Do columnar cell lesions exist in the male breast?

AIMS: In females, columnar cell lesions (CCLs) have been recognized as putative precursor lesions of low-grade breast cancer, but their role in male breast carcinogenesis is as yet unclear. METHODS AND RESULTS: We reviewed surgical resections from males with breast cancer (n = 89), gynaecomastia (n = 20) and normal breast specimens from autopsies (n = 5) for the presence of CCL. In addition, we performed immunohistochemistry for cytokeratin 5/6 (CK5/6), CK14 and oestrogen receptor alpha (ER). In 19 of 89 resections (two DCIS cases and 17 invasive carcinoma), some individual ducts were found to contain cells with snouts on the luminal border but lacking further typical columnar cell lesion features. We mainly found three-layered ductal epithelium, characteristic for gynaecomastia and confirmed by immunohistochemistry. Moreover, we found a few ducts in male breast cancer sections that were clonally negative for basal cytokeratins. CONCLUSION: We found no lesions with convincing CCL morphology at the periphery of invasive male breast cancers, in gynaecomastia or in normal male breast specimens. Although we cannot completely exclude the existence of CCLs in the male breast, these lesions seem to be very uncommon and are therefore unlikely to play a major role in male breast carcinogenesis.

Promoter hypermethylation in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

INTRODUCTION: Epigenetic events are, along with genetic alteration, important in the development and progression of cancer. Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. The role of promoter hypermethylation in male breast cancer has not yet been studied. METHODS: In a group of 108 male breast cancers, the methylation status of 25 genes was studied using methylation-specific multiplex ligation-dependent probe amplification. Methylation of more than 15% was regarded indicative for promoter hypermethylation. Methylation status was correlated with clinicopathological features, with patients' outcome and with 28 female breast cancer cases. RESULTS: Promoter hypermethylation of the genes MSH6, WT1, PAX5, CDH13, GATA5 and PAX6 was seen in more than 50% of the cases, but was uncommon or absent in normal male breast tissue. High overall methylation status was correlated with high grade (P = 0.003) and was an independent predictor of poor survival (P = 0.048; hazard ratio 2.5). ESR1 and GSTP1 hypermethylation were associated with high mitotic count (P = 0.037 and P = 0.002, respectively) and high grade (both P = 0.001). No correlation with survival was seen for individual genes. Compared with female breast cancers (logistic regression), promoter hypermethylation was less common in a variety of genes, particularly ESR1 (P = 0.005), BRCA1 (P = 0.010) and BRCA2 (P < 0.001). The most frequently hypermethylated genes (MSH6, CDH13, PAX5, PAX6 and WT1) were similar for male and female breast cancer. CONCLUSION: Promoter hypermethylation is common in male breast cancer and high methylation status correlates with aggressive phenotype and poor survival. ESR1 and GSTP1 promoter hypermethylation seem to be involved in development and/or progression of high-grade male breast cancer. Although female and male breast cancer share a set of commonly methylated genes, many of the studied genes are less frequently methylated in male breast cancer, pointing towards possible differences between male and female breast carcinogenesis.

Prognostic value of estrogen receptor α and progesterone receptor conversion in distant breast cancer metastases.

BACKGROUND: Changes in the receptor profile of primary breast cancers to their metastases (receptor conversion) have been described for the estrogen receptor α (ERα) and progesterone receptor (PR). The purpose of this study was to evaluate the impact of receptor conversion for ERα and PR on survival in a large group of distant non-bone breast cancer metastases. METHODS: Receptor conversion was studied by immunohistochemistry in a group of 233 metastatic breast cancer patients. Kaplan-Meier overall survival curves were plotted, and differences between the curves were analyzed by log-rank analysis. The additional prognostic value of conversion to established prognosticators was studied by Cox regression. RESULTS: Overall survival of patients showing conversion from positive to negative ERα or PR, or from negative to positive ERα or PR, or remaining receptor negative was comparable, and significantly worse than patients remaining receptor positive. ERα or PR receptor conversion from positive in the primary breast tumor to negative in distant metastases has independent negative prognostic value. CONCLUSIONS: ERα or PR receptor conversion from positive in the primary breast cancer to negative in distant metastases has negative prognostic value.

Oncogene amplification in male breast cancer: analysis by multiplex ligation-dependent probe amplification.

Gene amplification is an important mechanism for oncogene activation, a crucial step in carcinogenesis. Compared to female breast cancer, little is known on the genetic makeup of male breast cancer, because large series are lacking. Copy number changes of 21 breast cancer related genes were studied in 110 male breast cancers using multiplex ligation-dependent probe amplification. A ratio of >1.3 was regarded indicative for gene copy number gain and a ratio >2.0 for gene amplification. Data were correlated with clinicopathological features, prognosis and 17 genes were compared with a group of female breast cancers. Gene copy number gain of CCND1, TRAF4, CDC6 and MTDH was seen in >40 % of the male breast cancer cases, with also frequent amplification. The number of genes with copy number gain and several single genes were associated with high grade, but only CCND1 amplification was an independent predictor of adverse survival in Cox regression (p = 0.015; hazard ratio 3.0). In unsupervised hierarchical clustering a distinctive group of male breast cancer with poor prognosis (p = 0.009; hazard ratio 3.4) was identified, characterized by frequent CCND1, MTDH, CDC6, ADAM9, TRAF4 and MYC copy number gain. Compared to female breast cancers, EGFR (p = 0.005) and CCND1 (p = 0.041) copy number gain was more often seen in male breast cancer, while copy number gain of EMSY (p = 0.004) and CPD (p = 0.001) and amplification in general was less frequent. In conclusion, several female breast cancer genes also seem to be important in male breast carcinogenesis. However, there are also clear differences in copy number changes between male and female breast cancers, pointing toward differences in carcinogenesis between male and female breast cancer and emphasizing the importance of identifying biomarkers and therapeutic agents based on research in male breast cancer. In addition CCND1 amplification seems to be an independent prognosticator in male breast cancer.

Molecular subtyping of male breast cancer by immunohistochemistry.

Molecular subtyping of breast cancer by gene expression has proven its significance in females. Immunohistochemical surrogates have been used for this classification, because gene expression profiling is not yet routinely feasible. Male breast cancer is rare and large series are lacking. In this study, we used immunohistochemistry for molecular subtyping of male breast cancer. A total of 134 cases of male breast cancer were immunohistochemically stained on tissue microarrays for estrogen receptor (ER), progesterone receptor (PR), HER2 and epidermal growth factor receptor (EGFR), as well as for CK5/6, CK14, and Ki67. HER2 was also assessed by chromogen in situ hybridization. Cases were classified as luminal A (ER+ and/or PR+, and HER2- and Ki67 low), luminal B (ER+ and/or PR+, and HER2+ or Ki67 high), HER2 driven (ER-, PR-, HER2+), basal-like (ER-, PR-, HER2-, CK5/6+ and/or CK14+ and/or EGFR+), or unclassifiable triple-negative (negative for all six markers). Luminal type A was by far the most encountered type of male breast cancers, representing 75% of the cases. Luminal type B was seen in 21% and the remaining 4% of cases were classified as basal-like (n=4) and unclassifiable triple-negative (n=1). No HER2 driven cases were identified. Patients with basal-like cancer were significantly younger (P=0.034). Luminal B type cancers showed significantly higher histological grade (P<0.001), mitotic index (P<0.001), and PR negativity (P=0.005) compared with luminal type A cancers. In conclusion, most male breast cancers are luminal A and luminal B types, whereas basal-like, unclassifiable triple-negative, and HER2 driven male breast cancers are rare. Luminal type B seem to represent a subtype with an aggressive phenotype. This distribution of molecular subtypes in male breast cancer is clearly different compared with female breast cancers, pointing to possible important differences in carcinogenesis.

Fibrotic focus and hypoxia in male breast cancer.

Fibrotic focus is a scar-like lesion near the center of a carcinoma and has been associated with high-grade, lymph node metastases and poor survival in female breast cancers. Hypoxia is suggested to be the crucial link between fibrotic focus and aggressive tumor phenotype and is also itself a poor prognostic marker. We here set out to study fibrotic focus and hypoxia in male breast cancer for the first time. In a group of 134 male breast cancer patients, the presence and size of a fibrotic focus and the expression of three hypoxia-related immunohistochemical stainings, hypoxia-inducible factor-1α, carbonic anhydrase IX and Glut-1 were studied in correlation with clinicopathological features and prognosis. Fibrotic focus was seen in 25% of the male breast cancer cases and was correlated with hypoxia-inducible factor-1α overexpression (P=0.023), high grade (P=0.005), high mitotic activity (P=0.005) and lymph node metastases (P=0.037). Hypoxia-inducible factor-1α-positive tumors were more often high grade (P=0.003) and HER2 amplified (P=0.005). Glut-1 expression was also more common in grade 3 tumors (P=0.038), but no association between carbonic anhydrase IX and any clinicopathological feature was found. Fibrotic focus >8 mm and hypoxia-inducible factor-1α overexpression were correlated with decreased patients' outcome (P=0.035 and 0.008, respectively). Hypoxia-inducible factor-1α overexpression was an independent and the most powerful predictor of survival in multivariate analysis (P=0.029; hazard ratio 2.5). In conclusion, the presence of a fibrotic focus is associated with hypoxia-inducible factor-1α overexpression, and both are associated with aggressive tumor phenotype and poor survival in male breast cancer. These markers seem to have similar clinical importance as previously reported in female breast cancer.

Immunophenotyping of male breast cancer.

AIMS: Male breast cancer is a rare disease, and knowledge of carcinogenesis is limited. Conflicting results, based on small series, have been reported for clinically relevant biomarkers. METHODS AND RESULTS: One hundred and thirty-four cases of male breast cancer were immunohistochemically stained on tissue microarrays for oestrogen receptor (ER), progesterone receptor (PR), androgen receptor, human epidermal growth factor receptor 2 (HER2), BRST2, cyclin D1, bcl-2, p53, p16, p21, Ki67, cytokeratin (CK) 5/6, CK14, and epidermal growth factor receptor. Data were correlated with clinicopathological features and patient outcome. High mitotic count and high grade were correlated with high Ki67, HER2 amplification/overexpression, p53 accumulation, high p21 expression, low PR expression, and low bcl-2 expression. PR negativity (P=0.009) and p53 accumulation (P=0.042) were correlated with decreased 5-year survival and were independent markers for patient outcome in Cox regression. In unsupervised hierarchical clustering, four groups were identified that were correlated with distinctive clinicopathological features. The hormone negative/ER-positive/high-grade cluster was significantly associated with decreased survival (P=0.011) and was an independent prognostic factor in Cox regression. CONCLUSIONS: Several tissue biomarkers are associated with an aggressive phenotype in male breast cancer. PR and p53 are the most promising individual prognostic markers. On the basis of immunophenotype, four distinctive and prognostically relevant male breast cancer groups were identified, indicating that protein expression profiling may be clinically useful in male breast cancer.

Molecular targeted therapies for diffuse large B-cell lymphoma based on apoptosis profiles.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of adult non-Hodgkin lymphoma and is treated with chemotherapy in combination with rituximab. Despite this aggressive therapy, the disease is fatal in 30-40% of patients. Inhibition of the apoptosis signalling pathways is strongly related to response to chemotherapy and eventual clinical outcome. In order to survive, lymphoma cells depend on disruption of the apoptosis pathway by mutations in apoptosis inducing genes or by continuous expression of anti-apoptotic proteins. The development of molecules targeting these apoptosis inhibitors provides a very promising opportunity to specifically target tumour cells without toxicity to non-malignant cells in DLBCL patients. Sensitivity for most of these antagonists can be predicted based on biological markers, suggesting the possibility of pre-defining patients who will most likely benefit from these targeted therapies. Experimental therapies aimed at restoring the upstream apoptosis pathway or targeting apoptosis inhibitors are currently being tested in clinical trials and are expected to be effective particularly in chemotherapy-refractory DLBCL, providing hope for patients who are refractory to current therapies.

Receptor conversion in distant breast cancer metastases.

INTRODUCTION: When breast cancer patients develop distant metastases, the choice of systemic treatment is usually based on tissue characteristics of the primary tumor as determined by immunohistochemistry (IHC) and/or molecular analysis. Several previous studies have shown that the immunophenotype of distant breast cancer metastases may be different from that of the primary tumor ("receptor conversion"), leading to inappropriate choice of systemic treatment. The studies published so far are however small and/or methodologically suboptimal. Therefore, definite conclusions that may change clinical practice could not yet be drawn. We therefore aimed to study receptor conversion for estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in a large group of distant (non-bone) breast cancer metastases by re-staining all primary tumors and metastases with current optimal immunohistochemical and in situ hybridization methods on full sections. METHODS: 233 distant breast cancer metastases from different sites (76 skin, 63 liver, 43 lung, 44 brain and 7 gastro-intestinal) were IHC stained for ERα, PR and HER2, and expression was compared to that of the primary tumor. HER2 in situ hybridization (ISH) was done in cases of IHC conversion or when primary tumors or metastases showed an IHC 2+ result. RESULTS: Using a 10% threshold, receptor conversion by IHC for ERα, PR occurred in 10.3%, 30.0% of patients, respectively. In 10.7% of patients, conversion from "ER+ or PR+" to ER-/PR- and in 3.4% from ER-/PR- to "ER+ or PR+" was found. Using a 1% threshold, ERα and PR conversion rates were 15.1% and 32.6%. In 12.4% of patients conversion from "ER+ or PR+" to ER-/PR-, and 8.2% from ER-/PR- to "ER+ or PR+" occurred. HER2 conversion occurred in 5.2%. Of the 12 cases that showed HER2 conversion by IHC, 5 showed also conversion by ISH. One further case showed conversion by ISH, but not by IHC. Conversion was mainly from positive in the primary tumor to negative in the metastases for ERα and PR, while HER2 conversion occurred equally both ways. PR conversion occurred significantly more often in liver, brain and gastro-intestinal metastases. CONCLUSIONS: Receptor conversion by immunohistochemistry in (non-bone) distant breast cancer metastases does occur, is relatively uncommon for ERα and HER2, and more frequent for PR, especially in brain, liver and gastro-intestinal metastases.

Identification of genes putatively involved in the pathogenesis of diffuse large B-cell lymphomas by integrative genomics.

Diffuse large B-cell lymphomas (DLBCL) are highly heterogeneous with regard to clinical presentation and outcome. DLBCL copy number aberrations have been identified previously, of which the deletion at 6q21-24 was significantly associated with a highly favorable clinical response to chemotherapy. In this study, we aimed to identify genes implicated in this and other genomic regions with recurrent losses and/or gains. To identify implicated genes, we superimposed array comparative genomic hybridization (aCGH) data onto a microarray expression dataset of 42 clinically well-characterized primary nodal DLBCL biopsies. We confirmed that loss of 6q21-24 is significantly associated with a highly favorable clinical response to chemotherapy. Our approach identified 316 significant genes restricted to 32 chromosomal regions, including 24 genes identified at 6q21-24. In an independent dataset, 18% of overexpressed genes in gained regions and 55% of down-regulated genes in deleted regions were validated. In summary, using integrative genomics novel onco and tumor suppressor genes were identified in DLBCL that were not recognized by expression profiling alone.

Favorable outcome in clinically stage II melanoma patients is associated with the presence of activated tumor infiltrating T-lymphocytes and preserved MHC class I antigen expression.

In this study we investigated whether the presence of specific populations of tumor infiltrating lymphocytes (TILs) in diagnostic primary melanoma biopsies are related to outcome in clinically stage II melanoma patients. Moreover, we investigated whether the presence of TILs correlates with expression of MHC class I antigen and MHC class II antigen on tumor cells and/or tumor infiltrating antigen presenting cells. Diagnostic primary melanoma samples of 15 patients with an unfavorable outcome were compared with 20 patients with favorable outcome. Patients were matched for age, gender and Breslow thickness. Biopsies were examined for the presence of granzyme B+, CD8+, CD4+ and CD56+ TILs and for expression of MHC class I antigen and MHC class II antigen on tumor and/or tumor infiltrating cells. A favorable clinical outcome was strongly associated with the presence of GrB+ and CD4+ TILs, with expression of MHC class I antigen on tumor cells and with expression of MHC class II antigen on intratumoral antigen presenting cells. These data strongly support the notion that in melanoma patients the cellular immune response is a major factor in preventing melanoma cell dissemination.

Incidence of enteropathy--associated T-cell lymphoma: a nation-wide study of a population-based registry in The Netherlands.

OBJECTIVE: Enteropathy-associated T-cell lymphomas (EATLs) are T-cell non-Hodgkin lymphomas of the small bowel, which are specifically associated with coeliac disease (CD). To our knowledge no studies have previously reported on the overall incidence of EATLs in the general population. The aim of this study was to investigate the incidence of EATL and the demographic characteristics of patients with EATL in The Netherlands. MATERIAL AND METHODS: A survey of the nation-wide network and registry of histo- and cytopathology reports in The Netherlands (PALGA) was performed. We included all T-cell lymphomas detected between January 2000 and December 2006 that initially presented in the small bowel. Crude and world standardized incidence rates were computed as well as gender- and age-specific incidence rates. Finally, the distribution of characteristics such as the localization, the Marsh classification and method of diagnosis are described. RESULTS: Clinicopathological data were gathered for 116 cases of EATL. The mean age at primary presentation of EATL was 64 years. The crude incidence in the total Dutch population was 0.10/100,000 with an incidence of 2.08/100,000 in the over 50-year-olds. Age-specific incidences were 1.44/100,000 in the 50-59 years age group, 2.92/100,000 in the 60-69 years age group, and 2.53/100,000 in the 70-79 years age group. There was a significant predominance of males (64%, p=0.004, CI 54-72); above the age of 50 the gender-specific incidence was 2.95/100,000 in males versus 1.09/100,000 in females. Most EATLs were localized in the proximal small intestine and the diagnosis was made by surgical resection in the majority of cases. CONCLUSIONS: EATL is a rare disease with an incidence of 0.10 per 100,000 inhabitants per year, occurring in older age, with a peak incidence in the 7th decade. The tumour is mainly localized in the proximal small intestine. Although uncomplicated CD is twice as frequent in female patients, EATL is more prevalent in males.

Inhibition of the intrinsic apoptosis pathway downstream of caspase-9 activation causes chemotherapy resistance in diffuse large B-cell lymphoma.

PURPOSE: Inhibition of the apoptosis cascade is an important cause of therapy resistance in diffuse large B-cell lymphomas (DLBCL). In this study, we investigated possible mechanisms and expression levels of apoptosis-related genes in the apoptosis pathway that may be responsible for differences in chemotherapy sensitivity between DLBCL patients. EXPERIMENTAL DESIGN: Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9-mediated pathway was done using fluorescence-activated cell sorting analysis, Western blot analysis, and immunohistochemistry. RESULTS: Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a small-molecule XIAP antagonist. CONCLUSIONS: We conclude that the intrinsic, caspase-9-mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.

Expression of the polycomb-group gene BMI1 is related to an unfavourable prognosis in primary nodal DLBCL.

BACKGROUND: Clinical outcome in patients with diffuse large B cell lymphomas (DLBCL) is highly variable and poorly predictable. Microarray studies showed that patients with DLBCL with a germinal centre B cell-like (GCB) phenotype have a better prognosis than those with an activated B cell-like (ABC) phenotype. The BMI1 proto-oncogene was identified as one of the genes present in the signature of the ABC type of DLBCL, associated with a poor prognosis. OBJECTIVES: (1) To investigate, in primary nodal DLBCL, the expression of BMI1 and its association with clinical outcome and DLBCL signature; (2) to look for an association between BMI1 expression and the expression of its putative downstream targets p14ARF and p16INK4a. RESULTS: BMI1 expression was found to be associated with poor clinical outcome, but not clearly with an ABC-like phenotype of DLBCL. Expression of BMI1 was frequently, but not always, related to low levels of expression of p14ARF and p16INK4a. CONCLUSION: Expression of BMI1 is associated with an unfavourable clinical outcome of primary nodal DLBCL.

Frequent overexpression of aurora B kinase, a novel drug target, in non-small cell lung carcinoma patients.

The serine/threonine protein kinase aurora B, a key regulator of mitosis, is emerging as a novel drug target for cancer treatment. Aurora B overexpression has been previously documented by immunohistochemistry in several types of human tumors. We assessed aurora B expression in a series of 160 non-small cell lung cancer (NSCLC) samples (60% stage I, 21% stage II, 11% stage III, and 8% stage IV). In addition, we determined the expression of survivin and p16, two molecules also involved in cell cycle control. Aurora B was expressed selectively in tumor cells compared with normal epithelium. Aurora B expression was significantly correlated with expression of survivin in the nucleus (P < 0.0001), but not with expression of p16 (P = 0.134). High aurora B expression levels were significantly associated with older age (P = 0.012), male sex (P = 0.013), squamous cell carcinoma histology (P = 0.001), poor tumor differentiation grade (P = 0.007), and lymph node invasion (P = 0.037), in the subset of radically resected patients in our series. In addition, aurora B expression predicted shorter survival for the patients with adenocarcinoma histology, at both univariate (P = 0.020) and multivariate (P = 0.012) analysis. Survivin expression levels were neither associated with patient clinicopathologic characteristics nor with survival. However, expression of survivin in the nucleus was preferentially detected in stage I and II than in stage III and IV (P = 0.007) in the overall series of NSCLC samples. Taken together, our results suggest that aurora B may represent a valid target in NSCLC.

18F-FDG PET versus CT for the detection of enteropathy-associated T-cell lymphoma in refractory celiac disease.

UNLABELLED: Refractory celiac disease (RCD) can evolve into enteropathy-associated T-cell lymphoma (EATL). 18F-FDG PET has been reported to discriminate between RCD and EATL. Because prospective data are lacking, we designed a prospective study to evaluate the potential of 18F-FDG PET for detection of EATL in patients with RCD and compared the results with those obtained using abdominal CT in a referral center. METHODS: Between April 2003 and April 2005, 8 consecutive patients (median age, 66 y; range, 52-89 y) with EATL and 30 patients (median age, 61 y; range, 44-71 y) with RCD were included. CT and 18F-FDG PET were performed on all patients. Histologic evidence of EATL was identified in tissue samples obtained during upper gastrointestinal endoscopy or surgical resection. RESULTS: Villous atrophy was found in all patients with RCD and all (except 1) patients with EATL in nontumoral mucosa. Histologic examination of 1 patient with EATL localized in the duodenum showed intraepithelial lymphocytosis only. 18F-FDG PET could reveal sites histologically proven to be EATL in all 8 patients, whereas CT showed normal findings in 1 patient with EATL. 18F-FDG PET detected unsuspected extraintestinal sites affected by EATL in 2 patients. CT showed abnormalities such as a thickened small-bowel wall or lymphadenopathy in 14 patients with RCD lacking evidence of EATL at follow-up. 18F-FDG PET findings were positive in 3 and equivocal in another 3 patients with RCD. 18F-FDG PET was more sensitive and specific than CT (100% vs. 87% and 90% vs. 53%, respectively). CONCLUSION: Our data show that 18F-FDG PET is more sensitive in detecting EATL in patients with RCD than is CT. 18F-FDG PET, in addition to conventional CT, is recommended for evaluating patients with RCD.

The expression of TUCAN, an inhibitor of apoptosis protein, in patients with advanced non-small cell lung cancer treated with chemotherapy.

BACKGROUND: TUCAN is a caspase recruitment domain (CARD)-containing protein involved in tumor biology by regulating apoptosis and the NFkappaB pathway. Inhibition of caspase-9 may cause drug resistance. The pattern of expression, localization and prognostic value of TUCAN in the tumors of patients with non-small cell lung cancer (NSCLC) treated with chemotherapy were assessed in this study. MATERIALS AND METHODS: Using immunohistochemistry, the expression and localization of TUCAN was evaluated in forty-nine tumor specimens from patients with NSCLC who underwent neoadjuvant chemotherapy (32 stage IIB or IIIA), or palliative chemotherapy (17 stage IIIB or IV). The correlation between TUCAN expression and subcellular localization, major patient characteristics, response to the treatment and overall survival were assessed. RESULTS: TUCAN expression was detectable in 34 out of 49 (69%) tumor specimens. Among the positively-stained specimens, three patterns of localization were observed: 5 samples (11%) showed exclusive nuclear localization, 13 samples (27%) contained only cytoplasmic staining and 15 (31%) showed both cytoplasmic and nuclear localization. There was no significant correlation between the localization of TUCAN and response to chemotherapy. Although TUCAN expression was not correlated with outcome, interestingly, exclusive cytoplasmic localization of TUCAN predicted shorter survival (p = 0.027). CONCLUSION: Our results suggest that differential localization of TUCAN may be a prognostic factor for NSCLC, despite the lack of predictive value for response to chemotherapy.

Expression of the apoptosis inhibitor protease inhibitor 9 predicts clinical outcome in vaccinated patients with stage III and IV melanoma.

PURPOSE: There have been reports of successful treatment of metastatic melanoma patients with active specific immunotherapy (ASI) using irradiated autologous tumor cell vaccination. It is still unknown why some patients respond and others do not. Tumor cells can evade the immune system, for example through interference with antigen presentation by down-regulation of MHC molecules or expressing proteins interfering with cytotoxic lymphocyte-induced apoptosis like the granzyme B antagonist protease inhibitor 9 (PI-9). EXPERIMENTAL DESIGN: PI-9 expression was detected in melanoma cell lines. To investigated if PI-9 is important in the response to ASI, paraffin-embedded tissues from stage III or IV melanoma patients were stained. RESULTS: PI-9 is expressed in melanoma cells and expression in metastasized melanoma cells is, in this group of patients, an adverse prognostic marker with regard to overall and disease-free survival. Moreover, loss of MHC-1 expression frequently occurs during tumor progression but is not associated with poor clinical outcome. Interestingly, melanoma patients with a favorable clinical outcome after ASI therapy usually have high percentages of activated (granzyme B-positive) tumor-infiltrating lymphocytes at time of first diagnosis and low percentages of activated lymphocytes at time of recurrent tumor. CONCLUSIONS: Expression of PI-9 in metastatic melanoma cells is associated with unfavorable clinical outcome whereas MHC-1 down-regulation is not. Although it cannot be proven that PI-9 expression is directly responsible for failure of immunotherapy, these data suggest that expression of PI-9 could be an important immune escape mechanism and that modulation of this inhibitor may enhance the efficacy of immunotherapy.

FANCD2 expression in advanced non-small-cell lung cancer and response to platinum-based chemotherapy.

Fanconi's anemia (FA) is a genetically heterogeneous disease characterized by cancer susceptibility and hypersensitivity to cross-linking agents such as cisplatin. Recently, inactivation of the FA pathway has been proposed to contribute to genomic instability and an increased sensitivity to cisplatin-based therapy in a subset of ovarian tumors. Platinum-based chemotherapy constitutes standard systemic therapy for advanced non-small-cell lung cancer (NSCLC), but resistance to platinum chemotherapy is common. In this study, we evaluated the status of the FA pathway in tumor samples derived from patients with NSCLC in relation to their response to platinum-based chemotherapy. For this purpose, we assessed the expression of FANCD2 protein (a marker for FA pathway functioning) by immunohistochemistry in tumor specimens from 47 patients treated with platinum-based chemotherapy. FANCD2 expression could be detected in 32% of the cases (15 of 47). Expression of FANCD2 was not correlated with any patient or tumor characteristics, and FANCD2 expression was not a predictor of response to chemotherapy or patient survival. In conclusion, the activation status of the FA pathway had no value in predicting sensitivity to platinum-based chemotherapy in patients with advanced NSCLC.