MSH2 and CXCR4 involvement in malignant VIPoma

A 69-year-old man was referred from an outside hospital to our department with masses in the liver and therapy-resistent watery diarrhea. Since 1996 he had suffered from protracted watery diarrhea. At that time the patient received oral rehydration solutions and electrolyte supplementation without regression of the symptoms. In September 1999 ultrasonography was performed and several hepatic masses were detected. Subsequent biopsy of the hepatic masses revealed the diagnosis of metastases due to a malignant VIPoma without evidence of the location of the primary. For further conservative treatment the patient received Sandostatin without regression of the diarrhea. Thus, the patient was administered Rituximab and Roferon additionally, so the progress of the symptoms was impeded. During the course of treatment, minor relapses were compensated using Capecitabine and Cetuximab as additional medication. Regarding side effects of therapy,the patient developed enormous acne. In December 2005 the patient stopped responding to the performed conservative treatment and suffered from massive watery diarrhea (5 to 10 L/day), hypokalemia, achlorhydria, anemia, and severe metabolic acidosis with consecutive acute renal failure and requiring dialysis. Finally the patient was referred from an outside hospital and was admitted to our hospital and department in February 2006. At admission computed tomography (CT) and magnetic resonance imaging (MRI) were performed. In the liver a 4.7 × 3.3 cm mass in segment VII, a 6.6 cm mass in diameter in segment V/VI, and two lesions with 2 cm and 1.7 cm in diameter in segment V were detected. The findings showed an increase in size compared to a previous CT scan performed in July 2005. Since CT and MRI were not able to reveal the location of the primary a PET-CT with MBq 68-Gallium-DOTATOC was conducted. Enhancement of multiple lesions in the right lobe of the liver and a slight enhancement in the caudal pancreas without morphological circumscribable primary tumor were observed (see Figure 1). No infiltration of lymph nodes or other organs was detected. Due to the censorious condition of the patient, at first a tumor-debulking to decrease the amount of VIP causing the symptoms was suggested. Plasma levels of VIP were between 450 and 650 pmol. Accordingly a hemihepatectomy was conducted in March 2006 whereas the surgeon was able to palpate a lesion in the caudal pancreas intraoperatively. Based on the severe condition of the patient, a simultaneous hemihepatectomy and pancreasectomy was not able to be performed at that time without putting patient’s life at risk. A liver weighing 912 g and measuring 18.5 × 17.7 × 8.4 cm was resected. Pathologic examination and immunohistochemistry revealed metastases due to a malignant VIPoma and expression of Chromogranin-A and Synaptophysin. In time the condition of the patient stabilized. The symptoms improved and the plasma VIP decreased (18 pmol). After a reasonable recovery the patient underwent caudal pancreasectomy and splenectomy in January 2007 with successful resection of the primary tumor in sano. A pancreas specimen measuring 9 × 5 × 3 cm was resected. Pathologic examination and immunohistochemistry revealed a pancreatic neuroendocrine tumor confirming a malignant VIPoma with expression of Chromogranin-A and Synaptophysin. The postoperative course was uneventful and the patient did not show any further symptomatic. The follow-up examinations with CT and MRIdid not reveal any recurrence or clinical symptoms, and VIP plasma levels were low (2 pmol).

Histological proven tissue samples (primary pancreatic VIPoma with hepatic metastases and corresponding healthy tissue of the liver and the pancreas) of this patient were investigated. All tumor specimens were collected by the Department of General, Visceral and Transplant Surgery, University of Tübingen, Germany. Specimens were snap frozen in liquid nitrogen and stored at −80°C until use. Each tumor sample was cryosected, stained with hematoxylin and eosin, classified by two experience pathologists, and re-evaluated by an experienced surgical pathologist. To ensure a tumor content >80%, microdissection was performed regarding cancer specimens. The patients provided signed informed consent. The study was approved by the local Ethics Committee.

RNA was extracted using the NucleoSpin RNA II Kit (Macherey-Nagel, Dueren, Germany). The RNA quality and quantity were verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Material and Methods concerning production and analysis of our oligonucleotide microarrays have been described already by Zieker et al. [7, 8]. In short:

Microarray analysis was performed using oligonucleotide microarrays (65mer) produced at the Max Planck Institute, Tübingen, Germany. The arrays contained oligonucleotides for about 900 transcripts, and each oligonucleotide was printed twice. Using 5 oligonucleotide microarrays (including dye-swap) we compared the primary tumor, the metastases, the corresponding healthy tissue of the liver, and the pancreas. Amplification of the sample RNA was performed using Ambion’s Amino AllylMessageAmp™ II aRNA Amplification Kit (Ambion Inc., Austin, TX, USA). Dye-coupling reaction was performed using Amersham CyDye Post-labelling Reactive Dye Pack (GE Healthcare, Buckinghamshire, UK). After an aRNA fragmentation using Ambion’s Fragmentation Reagents (Ambion Inc., Austin, TX, USA), hybridization was carried out at 48°C for 14 h. The slides were scanned in a microarray scanner (Genetix Limited, Hampshire, UK). The photomultiplier tube voltage was set to 100% for both red and green channels. The two resulting green and red images were overlaid using ImaGene 5 (BioDiscovery, Inc., El Segundo, CA, USA). Raw data collection was performed using ImaGene v.5.0. Further statistical and bioinformatic analyses were performed using the R language (http://​www.​r-project.​org) and the ‘limma’ (Linear Models for Microarray) package from the Bioconductor project (http://​www.​bioconductor.​org/​). As a first step in signal extraction, for each channel we used the mean of the pixel distribution for the foreground signal and the median for the background of each spot as estimators of the raw signal values. All spots were used, regardless of their flag status. The data were normalized using loess normalization on the normexp-background corrected expression values, followed by a dye-swap normalization and in-between-array quantile normalization. Both the loess and quantile normalization methods were used as provided in the limma package. On the basis of the M values computed, differentially expressed genes were detected by applying the Welch one-sample t-test as implemented in R.

DNA was extracted from both tissue and patients’ blood using the Gentra-DNA-extraction kit (Biozym) according to the manufacturer’s instructions. The following DNA samples derived from patients’ material were used for microsatellite analysis:

Data derived from these samples were compared to results derived from blood DNA.

Microsatellite analysis was performed applying an extended marker panel for endocrine tumors which was previously established in our department [9]. In brief, markers BAT25, BAT26, D2S123, and D17S250 were amplified in a multiplex PCR (multiplex 1) using the QIAGEN Multiplex PCR Master Mix (Qiagen) under following conditions: final concentration of each forward primer (dye labeled) was 0.2 μM, of each reverse primer 1 μM, DNA concentration was 100 ng. Asymmetric PCR was used to reduce the ratio between primer signal and amplicon signal thereby improving the fluorescence detection threshold. Thermal cycling conditions were 94°C for 5 min (94°C for 30 s, 54°C for 90 s, 72°C for 60 s), 30 cycles, 60°C for 5 min. PCR products were purified using the QIAquick PCR purification kit (QIAGEN).

Microsatellite markers D2S443, D16S752, D21S1436 (multiplex 2) and D1S104, D3S1284 (multiplex 3) were amplified in two multiplex PCR reactions under following conditions: 200 ng DNA, 1 × PCR buffer, 0.08 mM dNTPs each, 2 mM MgCl₂, 1 U Taq, 0.8 μM primer each, ad 50 μL H2O; 94°C for 2 min (94°C for 40 s, 53°C (multiplex 2), and 61°C (multiplex 3) for 40 s, respectively, 72°C for 1.30 min), 40 cycles, 72°C for 2 min. PCR products derived from the same patients sample were combined and purified using the PCR-purification kit (Qiagen). LOH analysis was performed on an automated capillary sequencer CEQ 8000 (Beckman Coulter) and evaluated with the CEQ 8000 Fragment analysis software (Beckman Coulter).

The tumor was considered to be positive for LOH if the allele peak ratio was ≤0.7, in analogy with allelic signal reduction of at least 30% [10]. Homozygous peaks were classified as not informative and were not evaluated in LOH statistics.

A Medline search was conducted for the term ‘VIPoma’to December 2011. A total of 489 publications were retrieved. All English written publications reporting on oncogenesis were included. Articles dealing with clinical findings, diagnostics, therapy options, and clinical outcomes were excluded.

Gene expression analysis was performed using a custom-designed oligo microarray. We found a number of genes that were up- or downregulated comparing the primary tumor, the metastases, the corresponding healthy tissue of the liver, and the pancreas.

Concerning the expression ratio we found 36 genes to be differentially expressed (>log2). Eighteen genes upregulated and 18 genes were downregulated. Comparing the primary pancreatic tumor against its corresponding healthy tissue six genes were upregulated and eight genes were downregulated (>log2) (See Table 1). Five genes were upregulated and five genes were downregulated comparing the primary pancreatic tumor with the hepatic metastases (>log2) (see Table 2). Regarding the expression between the hepatic metastases and its corresponding healthy liver tissue, seven genes were upregulated and five genes were downregulated (>log2) (see Table 3).

Regarding the gene expression of MSH2 and CXCR4 we were able to detect a downregulation (−2.80-fold) of MSH2 in hepatic metastases and (−2.63-fold) of MSH2 in the primary pancreatic tumor compared to both corresponding healthy tissue. Comparing the hepatic metastases against the primary pancreatic tumor an upregulation (10.41-fold) was detected.

Ten microsatellite samples were used for MSI and LOH detection (see Table 4).

All experiments were repeated at least twice. LOH events were detected in several patient samples (see Figure 2). Altogether, allele peak ratios for LOH calculation varied between 0.60 and 0.12.

Sixty-three percent of markers analyzed showed LOH. Nine percent of markers were not informative for LOH calculation and 27% of markers showed microsatellite stability. LOH was detected in primary tumor DNA as well as in liver metastasis. Interestingly, two markers (D5S326 and D21S1436) revealed LOH in normal liver tissue. All tumor DNAs analyzed showed LOH for marker D2S123 which is located close to the MSH2.