Bozena Sarcevic

Cancer/testis tumour-associated antigens: immunohistochemical detection with monoclonal antibodies

Cancer/testis tumour-associated antigens (C/T TAA) were the first human tumour-associated antigens to be characterised at the molecular level. Specific genes are expressed in the testis and in tumours of varying histological origin. The tissue expression pattern supports the notion that these antigens could be targets for active specific immunotherapy. Specific serological reagents have been developed and have helped to clarify biochemical characteristics of C/T TAA and to assess their distribution within clinical tumour samples. We review immunohistochemical evidence of the expression of C/T TAA known to be recognised by specific cytotoxic T lymphocytes. The emerging picture is consistent with a mostly heterogeneous expression in human cancers. These findings support the concept of multiantigenic tumour vaccine preparations. Moreover, the wide range of tumours in which C/T TAA have been detected urges further efforts to develop effective specific immunotherapeutic procedures.

Expression of cancer/testis tumor associated antigens in cervical squamous cell carcinoma.

We investigated the expression of tumor-associated antigens (TAA) of the cancer/testis (C/T) gene family in cervical squamous cell carcinomas. First, we focused on the HeLa cervical cancer derived cell line, and we found that it expresses MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A12, GAGE-3/6, LAGE-1, and PRAME genes, encoding defined C/T TAA. In contrast, no expression of MAGE-A10, BAGE, GAGE-1/2, or NY-ESO-1 genes was observed. Corresponding gene products could also be detected by immunoblotting and immunocytochemistry, taking advantage of monoclonal antibodies recognizing discrete TAA. Capitalizing on these data, a monoclonal antibody predominantly recognizing MAGE-A4 TAA in paraffin-embedded sections (57B) was used to investigate the C/T gene expression in clinical tumor samples. A group of 60 patients was studied, and 57B positivity was detectable to different extents in 33% of the cases (20/60). In 13 of them (21%), staining of over 50% of the tumor cells was evident, whereas healthy cells always scored negative. Remarkably, MAGE-A4 expression was significantly (p < 0.05) more frequently detectable in poorly differentiated tumors (8/13) than in well-differentiated or moderately differentiated cancers (3/15 and 9/32, respectively) and in stage FIGO II as compared with stage FIGO Ib tumors (12/23 and 5/24, respectively, p = 0.04). Interestingly, staining was mostly nuclear in well-differentiated tumors, but involved both nuclei and cytoplasm in less differentiated cancers. Positivities of comparable frequency were also detectable in a smaller series of specimens upon staining with MAGE-A1- or NY-ESO-1/LAGE-1-specific reagents. Considering the high tumor specificity of C/T TAA, our data provide the rationale for the design of immunotherapy procedures targeting these antigens in cervical cancers.

Prognostic value of MAGE-A and NY-ESO-1 expression in pharyngeal cancer.

The prognostic value of cancer testis antigens in pharyngeal cancer is understudied.

Prognostic Significance of Carbonic Anhydrase IX (CA-IX), Endoglin (CD105) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in Breast Cancer Patients

The aim of this study was to examine the prognostic significance of carbonic anhydrase IX (CA-IX), an endogenous marker for tumor hypoxia; endoglin (CD105), a proliferation-associated and hypoxia-inducible glycoprotein and 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative DNA lesion, in breast cancer patients. Immunohistochemical expressions of CA-IX, CD105 and 8-OHdG, analyzed on paraffin-embedded tumor tissues from forty female breast cancer patients, were used to assess their prognostic implication on overall survival (OS) and relapse-free survival (RFS). Patients with high CA-IX expression (above cut-off value) had a higher occurrence of relapse (P = 0.002). High CA-IX expression was significantly associated with shorter RFS (P < 0.001, hazard ratio (HR) 0.21) and shorter OS (P < 0.001, HR 0.19). Lymph node negative patients with high CA-IX expression had worse RFS (P = 0.031, HR 0.14) and OS (P = 0.005, HR 0.05). Patients with grade I&II tumors and high CA-IX expression showed shorter RFS (P = 0.028, HR 0.28) and OS (P = 0.008, HR 0.20). Worse OS (P = 0.046, HR 0.28) was found in subgroup of patients with grade II tumors and high CA-IX expression. Among all three markers, only high CA-IX expression was strong independent prognostic indicator for shorter OS (HR 4.14, 95% CI 1.28–13.35, P = 0.018) and shorter RFS (HR 3.99, 95% CI 1.38–11.59, P = 0.011). Elevated expression of CA-IX was an independent prognostic factor for decreased RFS and OS and a significant marker for tumor aggressiveness. CD105 had week prognostic value; whereas, 8-OHdG, in this study, did not provide sufficient evidence as a prognostic indicator in breast cancer patients.

The prognostic significance of estrogen receptor β in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in the world. Although multimodal and targeted therapy is now used in therapeutic procedures, the survival of patients with HNSCC has remained unchanged over the last 30 years. A number of studies have demonstrated that the increased expression of intranuclear ERβ in breast, lung and colon cancer is a favorable prognostic marker associated with higher survival rates. However, the clinical significance of sex hormone receptors in HNSCC remains unclear. The current study aimed to assess the expression of ERβ in HNSCC immunohistochemically and investigate any possible association between ERβ expression, and clinical and histopathological factors, disease recurrence and patient survival. The present study included 174 patients (165 males and 9 females) with a median age of 60.8 years (range, 39–79) with HNSCC who were primary surgically treated between January 2000 and December 2006. Immunohistochemical reactions for ERβ demonstrated that 73 patients (42%) exhibited positive ERβ expression. Distribution of ERβ status among different head and neck subsites indicated that >40% of all negative cases were located in laryngeal primaries, while incidence of other sublocalization within positive cases was similar and comparable (P=0.04). Furthermore, a correlation was observed between ERβ immunopositivity and the survival of patients, with respect to the primary tumor site. Patients with ERβ positive oropharyngeal cancer had a survival rate of 35.3% at 5-years compared with 25% for patients with negative expression. However, ERβ status was not significantly correlated with any other clinical or histopathological parameter. After an average follow-up time of 38.5 months (range, 3–60 months), 54 patients (31.1%) had succumbed to disease recurrence while 50 (28.7%) succumbed to other causes. In conclusion, ERβ positivity indicates improved survival of patients with oropharyngeal cancer. Further research is required in order to implement novel therapeutic strategies.

Possible predictive role of cancer/testis antigens in breast ductal carcinoma in situ

Cancer/testis antigens (CTAs) are a large family of tumor-associated antigens expressed in human tumors of different histological origin, but not in normal tissues, with the exception of the testes and placenta. Numerous immunohistochemical studies have reported associations between CTA expression and a negative estrogen receptor (ER) status in breast tumors, and demonstrated that CTAs are frequently expressed in tumors with higher nuclear grade. The expression of CTAs has not been studied as extensively in ductal carcinoma in situ (DCIS) as it has been in invasive breast cancer. The present retrospective study included archived paraffin-embedded specimens from 83 patients diagnosed with DCIS in the period between January 2007 and December 2014. The follow-up time for local recurrence ranged between 1 and 8 years (mean, 5.02 years). Antigens from the melanoma-associated antigen gene (MAGE) family, namely multi-MAGE-A, MAGE-A1, MAGE-A10 and New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, were evaluated by immunostaining and their subcellular location was investigated. Presence of tumor-infiltrating lymphocytes (TILs) was evaluated on all sections, together with the histopathological variables of DCIS. Specific tested antigens exhibited associations with histopathological parameters for DCIS and all demonstrated statistically significant associations with nuclear staining, simultaneous cytoplasmic and nuclear staining, and local recurrence. Antigen MAGE-A10 demonstrated a significant association with higher expression of ER (P=0.005) and higher tumor nuclear grade (P=0.001), cytoplasmic staining (P=0.029) and antigen NY-ESO-1 with higher tumor size (P=0.001), expression of TILs (P=0.001) and R1 resection (P=0.001). A χ2 test revealed significant associations between simultaneous cytoplasmic and nuclear staining and local recurrence (P=0.005), central necrosis (P=0.016), and the expression of ER (P=0.003) and progesterone receptor (PR) (P=0.010). Additional analysis revealed an association between antigen MAGE-A10 and TILs (P=0.05). Additional analysis of TILs indicated that they were significantly associated with tumor grade (P=0.023), central necrosis (P<0.001), ER (P=0.003) and PR (P=0.029). Overall, CTAs from the MAGE family (MAGE-A1, multi-MAGE-A and MAGE-A10) and NY-ESO-1 associate with histopathological predictive variables of DCIS. The expression of antigens NY-ESO-1 and MAGE-A10 could serve an important role in the treatment of patients with negative histopathological predictive variables, but further analysis is required. Simultaneous cytoplasmic and nuclear protein expression of MAGE-A family and NY-ESO-1 CTAs may represent an independent marker for local recurrence. Taken together, the present data suggest that CTAs are not perfect indicators of invasiveness for DCIS, but could inform treatment strategies for patients when taken in combination with other histopathological predictive variables. However, this was a small study and further larger studies will be necessary to confirm the current findings.

Apoptosis regulator Bcl-2 is an independent prognostic marker for worse overall survival in triple-negative breast cancer patients

Background: The objective of this study was to examine the prognostic significance of carbonic anhydrase IX (CAIX), an endogenous marker for tumor hypoxia; the cellular tumor antigen p53; and the apoptosis regulator Bcl-2, in triple-negative breast cancer (TNBC) patients. Methods: Immunohistochemically determined expression of CAIX, p53, Bcl-2 and proliferation factor Ki-67, analyzed in 64 paraffin-embedded TNBC tissue samples, was used to assess their relation to clinicopathological variables and prognostic implications for overall survival (OS). Results: Bcl-2 expression was negatively correlated with histological grade of tumor, while expression of p53 was positively correlated with the same clinical variable (p = 0.036 and p = 0.033, respectively). The p53 expression was also positively correlated with tumor size (p = 0.010). Survival analysis showed that patients with high Bcl-2 expression (above cutoff value determined by receiver operator characteristic [ROC] curve analysis) had shorter OS (p = 0.020). The same was observed for patients with tumors larger than 5 cm (p = 0.034) or positive lymph nodes (p = 0.004). Among all 3 examined markers, multivariate analysis showed that only Bcl-2 expression was a strong independent prognostic indicator for decreased OS (hazard ratio [HR] = 15.16, 95% confidence interval [95% CI], 2.881-79.727, p = 0.001). Conclusions: Elevated expression of Bcl-2 was an independent prognostic factor for poorer OS in TNBC and as such a significant marker for tumor aggressiveness.

Expression of nm23-H1 and COX-2 in thyroid papillary carcinoma and microcarcinoma

The expression of non-metastatic expressed/non-metastatic 23 nucleoside diphosphate kinase 1 (nm23-H1) and cyclooxygenase 2 (COX-2) proteins in thyroid carcinoma have been analysed in a number of previous studies, but this requires further study. The current study focused on the expression levels of nm23-H1 and COX-2 in 130 human thyroid papillary carcinoma (PTC) tissues. Of the 130 PTC tissues, 55 were classified as microcarcinoma and may provide information on the development of the specific characteristics of this tumour type. Routine histopathological examination and immunohistochemical detection of nm23-H1 and COX-2 expression was performed on 130 PTC tissues from patients treated in the Clinical Hospital for Tumours (Zagreb, Croatia) between January 2000 and December 2007. The stain intensity of nm23-H1 and COX-2 proteins was compared with the characteristics of the patients and the tumour. The highest overall expression rate of nm23-H1 and COX-2 was 90 and 67.6%, respectively, and the joint expression of these proteins was statistically significant. The median expression level of nm23-H1 was significantly increased in the classical and follicular histological group of the PTC tissues compared with tissues from other histological groups. The median expression level of COX-2 was significantly increased in the follicular histological group, and reduced in the diffuse-sclerosing group of PTC tissues. All the metastatic microcarcinoma tissues had increased expression levels of the two proteins in comparison with microcarcinoma tissues without lymph node metastases; however, this variation was only statistically significant for COX-2 expression levels. Therefore the results of the current study indicate that COX-2 protein levels may be able to differentiate which thyroid papillary microcarcinoma tumours possess metastatic potential.

23 - Immunohistochemical Detection of Melanoma Antigen E (MAGE) Expression in Breast Carcinoma

U radu je istraživana imunohistokemijska izraženost gena MAGE u duktalnom invazivnom karcinomu dojke upotrebom misjeg monoklonskog protutijela 57B u obliku hibridoma supernatanta.