André Barghorn

Genetic Differences in Endocrine Pancreatic Tumor Subtypes Detected by Comparative Genomic Hybridization

The molecular pathogenesis as well as histogenesis of endocrine pancreatic tumors (EPTs) is not well understood, and the clinical behavior of EPTs is difficult to predict using current morphological criteria. Thus, more accurate markers of risk and better understanding of tumor initiation and progression are needed to allow a precise classification of EPTs. We have studied 44 benign and malignant EPTs by comparative genomic hybridization to correlate the overall number of genetic alterations with clinical and histopathological parameters and to identify chromosomal regions which might harbor genes involved in EPT pathogenesis and progression. Aberrations were found in 36 EPTs, and chromosomal losses (mean, 5.3) were slightly more frequent than gains (mean, 4. 6). The most frequent losses involved Y (45% of male EPTs), 6q (39%), 11q (36%), 3p, 3q, 11p (each 30%), 6p (27%), and 10q and Xq (each 25%), whereas most common gains included 7q (43%), 17q (41%), 5q and 14q (each 32%), 7p, 9q, 17p, 20q (each 27%), and 12q and Xp (each 25%). A correlation was found between the total number of genetic changes per tumor and both tumor size and disease stage. In particular, losses of 3p and 6 and gains of 14q and Xq were found to be associated with metastatic disease. Furthermore, characteristic patterns of genetic changes were found in the various EPT subtypes, eg, 6q loss in malignant insulinomas, indicating that these groups might evolve along genetically different pathways. The highlighted genetic aberrations, including the newly found involvement of 6q losses and sex chromosome alterations, should stimulate the further analysis of these chromosomal regions, which may lead to the discovery of novel genes important in the tumorigenesis and evolution of EPTs.

WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endocrine tumors.

Background It is difficult to predict the biologic behavior of pancreatic endocrine tumors in absence of metastases or invasion into adjacent organs. The World Health Organization (WHO) has proposed in 2004 size, angioinvasion, mitotic activity, and MIB1 proliferation index as prognostic criteria. Our aim was to test retrospectively the predictive value of these 2004 WHO criteria and of CK19, CD99, COX2, and p27 immunohistochemistry in a large series of patients with long-term follow-up. Design The histology of 216 pancreatic endocrine tumor specimens was reviewed and the tumors were reclassified according to the 2004 WHO classification. The prognostic value of the WHO classification and the histopathologic criteria necrosis and nodular fibrosis was tested in 113 patients. A tissue microarray was constructed for immunohistochemical staining. The staining results were scored quantitatively for MIB1 and semiquantitatively for CK19, COX2, p27, and CD99. The prognostic value of these markers was tested in 93 patients. Results The stratification of the patients into 4 risk groups according to the 2004 WHO classification was reliable with regard to both time span to relapse and tumor-specific death. In a multivariate analysis, the CK19 status was shown to be independent of the WHO criteria. By contrast, the prognostic significance of COX2, p27, and CD99 could not be confirmed. Conclusions The 2004 WHO classification with 4 risk groups is very reliable for predicting both disease-free survival and the time span until tumor-specific death. CK19 staining is a potential additional prognostic marker independent from the WHO criteria for pancreatic endocrine tumors.

Putative Tumor Suppressor Loci at 6q22 and 6q23-q24 Are Involved in the Malignant Progression of Sporadic Endocrine Pancreatic Tumors

Our previous comparative genomic hybridization study on sporadic endocrine pancreatic tumors (EPTs) revealed frequent losses on chromosomes 11q, 3p, and 6q. The aim of this study was to evaluate the importance of 6q losses in the oncogenesis of sporadic EPTs and to narrow down the smallest regions of allelic deletion. A multimodal approach combining polymerase chain reaction-based allelotyping, double-target fluorescence in situ hybridization, and comparative genomic hybridization was used in a collection of 109 sporadic EPTs from 93 patients. Nine polymorphic microsatellite markers (6q13 to 6q25-q27) were investigated, demonstrating a loss of heterozygosity (LOH) in 62.2% of the patients. A LOH was significantly more common in tumors >2 cm in diameter than below this threshold as well as in malignant than in benign tumors. We were able to narrow down the smallest regions of allelic deletion at 6q22.1 (D6S262) and 6q23-q24 (D6S310-UTRN) with LOH-frequencies of 50.0% and 41.2 to 56.3%, respectively. Several promising tumor suppressor candidates are located in these regions. Additional fluorescence in situ hybridization analysis on 46 EPTs using three locus-specific probes (6q21, 6q22, and 6q27) as well as a centromere 6-specific probe revealed complete loss of chromosome 6 especially in metastatic disease. We conclude that the two hot spots found on 6q may harbor putative tumor suppressor genes involved not only in the oncogenesis but maybe also in the malignant and metastatic progression of sporadic EPTs.

Deletion at 3p25.3-p23 is frequently encountered in endocrine pancreatic tumours and is associated with metastatic progression.

For several reasons, chromosome 3p is thought to be involved in the pathogenesis of sporadic endocrine pancreatic tumours (EPTs): von Hippel–Lindaus disease (VHL gene at 3p25.5) is associated with EPTs; 3p is frequently involved in solid human tumours; and comparative genomic hybridization has identified frequent losses at 3p in EPTs. This study investigated 99 benign and malignant tumours, including 20 metastases, from 82 patients, by microsatellite loss of heterozygosity (LOH) analysis and fluorescence in situ hybridization (FISH) in order to evaluate the importance of chromosome 3p deletions in the molecular pathogenesis and biological behaviour of EPTs, to elaborate a common region of deletion, and to narrow down putative tumour suppressor gene loci. Allelic losses of 3p were found in 58/99 (58.6%) of tumours in 45/82 (54.9%) patients; analysis of seven microsatellite markers (3p26–p21) revealed a common region of LOH at 3p25.3–p23. The LOH frequency was significantly higher in malignant than in benign neoplasms (70.2% versus 28.0%; p=0.001). In addition, a strong correlation was found between the loss of alleles on chromosome 3p and clinically metastatic disease (LOH of 73.7% in metastasizing versus 41.5% in non‐metastasizing tumours; p=0.008). EPTs from these patients showed a tendency towards losing large parts or the entire short arm of chromosome 3 with tumour progression. Furthermore, FISH analysis revealed complete loss of chromosome 3 in ten out of 37 EPTs (27%). These results indicate that a putative tumour suppressor gene at 3p25.3–p23 may play a role in the oncogenesis of sporadic EPTs and that losses of larger centromeric regions are associated with metastatic progression. Copyright

Noninflammatory Chronic Pelvic Pain Syndrome: Immunological Study in Blood, Ejaculate and Prostate Tissue1

Objectives: The aim of this prospective study was to observe immunophenotypic patterns in patients with noninflammatory chronic pelvic pain syndrome (Cat IIIB CPPS) for further description and as possible surrogate markers for diagnosis and treatment. Methods: Eighty–eight patients with a referral diagnosis of chronic prostatitis underwent fractionated urinary cultures including expressed prostate secretion (EPS) and ejaculate analysis twice on two occasions. Monthly serum analyses included C3c, C4, IL–1α, sIL–2R, and IL–6. One hundred samples from healthy individuals were used as the control group for serum analysis. Monthly ejaculate testing was done for IgG, IgA, IgM, IL–1α, sIL–2R, and IL–6. The control group for ejaculate analysis was composed of 96 normal ejaculates (according to the WHO criteria). Immunohistochemical detection of CD3 cells (T lymphocytes) and CD20 cells (B lymphocytes) was performed in 71 biopsy cylinders of Cat IIIB CPPS patients and in 25 prostate biopsy cylinders of men without symptoms or obstruction. Results: Complete sampling of urinary, serum and ejaculate specimens was achieved in 50/88 (57%) patients. Cat IIIB CPPS was observed in 44/50 (88%) patients. Intra–acinar T–lymphocytic infiltrates were dominated by T cytotoxic cells (p = 0.05). Immunohistochemical studies showed inflammatory expression in serum complement, serum interleukin, and ejaculate interleukin concentrations in relation to the presence of large numbers of T cells (all p values ≤0.01). No difference was found in the proportion of B lymphocytes in patients with Cat IIIB CPPS compared to the control group. Serum and ejaculate IL–6 and ejaculate IgA increased significantly and dropped again, correlating with a release of clinical symptoms. Conclusions: Interleukin, complement and immunoglobulin determinations in serum and ejaculate reveal an inflammatory process even in Cat IIIB CPPS. The findings of intra–acinar T–cell–rich infiltrates and the associated inflammatory reaction may be a significant advance in defining Cat IIIB CPPS caused by a possible autoimmune component. Serum and ejaculate IL–6 and ejaculate IgA are possible surrogate markers for the diagnosis and treatment of Cat IIIB CPPS.

Absence of somatic SDHD mutations in sporadic neuroendocrine tumors and detection of two germline variants in paraganglioma patients

Allelic loss of the long arm of chromosome 11 is frequent in neuroendocrine tumors (NET) of different organs. However, the MEN1 gene on 11q13 is mutated only in a subset of NET and allelic losses on 11q frequently extend to the telomere. In this genetic region lies the tumor suppressor gene SDHD which is associated with hereditary paragangliomas (PGL1). We sought to determine whether SDHD plays a role in the development of sporadic NET. By mutation and deletion analysis of SDHD we were unable to detect any SDHD mutation in 45 NET of the lung, gastrointestinal tract, pancreas or parathyroid. However, we found allelic deletions in 20 to 50% of all tumors but parathyroid adenomas. Furthermore, we found heterozygous germline variants in 2/8 paragangliomas. A first case of variant c.149 A>G (H50R) was found in a patient with an extra-adrenal pheochromocytoma, the other variant c.34 G>A (G12S) in a patient with a paratracheal paraganglioma, C-cell hyperplasia of the thyroid and hyperplasia of ACTH-producing cells of the pituitary gland. Both variants were absent in 93 controls. Our results demonstrate that somatic SDHD mutations are rare in sporadic NET. However, LOH alone could lead to a complete loss of function since SDHD is an imprinted gene. Furthermore, we describe two germline variants possibly causing hereditary paragangliomas.

Immunological alterations in the ejaculate of chronic prostatitis patients: clues for autoimmunity

Summary.  The aim of this prospective study was to observe immunophenotypic patterns in the ejaculate of patients with noninflammatory chronic pelvic pain syndrome (Cat IIIB CPPS) and to test for a possible autoimmune aetiology. Thirty‐five patients of a total of 88 patients with chronic prostatitis Cat IIIB were consecutively selected. Monthly ejaculate testing was carried out for IgG, IgA, IgM, IL‐1α, sIL‐2R and IL‐6. The control group for ejaculate analysis was composed of 96 normal ejaculates (according to the WHO criteria). Immunohistochemical detection of CD3 cells (T lymphocytes) and CD20 cells (B lymphocytes) was performed in 71 biopsy cylinders of Cat IIIB CPPS patients and in 25 prostate biopsy cylinders of subjects without symptoms or obstruction. Intra‐acinar T‐lymphocytic infiltrates were dominated by T‐cytotoxic cells (P = 0.05). Ejaculate IL‐6 and ejaculate IgA increased significantly and dropped again, correlating with a release of clinical symptoms. Inflammatory ejaculate interleukin concentrations correlated with the immunohistochemical findings with presence of large numbers of T cells (all P‐values ≤ 0.01). Immunomodulation was performed in a pilot series of three patients by five monthly cycles of IgG (Sandoglobulin®), 1 g kg−1 body weight. Immunomodulation with IgG decreased pain moderately and did not change ejaculate interleukin and immunoglobulin concentrations. In summary, interleukin and immunoglobulin determinations in the ejaculate revealed an inflammatory process even in Cat IIIB CPPS. The findings of intra‐acinar T‐cell rich infiltrates and the associated inflammatory reaction may indicate a possible autoimmune component in the aetiology of CPPS. Exact origin and role of interleukin changes in the ejaculate of CPPS patients need to be further evaluated. Unfortunately, pilot series with immunomodulation with IgG do not seem to provide clear clinical benefit.

MAGEC2 is a sensitive and novel marker for seminoma: a tissue microarray analysis of 325 testicular germ cell tumors

Melanoma-associated gene C2 (MAGEC2) is a recently identified cancer testis antigen expressed in normal testicular and placental tissue. It has been detected in some human carcinomas, but its expression in primary testicular germ cell tumors is unknown. Immunohistochemistry was used to study MAGEC2 protein in 325 primary testicular germ cell tumors, including 94 mixed germ cell tumors. Seminomatous and non-seminomatous components were separately arranged and evaluated on tissue microarrays. MAGEC2 expression was compared with POU5F1 (OCT3/4), SOX2, SOX17, KIT and TNFRSF8 (CD30). The mouse monoclonal anti-MAGEC2 antibody (clone LX-CT10.5) revealed a nuclear MAGEC2 expression with little or no background staining. MAGEC2 expression was found in 238 of 254 seminomas (94%), but not in embryonal carcinomas (n=89). POU5F1 (OCT3/4) was positive in 97% of seminomas and all embryonal carcinomas. In contrast, KIT was positive in 94% of seminoma but also in 8% of embryonal carcinomas. TNFRSF8 (CD30) and SOX2 were negative in seminoma and positive in embryonal carcinoma (96 and 90%, respectively). SOX17 was positive in 94% of seminoma and negative in embryonal carcinoma. We conclude that MAGEC2 allows a reliable distinction of seminoma from embryonal carcinomas. Therefore, MAGEC2 represents an additional tool for the differential diagnosis of testicular germ cell tumors.

Distinct expression patterns of the immunogenic differentiation antigen NY‐BR‐1 in normal breast, testis and their malignant counterparts

NY‐BR‐1 is a differentiation antigen and a potential target for cancer immunotherapy. Its mRNA expression is restricted to breast, testis, prostate and breast cancer by RT‐PCR. In this study, we correlated NY‐BR‐1 protein and mRNA expression on tissue microarrays of mammary, prostatic and testicular malignancies using immunohistochemisty and in situ hybridization with probes for exon 4–7 and 30–33. NY‐BR‐1 mRNA was confined to primary spermatocytes, suggesting a role in spermatogenesis. Exon 4–7 and 30–33 were equally expressed this cell type. However, NY‐BR‐1 was absent in all germ cell tumours analyzed (n = 475) and present in one of 56 (2%) prostate carcinomas. In breast, NY‐BR‐1 mRNA expression was detected in 307 of 442 (70%) primary carcinomas, with strong correlation to its protein expression (p < 0.0001). mRNA expression was significantly stronger and more frequently detected by the exon 30–33 probe than by the exon 4–7 probe (70% vs. 35%, p < 0.0001), indicating the presence of alternative splice variants that lack 5‐prime sequences. A similar restricted mRNA pattern was also observed in the normal breast epithelium. NY‐BR‐1 protein and mRNA correlated significantly with estrogen receptor α (ERα) protein expression (p < 0.0001), with stronger association to NY‐BR‐1 mRNA than protein (odds ratio 7.7 compared to 4.6). We identified 4 estrogen response elements (ERE)‐like sequences nearby the promoter region, suggesting that NY‐BR‐1 transcription might be controlled by ERα. Accordingly, analysis of matching pairs of primary tumors with their recurrences showed a marked decrease of NY‐BR‐1 expression in recurrences after tamoxifen treatment (p < 0.0001).

Cancer testis antigen expression in testicular germ cell tumorigenesis.

Cancer testis antigens are encoded by germ line-associated genes that are present in normal germ cells of testis and ovary but not in differentiated tissues. Their expression in various human cancer types has been interpreted as ‘re-expression’ or as intratumoral progenitor cell signature. Cancer testis antigen expression patterns have not yet been studied in germ cell tumorigenesis with specific emphasis on intratubular germ cell neoplasia unclassified as a precursor lesion for testicular germ cell tumors. Immunohistochemistry was used to study MAGEA3, MAGEA4, MAGEC1, GAGE1 and CTAG1B expression in 325 primary testicular germ cell tumors, including 94 mixed germ cell tumors. Seminomatous and non-seminomatous components were separately arranged and evaluated on tissue microarrays. Spermatogonia in the normal testis were positive, whereas intratubular germ cell neoplasia unclassified was negative for all five CT antigens. Cancer testis antigen expression was only found in 3% (CTAG1B), 10% (GAGE1, MAGEA4), 33% (MAGEA3) and 40% (MAGEC1) of classic seminoma but not in non-seminomatous testicular germ cell tumors. In contrast, all spermatocytic seminomas were positive for cancer testis antigens. These data are consistent with a different cell origin in spermatocytic seminoma compared with classic seminoma and support a progression model with loss of cancer testis antigens in early tumorigenesis of testicular germ cell tumors and later re-expression in a subset of seminomas.