Hypermethylation of DAPK1 is an independent prognostic factor predicting survival in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin''s lymphoma. Improvements in overall survival have been observed with the introduction of rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), however, prognostic markers are still needed. Methylation of the death associated protein kinase (DAPK or DAPK1) gene and TP53 mutations are likely to have prognostic value in DLBCL. We have assessed TP53 mutations and allelic DAPK1 methylation patterns in a cohort of 119 DLBCL patients uniformly treated with R-CHOP-like regimens. We found that DAPK1 promoter methylation was associated with shorter overall survival (p=0.017) and disease-specific survival (p=0.023). In multivariate analyses DAPK1 methylation remained as an independent prognostic factor predicting disease-specific survival (p=0.038). When only considering individuals heterozygous for the rs13300553 SNP monoallelic methylation of the A-allele was associated with shorter overall- and disease-specific survival (p<0.001). Patients carrying both DAPK1 methylation and a TP53 mutation had an inferior survival compared to patients carrying only one of these molecular alterations, however, this was borderline statistically significant. Allele-specific DAPK1 methylation patterns were also studied in a cohort of 67 multiple myeloma patients, and all of the methylated multiple myeloma samples heterozygous for the rs13300553 SNP were methylated on both alleles.     

Tumour DNA ploidy as an independent prognostic factor in breast cancer

We determined nuclear DNA content from 308 archival paraffin-embedded malignant breast tumours and evaluated the survival of the patients by univariate and multivariate statistical analyses. The overall 8-year survival rate of stage I-III breast cancer patients was 74.3% in DNA-diploid and 51.2% in DNA-aneuploid tumours (P less than 0.0001). DNA ploidy had prognostic significance in both node-negative and node-positive breast cancer, primarily in cases with steroid receptor-positive tumours. In a Cox multivariate analysis DNA ploidy (P = 0.001), primary tumour size (P = 0.0007), nodal status (P = 0.04) and the content of progesterone receptors (P = 0.0008) emerged as significant independent prognostic factors, whereas oestrogen receptor status, age and menopausal status of the patients had no significant independent prognostic value. If the histological grade of ductal carcinomas was also included in the Cox model, both grade and DNA ploidy had independent prognostic effect. In conclusion, our results indicate that the analysis of DNA ploidy is a useful adjunct in the assessment of prognosis for breast cancer patients.     

Telomere length is a prognostic factor in neuroblastoma

BACKGROUND: Maintenance of telomeres, in most instances by reactivation of telomerase, is obligatory for the indefinite proliferation of tumor cells. The objective of this study was to evaluate telomere length and telomerase activity (TA) as markers for progression and prognosis in neuroblastoma. METHODS: Primary tumor samples from 51 patients were analyzed for telomere length and TA and were correlated with known prognostic parameters and outcome. RESULTS: Telomere length had a highly significant correlation with prognosis (P = .007). Short telomeres were predictive of a favorable prognosis, whereas long or unchanged telomeres were predictive of a poor outcome. For the first time to their knowledge, the authors have shown that, within the high-risk group patients, telomere length could define a favorable subgroup that had a progression-free survival (PFS) rate of 86% compared with a PFS rate of 36% for patients with more adverse disease, which is the expected PFS rate for such patients (P = .04). In a multivariate analysis, telomere length was the most significant prognostic parameter (P = .032). TA was correlated significantly with outcome and with known prognostic factors. High TA and low TA were associated with adverse and favorable outcomes, respectively (P = .01). CONCLUSION: The results of this investigation suggested that telomere length is a highly significant prognostic parameter of clinical relevance in patients with neuroblastoma. In high-risk patients, telomere length was the sole significant parameter that identified a group of patients who had a favorable prognosis. The authors suggest that telomere length should be included in the recommended diagnostic investigations for patients with neuroblastoma.     

Topoisomerase IIalpha expression as an independent prognostic factor in Hodgkin's lymphoma.

PURPOSE: To correlate the immunohistochemical expression of topoisomerase IIalpha (topoIIalpha) in Hodgkin's lymphoma (HL) with clinicopathological parameters, the expression of Ki-67 and the outcome of patients, who had been homogenously treated with ABVD or equivalent regimens. EXPERIMENTAL DESIGN: Immunohistochemistry using the monoclonal antibody Ki-S1 (topoIIalpha) was performed in 238 HL patients. MiB1 (Ki-67) expression was evaluated in 211/238. RESULTS: The mean +/- SD percentage of topoIIalpha- and Ki-67-positive Hodgkin-Reed-Sternberg (HRS) cells was 63 +/- 19% (5%-98%) and 73 +/- 19% (8%-99%), respectively. The median percentage of topoIIalpha-positive HRS cells was 64% (interquartile range, 51-78%). There was no correlation between topoIIalpha expression and patient characteristics. TopoIIalpha and Ki-67 expression were correlated (Spearman's Rho 0.255, P < 0.001). TopoIlalpha expression within the highest quartile of this patient population was predictive of failure free survival (FFS) (10-year rates 82 +/- 3% vs 68 +/- 7%, P = 0.02 for patients falling into the quartiles 1-3 and 4 respectively). In multivariate analysis topoIIalpha expression was independently predictive of FFS. CONCLUSION: TopoIIalpha was expressed in all cases of HL showing a correlation with Ki-67 expression. Under current standard therapy including drugs inhibiting its activity, topoIIalpha was an independent adverse predictor of FFS with no statistically significant correlation with other established prognostic factors.     

International neuroblastoma pathology classification adds independent prognostic information beyond the prognostic contribution of age

Age has been used as a prognostic factor for patients with peripheral neuroblastic tumours (pNTs). The latest analysis disclosed a cut-off around 18 months for the optimal prognostic distinction. The International Neuroblastoma Pathology Classification (INPC) distinguishes favourable and unfavourable histology based on the age-appropriate evaluation of histologic indicators (grade of neuroblastic differentiation, mitosis-karyorrhexis index) in the categories of neuroblastoma and ganglioneuroblastoma, nodular. This study showed that age tested by using 3 different cut-offs (12, 18, 24 months) was prognostically significant. INPC remained prognostically significant regardless of the age group to which it was applied. Prognostic effects of age and histologic indicators were independently significant, i.e., age had prognostic ability beyond that of histologic indicators, and histologic indicators had prognostic ability beyond that of age. Due to the fact that INPC incorporated age factor (18, 60 months) in the system, it served better than age by itself for prognostic distinction of pNT patients.     

Apoptotic index as a prognostic factor in Hodgkin's disease

Hodgkin's disease (HD) is an unusual malignant neoplasm, mainly because of the rarity of tumor cells in the diseased tissues, but also due to a relatively favorable response to treatment. In a previous study, we have shown a variable degree of apoptosis in lymph nodes from HD patients. We now looked for clinicopathological correlations of apoptosis with special emphasis on the prognosis in this disease. A retrospective study of 92 patients was carried out, using in situ end labelling of DNA fragments and an apoptosis detection kit. An apoptotic index (Al) was calculated in each case, as the percentage of apoptotic Hodgkin-Reed-Sternberg cells out of the total number of tumor cells in 10 selected high power fields. An association between a high Al and advanced stages was noted. A Kaplan-Meier analysis showed a negative correlation between Al and survival (p=0.05). In a multivariable analysis adjusting for Ann Arbor stage, a high Al carried a 3.27 fold risk of dying of HD (OR=3.27; Cl=0.89-11.94). However, in our limited cohort of HD patients, Al was not an independent prognostic factor. The results of this study confirm the important role played by apoptosis in HD and suggest that the apoptotic index is probably a negative prognostic marker in this disease. Its assessment in patients with HD may provide a new, important clinical tool.     

High levels of thymidine phosphorylase as an independent prognostic factor in renal cell carcinoma

BACKGROUND: We investigated whether thymidine phosphorylase (TP) protein level in renal cell carcinoma (RCC) correlates with clinicopathological characteristics and clinical outcomes. METHODS: TP protein level was measured in 116 RCC specimens and in 90 non-neoplastic kidney tissues using a sandwich-type enzyme-linked immunosolvent assay. RESULTS: The median TP protein level in RCC tissues was 9.76-fold (range, 3.2-933.9) higher than those in non-neoplastic kidney tissues (P < 0.0001). TP protein level was correlated with T classification, histological grade and mode of infiltration. TP as a prognostic variable was studied using a logistic regression model. TP at higher levels (128 U/mg protein or greater) would play a role as an independent prognostic factor (odds ratio, 13.73; 95% confidence interval, 2.09-90.41; P = 0.0064). CONCLUSION: TP at high levels can be regarded as an unfavorable independent prognostic factor. These results may pave a way for a novel approach to effective treatment of RCC.     

Age as an independent prognostic factor for survival of localised synovial sarcoma patients

Background:

We performed a retrospective nationwide study to explore age as a prognostic factor in synovial sarcoma patients.

Methods:

Data on 613 synovial sarcoma patients were obtained from the Netherlands Cancer Registry. The prognostic relevance of age groups (children, adolescent and young adults (AYAs), adults, and elderly) was estimated by Kaplan–Meier survival curves and multivariable Cox-proportional hazards modelling.

Results:

A total of 461 patients had localised disease at diagnosis. The 5-year overall survival (OS) was 89.3±4.6%, 73.0±3.8%, 54.7±3.6%, and 43.0±7.0% in children (n=54), AYAs (n=148), adults (n=204), and elderly (n=55), respectively. Treatment modalities had no significant effect on survival in the univariable analysis. Multivariable analysis identified age at diagnosis, tumour localisation, and tumour size as significant factors affecting OS. Both tumour localisation and size were equally distributed over the age groups.

Conclusions:

We show that outcome of synovial sarcoma patients significantly decreases with age regardless of primary tumour site, size, and treatment.     

CD44 expression: a new prognostic factor in neuroblastoma

CD44 gene products are potential markers of aggressiveness in different tumor models, a result which prompted us to study clinical neuroblastoma (NB) specimens. CD44 expression was determined by immunostaining of 52 NB with a monoclonal antibody (J173) directed against an epitope common to all CD44 isoforms. All tumors were from patients (pts) with newly diagnosed NB treated with standardized protocols. They were classified according to international criteria [11]. CD44 immunoreactivity was detected in 37 tumors (71%). CD44 was expressed in 100% of favorable NB stages (1, 2 or 4S), but only 50% of advanced NB (stages 3 and 4) (p = 0.0001), suggesting that the absence rather that the overexpression of CD44 is a signal of tumor aggressiveness. The cumulative event-free survival was significantly longer in pts with CD44-positive tumors as compared to pts with CD44-negative tumors (p < 10(-5)). More importantly, progression-free survival was also significantly higher in CD44-positive pts within the high-risk group (p < 0.01). In univariate analyses, we tested the prognostic value of tumor expression of CD44 in comparison with tumor stage, age, tumor histology and presence or absence of N-myc proto-oncogene amplification. All five measures had significant prognostic value. The expression of CD44 and the absence of N-myc amplification were the most powerful predictors of a favorable clinical outcome. In a multivariate analysis of these measures, CD44 expression and tumor stage were the only independent prognostic factors for the prediction of patient survival. NB is the first clinical model described in which tumor aggressiveness correlates with a repression rather than a stimulation of CD44 expression. We recommend the use of CD44 as an additional biological marker in the initial staging of neuroblastoma.     

Young age as an independent adverse prognostic factor in premenopausal patients with breast cancer

Breast cancer in younger patients appears to be more aggressive than disease occurring in older patients. Even though large population-based studies suggest poorer survival of patients younger than 35 years, data demonstrating the relationship of age and prognosis within premenopausal cohorts are much more scarce and conflicting. In this retrospective analysis of 885 premenopausal patients, the relationship between age, typical prognostic factors, treatment, and patient outcome was investigated. Eight hundred four patients (90.8%) > 35 years and 81 patients (9.2%) = 35 years who had been treated for stage I/II breast cancer were evaluated. Median follow-up time was 71 months. The prevalence of adverse prognostic features such as tumor size, tumor type, tumor grading, pathologic lymph node status, and hormone receptor status were evenly distributed between the two age groups. Age = 35 years proved to be a powerful independent prognostic factor in multivariate analyses of recurrence-free (P < 0.0001; relative risk [RR] = 2.5) and overall survival (P < 0.0039, RR = 2.2). Thus, in the face of evenly distributed risk factors in this strictly premenopausal, homogeneous population, young age was seen as the second most powerful risk factor after lymph node status. According to these findings, patients diagnosed with breast cancer at = 35 years of age have a worse prognosis compared to premenopausal women above this age. Future studies should focus on unveiling the young age surrogate in order to improve treatment and prognosis.     

Hypermethylation of CpG islands in p16 as a prognostic factor for diffuse large B-cell lymphoma in a high-risk group

PURPOSE: The aim of the study was to analyze the methylation status of the promoter regions of p15 and p16 and to assess the prognostic significance of promoter hypermethylation in diffuse large B-cell lymphoma (DLBCL). EXPERIMENTAL DESIGN: DLBCL was diagnosed by morphology and immunohistochemical analysis according to the World Health Organization (WHO) classification. The methylation status of CpG islands in the p15 and p16 promoters was analyzed by methylation-specific polymerase chain reaction in 49 DLBCLs. RESULTS: Hypermethylation of the p15 and p16 promoters was detected in 20 (41%) and 22 (45%) of the 49 DLBCLs, respectively. Among all patients with DLBCL, there was no significant difference in the overall survival between those with hypermethylated and unmethylated p15 (P=0.442) or between those with hypermethylated and unmethylated p16 (P=0.468). Therefore, methylation was analyzed in combination with evaluation of clinical features using the international prognostic index (IPI). In the high-intermediate-risk and high-risk groups, patients with hypermethylated p16 had significantly lower survival rates than those of patients in the same risk group with unmethylated p16 (P=0.010). CONCLUSIONS: Our results suggest that hypermethylation of the p16 promoter indicates a poor prognosis in high-intermediate-risk and high-risk DLBCL patients, and may be a useful marker for selection of appropriate treatment when used in conjunction with the IPI.     

Calreticulin expression in neuroblastoma--a novel independent prognostic factor.

BACKGROUND: Calreticulin (CRT), an endoplasmic reticulum protein, has been reported to be essential for the differentiation of neuroblastoma (NB) cells, suggesting that CRT may affect the tumor behavior of neuroblastoma. The aim of this study was to evaluate the association of clinicopathologic factors and patient survival with the expression of CRT in patients with NB. PATIENTS AND METHODS: Sixty-eight NBs were investigated by immunohistochemical staining against CRT, and were divided into positive and negative immunostaining groups. Correlations between calreticulin expression, various clinicopathologic and biologic factors, and patient survival were studied. In seven tumor samples, CRT mRNAs and proteins were evaluated with real-time PCR and western blot, respectively, and correlated with immunohistochemical findings. RESULTS: Among 68 NBs, 32 (47.1%) showed positive CRT expression. Positive CRT immunostaining strongly correlated with differentiated histologies, as well as known favorable prognostic factors such as detected from mass screening, younger age (< or =1 year) at diagnosis and early clinical stages, but inversely correlated with MYCN amplification. Kaplan-Meier analysis revealed that NB patients with CRT expression did have better survival. Multivariate analysis demonstrated CRT expression to be an independent prognostic factor. Moreover, CRT expression also predicted better survival in patients with advanced-stage NBs, and its absence predicted poorer survival in patients whose tumor had no MYCN amplification. The amount of CRT mRNAs and proteins in NB tumor samples tested correlated well with the immunohistochemical expressions. CONCLUSIONS: CRT expression correlates with the differentiation of NB and predicts favorable survival, thereby suggesting CRT to be a useful indicator for planning treatment of NB.     

Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is highly malignant and prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumour appearing is of clinical importance. At this point, no convenient method is available to determine the origin of these HCCs. Tissue samples were obtained from 19 patients and analysed for the promoter hypermethylation status of multiple tumour suppressor genes (p16, DAP-Kinase, MGMT, GSTP1, APC, RIZ1, SFRP1, SFRP2, SFRP5, RUNX3, and SOCS1) using methylation-specific PCR (MSP). Methylation status was used to determine tumour clonality. In each of the 19 cases, at least one tumour was recognised as having an aberrantly methylated gene. The frequency of the methylation in tumour tissue was 57.1% in p16, 2.4% in DAP-kinase, 23.8% in GSTP1, 90.5% in APC, 45.2% in RIZ1, 64.3% in SFRP1, 59.5% in SFRP2, 28.6% in SFRP5, 47.6% in RUNX3, and 54.8% in SOCS1, while in MGMT, no aberrant methylation was detected. The methylation status of these genes was assessed using MSP as being either positive or negative, and was used to determine the tumour clonality. The clonality of every tumour could be decided even with lesions that could not be judged by clinical diagnosis or by another molecular method (mt DNA mutation). Determining the methylation status of multiple genes in multicentric HCC was useful as a clonal marker and provided useful information for characterising the tumour. From our findings, multicentric HCCs tend to occur more independently than metastatically from the original tumour. Expanded study should be pursued further for a better understanding of the molecular mechanism of hepatocarcinogenesis.     

Angiogenesis is an independent prognostic factor in malignant mesothelioma

Angiogenesis is essential for tumour growth beyond 1 to 2 mm in diameter. The clinical relevance of angiogenesis, as assessed by microvessel density (MVD), is unclear in malignant mesothelioma (MM). Immunohistochemistry was performed on 104 archival, paraffin-embedded, surgically resected MM samples with an anti-CD34 monoclonal antibody, using the Streptavidin-biotin complex immunoperoxidase technique. 93 cases were suitable for microvessel quantification. MVD was obtained from 3 intratumoural hotspots, using a Chalkley eyepiece graticule at x 250 power. MVD was correlated with survival by Kaplan-Meier and log-rank analysis. A stepwise, multivariate Cox model was used to compare MVD with known prognostic factors and the EORTC and CALGB prognostic scoring systems. Overall median survival from the date of diagnosis was 5.0 months. Increasing MVD was a poor prognostic factor in univariate analysis (P = 0.02). Independent indicators of poor prognosis in multivariate analysis were non-epithelial cell type (P = 0.002), performance status > 0 (P = 0.003) and increasing MVD (P = 0.01). In multivariate Cox analysis, MVD contributed independently to the EORTC (P = 0.006), but not to the CALGB (P = 0.1), prognostic groups. Angiogenesis, as assessed by MVD, is a poor prognostic factor in MM, independent of other clinicopathological variables and the EORTC prognostic scoring system. Further work is required to assess the prognostic importance of angiogenic regulatory factors in this disease.