Tvrtko Hudolin

NY-ESO-1/LAGE-1 coexpression with MAGE-A cancer/testis antigens: a tissue microarray study.

The characterization of the expression pattern of different families of cancer/testis (C/T) antigens in different tumors, at the protein level, might be of relevance in the development of multiantigen vaccine preparations for active specific immunotherapy. We have used tissue microarray (TMA) technology to explore in large numbers of tumor specimens the expression of NY‐ESO‐1/LAGE‐1 C/T antigens and its correlation with MAGE‐A expression by using D8.38 and 57B monoclonal antibodies (MAb). The epitopes recognized by these reagents in C/T antigens were identified by molecular mapping by using a bacterial expression system. Out of 2,052 samples, 119 (5.8%) scored positive upon staining with D8.38 NY‐ESO‐1/LAGE‐1‐specific MAb. Expression in >10% of cases was detectable in melanoma and basalioma (31.6 and 18.2%, respectively), large cell carcinomas and adenocarcinomas of the lung (17.8 and 10.5%, respectively), stomach adenocarcinomas of the intestinal type (13.2%), pT2‐4 bladder TCC (18.2%), nonseminomatous carcinomas of the testis (10.4%) and liposarcomas (15.4%). Simultaneous expression of NY‐ESO‐1/LAGE‐1 and MAGE‐A C/T antigens was then addressed in a TMA where 101/845 and 73/845 samples (12 and 8.6%, respectively) showed evidence of MAGE‐A or NY‐ESO‐1/LAGE‐1 specific staining, respectively. In 35/845 specimens (4.1%) concomitant expression of MAGE‐A and NY‐ESO‐1/LAGE‐1 was observed (p = 0.0002). Discrepancies in the expression of NY‐ESO‐1/LAGE‐1 and MAGE‐A were conspicuously detectable in squamous cell carcinomas of the skin (MAGE‐A positive but NY‐ESO‐1/LAGE‐1 negative) and in liposarcomas (NY‐ESO‐1/LAGE‐1 positive, but MAGE‐A negative). Taken together, these data suggest novel areas of application of C/T antigens targeted active specific immunotherapy possibly based on multiantigen vaccine preparations.

High expression of indoleamine 2,3-dioxygenase gene in prostate cancer

Arginase 2, inducible- and endothelial-nitric-oxide synthase (iNOS and eNOS), indoleamine 2,3-dioxygenase (IDO) and TGF-beta, might impair immune functions in prostate cancer (PCA) patients. However, their expression was not comparatively analysed in PCA and benign prostatic hyperplasia (BPH). We evaluated the expression of these genes in PCA and BPH tissues. Seventy-six patients (42 BPH, 34 PCA) were enrolled. Arginase 2, eNOS and iNOS gene expression was similar in BPH and PCA tissues. TGF-beta1 gene expression was higher in BPH than in PCA tissues (p=0.035). IDO gene expression was more frequently detectable (p=0.00007) and quantitatively higher (p=0.00001) in PCA tissues than in BPH. IDO protein, expressed in endothelial cells from both BPH and PCA, was detectable in tumour cells in PCA showing evidence of high specific gene expression. In these patients, IDO gene expression correlated with kynurenine/tryptophan ratio in sera. Thus high expression of IDO gene is specifically detectable in PCA.

Myofibroblastic stromal reaction and expression of tenascin-C and laminin in prostate adenocarcinoma

The aim of this study was to analyse relationship between changes of the stroma and expression of tenascin-C (TN-C) and laminin in prostate carcinoma. Tenascin-C immunostaining was increased, and laminin decreased in carcinomas compared with peritumoural tissue and benign prostate hyperplasia (P<0.05). Statistical analysis confirmed connection between stromal changes and TN-C expression in prostate carcinoma (P<0.05). Gleason pattern 3 carcinomas showed more pronounced stromal reaction and TN-C expression compared with Gleason pattern 4 carcinomas (P<0.05). The main cells in prostate cancer stroma are myofibroblasts that are also responsible for tenascin production. Degradation of laminin was not connected with myofibroblastic stromal changes.

MAGE‐A10 cancer/testis antigen is highly expressed in high‐grade non‐muscle‐invasive bladder carcinomas

Bladder cancer is a common urinary malignancy and a prevalent cause of cancer‐related death. Current therapies of early stage non‐muscle‐invasive bladder cancer (NMIBC) are frequently associated with undesirable toxicities and recurrence. Active antigen‐specific immunotherapy may provide a valid therapeutic option for patients with NMIBC. Cancer‐testis antigens (CTA) expressed in various tumour types and in a limited range of healthy tissues may represent potential targets for specific immunotherapy. MAGE‐A10 is probably the most immunogenic antigen of the MAGE‐A family. We evaluated the expression of MAGE‐A10 in NMIBC. Seventy‐nine patients undergoing surgical treatment for NMIBC were enrolled in the study. MAGE‐A10 gene expression was assessed by quantitative real‐time polymerase chain reaction. Immunohistochemistry was performed on paraffin‐embedded sections. MAGE‐A10 gene was specifically expressed in one‐third of NMIBC (n = 24: 32.43%). Gene expression was correlated with high tumour grade. MAGE‐A10 protein was exclusively detectable in nuclei of tumour cells. More importantly, MAGE‐A10 protein was also more frequently detectable in high‐grade tumours (p = 0.0001) and in stage T1 tumours invading subepithelial tissue or lamina propria (p = 0.01). A strong correlation between MAGE‐A10 staining score and tumour grade and stage could accordingly be observed. These data indicate that MAGE‐A10 expression is a feature of aggressive NMIBC and might be used as a novel target for specific immunotherapy of these cancers.

Differential Patterns of Large Tumor Antigen-Specific Immune Responsiveness in Patients with BK Polyomavirus-Positive Prostate Cancer or Benign Prostatic Hyperplasia

ABSTRACT The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor β1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4+ CD25+(high) CD127− FoxP3+ T cell population with an effector memory phenotype (CD103+) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4+ CD25− T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV+ PCa.

Immunohistochemical expression of tumor antigens MAGE-A3/4 and NY-ESO-1 in renal oncocytoma and chromophobe renal cell carcinoma

The distinction between renal oncocytoma (RO) and chromophobe renal cell carcinoma (ChRCC), especially the eosinophilic variant, can often be difficult. Our study has documented for the first time the expression of MAGE-A3/4 and NY-ESO-1 cancer testis antigens (CTAs) in these tumors. A total of 35 patients (17 ROs and 18 ChRCCs) were included in the study. Two antibodies were used for immunohistochemical staining: 57B recognizing multiple MAGE-A and D8.38 recognizing NY-ESO-1 CTAs. Fifteen (88.2%) samples of RO stained positively for both MAGE-A3/4 and NY-ESO-1 antigens. Regarding ChRCC, seven (38.9%) stained positively for MAGE-A3/4 and six (33.3%) for NY-ESO-1 antigens. Median MAGE-A3/4 expression was moderately positive in RO and negative in ChRCC. The difference in MAGE-A3/4 expression between two tumor groups was significant (P=0.0013). Median NY-ESO-1 expression was strongly positive in RO and negative in ChRCC. The difference in NY-ESO-1 expression between two tumor groups was also significant (P=0.0008). Our study has shown that RO had a significantly higher expression of both CTAs. However, additional research is needed to clarify their potential diagnostic implications.

Immunohistochemical analysis of the expression of MAGE-A and NY-ESO-1 cancer/testis antigens in diffuse large B-cell testicular lymphoma

BackgroundPrimary testicular lymphoma (PTL) is a rare and lethal disease. The most common histological subtype is diffuse large B-cell lymphoma (DLBCL). Standard treatments are frequently ineffective. Thus, the development of novel forms of therapy is urgently required. Specific immunotherapy generating immune responses directed against antigen predominantly expressed by cancer cells such as cancer-testis antigens (CTA) may provide a valid alternative treatment for patients bearing PTL, alone or in combination with current therapies.MethodsThree monoclonal antibodies (mAbs), 77B recognizing MAGE-A1, 57B recognizing an epitope shared by multiple MAGE-A CTA (multi-MAGE-A specific) and D8.38 recognizing NY-ESO-1/LAGE-1 were used for immunohistochemical staining of 27 PTL, including 24 DLBCL.ResultsExpression of MAGE-A1 was infrequently detectable in DLBCL specimens (12.50%), whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained the cytoplasms of tumor cells in DLBCL specimens with higher frequencies (54.17% and 37.50%, respectively) with different expression levels.ConclusionsThese results suggest that MAGE-A and NY-ESO-1/LAGE-1, possibly in combination with other CTA, might be used as targets for specific immunotherapy in DLBCL.

Correlation between retroperitoneal lymph node size and presence of metastases in nonseminomatous germ cell tumors

Eighty-five patients had staging laparoscopic retroperitoneal lymph node dissection (L-RPLND) for nonseminomatous germ cell tumors at our institution. The largest lymph node size was measured and presence or absence of metastatic disease was determined. A total of 1139 lymph nodes have been removed and in 27 (31.8%) patients, metastases in one or more lymph nodes were detected. There were 338 (29.7%) hilar, 259 (22.7%) paraaortic, 221 (19.4%) interaortocaval, 171 (15%) paracaval, 133 (11.7%) preaortic and 17 (1.5%) precaval lymph nodes. The total number of lymph nodes with metastases was 74 (6.5%), and 1065 (93.5%) nodes did not have any metastases. The average size of a lymph node with metastases was 1.05 (0.3-3), and without metastases it was 0.55 (0.1-2.5) cm, (p<0.001). If we use > 1 cm size of a lymph node as a “cut-off” value for enlargement and presence of metastases, 60% of metastatic lymph nodes would be missed since they were all ≤ 1 cm. Our results have shown that decreasing size of lymph nodes which are considered positive from > 1 cm to 0.7 -0.8 cm can be recommended, with specificity and sensitivity equal 70%.

Immunolocalization and mRNA Expression of Bone Morphogenetic Protein-6 in Human Clear Cell Renal Carcinoma

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily of proteins. Dysregulation of BMP signaling has been suggested in the carcinogenesis of different organs. We determined BMP-6 mRNA and protein expression in localized human clear cell renal carcinoma (CCRC), obtained from 20 patients who underwent nephrectomy, by the real-time polymerase chain reaction and immunohistochemistry. 15/20 patients exhibited higher BMP-6 mRNA expression in malignant than in healthy renal tissue relative to the PBGD expression (p < 0.05). Immunostaining intensity for BMP-6 in healthy renal tissue ranged from 0 to 2 (average 0.9), as well as in renal clear cell carcinoma (average 1.1). Seven of 20 (35%) healthy tissue samples failed to stain with BMP-6 antibody, compared to 2/20 (10%) tumor samples (p < 0.05). BMP-6 immunostaining was positive in 18/20 CCRC samples. Staining was localized in the cytoplasm and/or membrane of malignant cells. Malignant tissue had significantly higher BMP-6 mRNA expression than healthy tissue. There was no significant correlation between BMP-6 mRNA and protein expression with disease presentation, disease progression and patients’ characteristics. Long-term follow-up of our patients is needed to determine the possible role of increased expression of BMP-6 in CCRC.

Lidocaine Suppository for Transrectal Ultrasound-Guided Biopsy of the Prostate: A Prospective, Double-Blind, Randomized Study

Aims: To investigate analgesia using lidocaine suppositories for prostate biopsy. Methods: From 2007 to 2009, 160 patients underwent transrectal ultrasound-guided prostate biopsy at the Department of Urology, KBC Zagreb. 80 patients received a 60-mg lidocaine suppository intrarectally at different time points from 15 to 120 min before biopsy and 80 patients received a glycerin suppository as placebo. The pain level was evaluated using a visual analogue scale (VAS). Results: There were no statistically significant differences between the groups, i.e. they were similar regarding patients’ age, prostate-specific antigen levels, prostate volume and the incidence of diagnosis of malignancy on biopsy. The mean pain score in the lidocaine group (3 ± 1) was significantly lower than the mean pain score in the glycerin group (4.1 ± 1.3) (p < 0.001). A noticeable trend towards lower pain scores in the lidocaine group was observed with more time elapsing from placing the suppository till the biopsy and the optimal time for performing biopsy starting approximately 1 h after placing the suppository. Conclusions: Lidocaine suppositories are an easy-to-use, self-applicable (by the patient) and cheap method of local analgesia, with acceptable results. Possible complications related to this procedure are insignificant.