethylating agent 5‐aza‐2′‐deoxycytidine, indicating that loss of expression is caused by epigenetic inactivation in colon carcinogenesis. This is the first demonstration of frequent de novo hypermethylation of the RUNX3 promoter in sporadic colon cancers. The significant association of RUNX3 promoter hypermethylation with MSI‐H colon cancers suggests that RUNX3 is a novel target of methylation, along with the hMLH1 gene, in the evolution of MSI‐H colorectal cancers.

APC promoter hypermethylation contributes to the loss of APC expression in colorectal cancers with allelic loss on 5q1

Introduction: Germ-line mutations of the APC gene are associated with familial adenomatous polyposis, and somatic mutations occur frequently in sporadic colorectal cancer. However, to abrogate APC function, both alleles must be inactivated. Recently, it has been demonstrated that epigenetic modification of the APC promoter influences APC silencing. Here we examined the influence of APC methylation on APC expression in tumors with and without LOH at the APC locus. Material and Methods: 137 sporadic colorectal cancer specimens were investigated for LOH at the 5q locus. The methylation status of the APC promoter was determined by methylation-specific PCR. APC expression was performed by immunohistochemistry. Results: APC expression was reduced or lost in 110 of 137 (80%) tumors and LOH at 5q was found in 13 of 132 (10%) tumors. There was no difference in 5q LOH between tumors with or without intact APC expression. Vice versa, there was no difference in the APC expression in tumors with 5q LOH. Aberrant APC promoter methylation was detected in 33 of 118 (28%) tumors investigated. Of the tumors with 5q LOH for which methylation data were available, 4 of 11 (36%) were methylated versus 28 of 105 (27%) of those without LOH. No difference in methylation was observed in tumors without 5q LOH and normal APC expression and those without 5q LOH and reduced or missing APC expression. Importantly, none of the tumors with 5q LOH and normal APC staining were aberrantly methylated, whereas 50% of the cancers with LOH at 5q and reduced or absent staining were hypermethylated. Conclusions: This report suggests that tumors with 5q LOH and reduced APC expression are more frequently hypermethylated at the APC promoter compared to those tumors with 5q LOH and normal APC expression. The association among APC promoter methylation status, 5q LOH, and reduced or lost APC expression suggests that de novo methylation plays an important role as a “second hit” in silencing APC expression in colorectal neoplasia.

Analysis of molecular pathways in sporadic neuroendocrine tumors of the gastro-entero-pancreatic system

Little is known about the molecular pathogenesis of neuroendocrine tumors (NET) of the gastro‐entero‐pancreatic (GEP) system. We analyzed genetic and epigenetic alterations as well as the CpG island methylator phenotype (CIMP). The study comprised 118 well‐differentiated fore‐ and mid‐gut GEP‐NET from 71 patients. In addition to loss of heterozygosity (LOH), microsatellite instability (MSI) and the methylation status of various tumor associated genes were examined. The expression profile of p16, APC and MENIN was investigated by immunohistochemistry. None of the tumors was highly microsatellite unstable, LOH was found in 22.2%. Significant differences in promoter hypermethylation were identified in the RUNX3 and the O6‐MGMT genes. We found a significant loss of p16 expression in insulinomas (p = 0.05) and functional NET (p = 0.01), respectively. APC was expressed less in gastrinomas (p = 0.01) and functional GEP‐NET (p = 0.05) vs. nonfunctional tumors. MENIN expression was reduced in pancreatic vs. extrapancreatic NET (p = 0.008) and in insulinomas vs. nonfunctional GEP‐NET (p = 0.019) and NET associated with the carcinoid syndrome (p = 0.029). Further CIMP and a Ki‐67 index >10% showed a close correlation. Outcome analysis of 19 patients showed a better survival for CIMP‐negative patients. The analyses identified significant genetic and epigenetic alterations in well‐differentiated fore‐ and mid‐gut NET. CIMP, similar to Ki‐67, might turn out to be of prognostic relevance.

Nuclear Survivin Is a Powerful Novel Prognostic Marker in Gastroenteropancreatic Neuroendocrine Tumor Disease

Gastroenteropancreatic neuroendocrine tumors represent a heterogeneous tumor entity. The growth pattern ranges from very slowly to fast growing, aggressive types of tumors. Survivin, a member of the family of apoptosis inhibitors, is a bifunctional protein that suppresses apoptosis and regulates cell division. In this study we determined the prognostic value of survivin in this tumor entity. Tumor specimens from 104 patients (38 foregut, 53 midgut, 13 hindgut) were studied immunohistochemically for cytoplasmic and nuclear survivin expression as well as for ki-67 antigen expression. 5-year-follow-up was complete in 89 patients. 29 patients with localized, well-differentiated gastroenteropancreatic neuroendocrine tumors (WDET, WHO class 1) had been curatively treated by surgical or endoscopic tumor resection. 50 patients suffered from well-differentiated endocrine carcinomas (WDEC, WHO class 2), 10 patients were diagnosed with poorly differentiated neuroendocrine carcinomas (PDEC, WHO class 3). Survivin expression was correlated with survival for the 50 patients with metastatic WDEC disease. All 29 WDETs were negative for nuclear survivin, whereas all 10 PDECs stained positive for nuclear survivin. In the 50 patients with metastatic WDECs, 5/50 (10%) tumors were nuclear survivin positive. Those 5 patients had a statistically significant worse prognosis (survival of 41 vs. 103 months, p = 0.01). ki-67 was not a prognostic factor in this subgroup of patients. Nuclear survivin expression thus appears to be upregulated during progression of gastroenteropancreatic neuroendocrine tumors. The analysis of nuclear survivin expression identifies subgroups in metastatic disease (WHO class 2) with good (survivin–) or with less favorable prognosis (survivin+). We propose that the determination of nuclear survivin expression could be used to individualize therapeutic strategies in this tumor entity in the future.

Acute hemoperitoneum after large-volume paracentesis.

Hemoperitoneum resulting from rupture of mesenteric varices is a rare complication of portal hypertension with a high mortality of up to 70%. This case report describes the symptoms, clinical course, and treatment of 4 patients with acute hemoperitoneum caused by mesenteric variceal bleeding after large-volume paracentesis. Abdominal pain and/or hemorrhagic shock developed in 4 patients (age, 48-68 years), admitted for refractory ascites, 3 hours to 4 days after 1-4 large-volume paracenteses (> 4000 mL). Duplex sonography, performed in 3 of the 4 patients before onset of bleeding, showed retrograde flow in the mesenteric veins, suggesting large-caliber mesenteric collateralization. Treatment consisted of surgical ligation followed by transjugular intrahepatic portosystemic shunt (TIPS) (2 patients) and emergency TIPS with embolization of the bleeding vessel (1 patient). One patient died before any intervention could be initiated. In these 4 patients, the concurrence of large-volume paracentesis and hemoperitoneum suggests their causal relationship. The mechanism may be a sudden reduction in intraperitoneal pressure increasing the pressure gradient across the wall of the mesenteric varices, resulting in rupture and bleeding. The awareness of this complication may accelerate the diagnostic process and treatment.

Tuberculous colitis mimicking Crohn's disease.

Intestinal tuberculosis is a rare disease in western countries and may mimic a variety of gastrointestinal disorders. Here, we report the case of a 63-yr-old patient who presented with profuse bleeding from a deep rectal ulcer. Similar lesions were found in different parts of the colon. Multilocular colorectal carcinoma was suspected based on the macroscopic appearance. Histology, however, suggested Crohns disease. Intestinal tuberculosis was initially ruled out by negative staining for acid-fast bacilli, mycobacterial culture, and polymerase chain reaction analysis. A treatment for Crohns disease was started. Endoscopic reexamination revealed progressive disease with extensive ulcerations of the terminal ileum. Histopathological examination then revealed acid-fast bacilli in the colonic mucosa typical for mycobacterium tuberculosis infection. This case emphasizes the need to include intestinal tuberculosis in the initial differential diagnosis of ulcerative colorectal lesions also in the western population.

Loss of Nuclear p27 Expression and Its Prognostic Role in Relation to Cyclin E and p53 Mutation in Gastroenteropancreatic Neuroendocrine Tumors

Purpose: Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are classified by the WHO, yet its prognostic value needs to be confirmed. Therefore, we aimed to determine the prognostic role of cell cycle key regulatory genes p53, p27kip1 (p27), and cyclin E in this tumor entity. Experimental Design: Tumor specimen from 89 patients with a complete follow-up were studied immunohistochemically for p27 and cyclin E expression and for p53 mutations. The functional relevance of p27 was evaluated in the neuroendocrine cell lines BON1 (human) and INS1 (rat) by the use of small interfering RNA. Results: Twenty-six of 29 benign, well-differentiated endocrine tumors (WHO class 1) showed a high expression (>50%) of p27, whereas all 10 poorly differentiated endocrine carcinomas (WHO class 3) displayed a low expression of p27. Metastatic well-differentiated endocrine carcinomas (WHO class 2) showed a low p27 expression in 20 of 50 (40%) patients, which conferred a poor prognosis (median survival, 57 versus 140 months; P = 0.037). This prognostic dichotomy was improved by the use of a combination of p27 and cyclin E (high cyclin E/low p27 versus low cyclin E/high p27: median survival 53 months versus not reached; P = 0.0044). p53 mutations were rare (1 of 10 poorly differentiated endocrine carcinomas). Conclusions: Loss of p27 and overexpression of cyclin E play a critical role in the aggressiveness of gastroenteropancreatic neuroendocrine tumors. This coincides with increased cell cycle progression. We propose a discussion whether to incorporate the immunohistochemical expression of p27 into a revised classification to individualize therapeutic strategies in this tumor entity.