A super-SILAC based proteomics analysis of diffuse large B-cell lymphoma-NOS patient samples to identify new proteins that discriminate GCB and non-GCB lymphomas.

Diffuse large B-cell lymphoma-not otherwise specified (DLBCL-NOS) is a large and heterogeneous subgroup of non-Hodgkin lymphoma. DLBCL can be subdivided into germinal centre B-cell like (GCB) and activated B-cell like (ABC or non-GCB) using a gene-expression based or an immunohistochemical approach. In this study we aimed to identify additional proteins that are differentially expressed between GCB and non-GCB DLBCL. A reference super-SILAC mix, including proteins of eight B-cell lymphoma cell lines, was mixed with proteins isolated from seven non-GCB DLBCL and five GCB DLBCL patient tissue samples to quantify protein levels. Protein identification and quantification was performed by LC-MS. We identified a total of 4289 proteins, with a four-fold significant difference in expression between non-GCB and GCB DLBCL for 37 proteins. Four proteins were selected for validation in the same cases and replication in an independent cohort of 47 DLBCL patients by immunohistochemistry. In the validation cohort, we observed a non-significant trend towards the same differential expression pattern as observed in the proteomics. The replication study showed significant and consistent differences for two of the proteins: expression of glomulin (GLMN) was higher in GCB DLBCL, while expression of ribosomal protein L23 (RPL23) was higher in non-GCB DLBCL. These proteins are functionally linked to important pathways involving MYC, p53 and angiogenesis. In summary, we showed increased expression of RPL23 and decreased expression of GLMN in non-GCB compared to GCB DLBCL on purified primary DLBCL patient samples and replicated these results in an independent patient cohort.

Treatment of sporadic Burkitt lymphoma in adults, a retrospective comparison of four treatment regimens.

Burkitt lymphoma is an aggressive B cell malignancy accounting for 1-2% of all adult lymphomas. Treatment with dose-intensive, multi-agent chemotherapy is effective but associated with considerable toxicity. In this observational study, we compared real-world efficacy, toxicity, and costs of four frequently employed treatment strategies for Burkitt lymphoma: the Lymphome Malins B (LMB), the Berlin-Frankfurt-Münster (BFM), the HOVON, and the CODOX-M/IVAC regimens. We collected data from 147 adult patients treated in eight referral centers. Following central pathology assessment, 105 of these cases were accepted as Burkitt lymphoma, resulting in the following treatment groups: LMB 36 patients, BFM 19 patients, HOVON 29 patients, and CODOX-M/IVAC 21 patients (median age 39 years, range 14-74; mean duration of follow-up 47 months). There was no significant difference between age, sex ratio, disease stage, or percentage HIV-positive patients between the treatment groups. Five-year progression-free survival (69%, p = 0.966) and 5-year overall survival (69%, p = 0.981) were comparable for all treatment groups. Treatment-related toxicity was also comparable with only hepatotoxicity seen more frequently in the CODOX/M-IVAC group (p = 0.004). Costs were determined by the number of rituximab gifts and the number of inpatients days. Overall, CODOX-M/IVAC had the most beneficial profile with regards to costs, treatment duration, and percentage of patients completing planned treatment. We conclude that the four treatment protocols for Burkitt lymphoma yield nearly identical results with regards to efficacy and safety but differ in treatment duration and costs. These differences may help guide future choice of treatment.

High prevalence of oncogenic MYD88 and CD79B mutations in diffuse large B-cell lymphomas presenting at immune-privileged sites.

Activating mutations in CD79 and MYD88 have recently been found in a subset of diffuse large B-cell lymphoma (DLBCL), identifying B-cell receptor and MYD88 signalling as potential therapeutic targets for personalized treatment. Here, we report the prevalence of CD79B and MYD88 mutations and their relation to established clinical, phenotypic and molecular parameters in a large panel of DLBCLs. We show that these mutations often coexist and demonstrate that their presence is almost mutually exclusive with translocations of BCL2, BCL6 and cMYC, or Epstein-Bar virus infection. Intriguingly, MYD88 mutations were by far most prevalent in immune-privileged site-associated DLBCL (IP-DLBCL), presenting in central nervous system (75%) or testis (71%) and relatively uncommon in nodal (17%) and gastrointestinal tract lymphomas (11%). Our results suggest that MYD88 and CD79B mutations are important drivers of IP-DLBCLs and endow lymphoma-initiating cells with tissue-specific homing properties or a growth advantage in these barrier-protected tissues.

FADD expression is associated with regional and distant metastasis in squamous cell carcinoma of the head and neck.

AIMS: The Fas-associated death domain gene (FADD) is often overexpressed in squamous cell carcinoma of the head and neck (HNSCC), and is considered to be a driver gene in amplification of the chromosomal 11q13.3 region. Amplification of 11q13.3 is associated with increased metastasis in HNSCC and breast cancer. The aim of this study was to investigate the association between FADD protein expression in advanced-stage HNSCC and clinicopathological features and outcome. METHODS AND RESULTS: Tumour tissues of 177 HNSCC patients uniformly treated with primary surgery and postoperative radiotherapy were collected. FADD expression was assessed on pretreatment tumour biopsies using immunohistochemistry. High FADD expression was detected in 44% of the HNSCC patients. High expression was associated with an increased rate of lymph node metastasis (P = 0.001) and with a shorter distant metastasis-free interval (DMFI) (HR 2.6, 95% CI 1.0-6.7, P = 0.046) when lymph node metastases were present. CONCLUSIONS: Our data show that an increase in FADD expression is associated with a higher incidence of lymph node metastasis at presentation, and with shorter DMFI when lymph node metastases are present. High FADD expression in the primary tumour could be a useful marker to select patients for systemic treatment strategies that reduce the risk of distant metastases.

Tryptase and histamine metabolites as diagnostic indicators of indolent systemic mastocytosis without skin lesions.

BACKGROUND: Risk indicators of indolent systemic mastocytosis (ISM) in adults with clinical suspicion of ISM without accompanying skin lesions [urticaria pigmentosa (UP)] are lacking. This study aimed at creating a decision tree using clinical characteristics, serum tryptase, and the urinary histamine metabolites methylimidazole acetic acid (MIMA) and methylhistamine (MH) to select patients for bone marrow investigations to diagnose ISM. METHODS: Retrospective data analysis of all adults, in whom bone marrow investigations were performed to diagnose ISM, was carried out. RESULTS: In total, 142 patients were included. SM was absent in all 44 patients with tryptase <10 µg/l, in 45 of 98 (46%) patients with tryptase ≥10 µg/l and in 18 of 52 patients (35%) with tryptase >20 µg/l. Above 43 µg/l, all patients had ISM (n = 11). Male gender, insect venom anaphylaxis as presenting symptom, tryptase, MIMA, and MH were independent ISM predictors. If tryptase was ≥10 µg/l, the diagnostic accuracy of MIMA and MH was high (areas under the ROC curve 0.92). CONCLUSIONS: In suspected patients without UP, the ISM risk is very low (if present at all) if tryptase is <10 µg/l. If tryptase is ≥10 µg/l, this risk depends on MIMA and MH, being low if these are normal, but high if these are elevated. Male gender and insect venom anaphylaxis are additional risk indicators. We recommend refraining from bone marrow examinations in suspected patients without UP if tryptase is <10 µg/l. Our results question the reliability of the minor diagnostic World Health Organization criterion of tryptase >20 µg/l.

Gene expression profiling in the leukemic stem cell-enriched CD34+ fraction identifies target genes that predict prognosis in normal karyotype AML.

In order to identify acute myeloid leukemia (AML) CD34(+)-specific gene expression profiles, mononuclear cells from AML patients (n=46) were sorted into CD34(+) and CD34(-) subfractions, and genome-wide expression analysis was performed using Illumina BeadChip Arrays. AML CD34(+) and CD34(-) gene expression was compared with a large group of normal CD34(+) bone marrow (BM) cells (n=31). Unsupervised hierarchical clustering analysis showed that CD34(+) AML samples belonged to a distinct cluster compared with normal BM and that in 61% of the cases the AML CD34(+) transcriptome did not cluster together with the paired CD34(-) transcriptome. The top 50 of AML CD34(+)-specific genes was selected by comparing the AML CD34(+) transcriptome with the AML CD34(-) and CD34(+) normal BM transcriptomes. Interestingly, for three of these genes, that is, ankyrin repeat domain 28 (ANKRD28), guanine nucleotide binding protein, alpha 15 (GNA15) and UDP-glucose pyrophosphorylase 2 (UGP2), a high transcript level was associated with a significant poorer overall survival (OS) in two independent cohorts (n=163 and n=218) of normal karyotype AML. Importantly, the prognostic value of the continuous transcript levels of ANKRD28 (OS hazard ratio (HR): 1.32, P=0.008), GNA15 (OS HR: 1.22, P=0.033) and UGP2 (OS HR: 1.86, P=0.009) was shown to be independent from the well-known risk factors FLT3-ITD, NPM1c(+) and CEBPA mutation status.

Translocations involving 8q24 in Burkitt lymphoma and other malignant lymphomas: a historical review of cytogenetics in the light of todays knowledge.

Burkitt lymphoma (BL) has a characteristic clinical presentation, morphology, immunophenotype and primary chromosomal aberration, that is, the translocation t(8;14)(q24;q32) or its variants. However, diagnostic dilemmas may arise in daily practice due to overlap of BL with subsets of other aggressive, mature B-cell lymphomas such as diffuse large B-cell lymphomas (DLBCL). Recently, two gene expression studies have described a distinct molecular profile for BL, but also showed the persistence of some cases intermediate between BL and DLBCL. An alternative approach to define BL is to consider (cyto)genetic data, in particular chromosomal abnormalities other than the t(8;14) or its variants. In this review the 'Mitelman Database of Chromosome Aberrations in Cancer,' harboring the majority of all published neoplasia-related karyotypes, was explored to define a cytogenetic profile of 'true' BL. This core subset of BL showed a very low complexity of chromosomal abnormalities with 40% of the cases having the IG-MYC fusion as the sole abnormality. In the remaining cases, additional recurrent but partially exclusive abnormalities included gains at chromosomes 1q, 7 and 12, and losses of 6q, 13q32-34 and 17p. Within the core subset, no differences were found between pediatric and adult patients. In addition, the genetic profile of the core subset was significantly different from BL with an 8q24 breakpoint not affecting one of the three immunoglobulin loci, BL with a translocation involving 18q21/BCL2, 3q27/BCL6 or 11q13/BCL1, additionally to a breakpoint at 8q24/MYC, and from other morphological types of lymphomas with an 8q24/MYC breakpoint. These groups showed a higher cytogenetic complexity than the core subset of BL. BL without a detectable 8q24/MYC breakpoint might be heterogeneous and deserves further studies. We suggest that, concordant with the WHO classification to be published in 2008, the diagnosis of BL should be restricted to cases with expression of CD10 and BCL6, absence or very weak expression of BCL2 protein, a homogeneously very high proliferation index and a proven IG-MYC translocation without evidence of a chromosomal translocation typical for other lymphoma entities. In addition, a high number of nonspecific cytogenetic abnormalities should suggest need for a critical review of the diagnosis of BL.

Malignant aldosterone-producing adrenal tumour: reoccurrence with glucocorticoid excess without hyperaldosteronism.

We describe a case of hypokalaemic hypertension due to hyperaldosteronism caused by a unilateral adrenocortical tumour with unfavourable histopathology suggestive of malignancy. After removal, the aldosterone excess disappeared. The patient's clinical course was uneventful, until she presented with extensive metastases of adrenal carcinoma four years later. Biochemical abnormalities were now consistent with glucocorticoid excess without hyperaldosteronism. She died four months later. Although malignant aldosterone-producing adrenal tumours are very rare, the present case underscores that clinicians should be aware that primary hyperaldosteronism can occur in the context of adrenocortical carcinoma.

Overexpression of intrinsic hypoxia markers HIF1alpha and CA-IX predict for local recurrence in stage T1-T2 glottic laryngeal carcinoma treated with radiotherapy.

PURPOSE: To examine the prognostic value of three endogenous hypoxia markers (hypoxia inducible factor 1 alpha subunit [HIF1 alpha], carbonic anhydrase IX [CA-IX], and glucose transporter type 1 [GLUT-1]) on the clinical outcome in patients with early-stage glottic carcinoma primarily treated with radiotherapy (RT) and to determine the predictive hypoxic profile to choose the optimal treatment of early-stage laryngeal carcinoma. METHODS AND MATERIALS: Immunohistochemistry for HIF1 alpha, CA-IX, and GLUT-1 was performed on formalin-fixed, paraffin-embedded, pretreatment tissue samples of 91 glottic squamous cell carcinoma specimens. The patient group consisted only of those with early-stage (T1-T2) glottic carcinoma, and all patients were treated with RT only. Relative tumor staining was scored on the tissue samples. Receiver operating curve analysis was performed to determine the optimal cutoff value for each tumor marker. Cox regression analyses for the variables HIF1 alpha, CA-IX, GLUT-1, gender, age, hemoglobin level, T category, N category, tobacco use, and alcohol use were performed with local control and overall survival as endpoints. RESULTS: HIF1 alpha overexpression in early-stage glottic carcinoma correlated significantly with worse local control (hazard ratio [HR], 3.05; p = 0.021) and overall survival (HR, 2.92; p = 0.016). CA-IX overexpression correlated significantly with worse local control (HR, 2.93; p = 0.020). GLUT-1 overexpression did not show any correlation with the clinical outcome parameters. Tumors with a nonhypoxic profile (defined as low HIF1 alpha and low CA-IX expression) had significantly better local control (HR, 6.32; p = 0.013). CONCLUSION: The results of our study have shown that early-stage glottic laryngeal carcinomas with low HIF1 alpha and CA-IX expression are highly curable with RT. For this group, RT is a good treatment option. For tumors with HIF1 alpha or CA-IX overexpression, hypoxic modification before RT or primary surgical treatment should be considered.

miRNA analysis in B-cell chronic lymphocytic leukaemia: proliferation centres characterized by low miR-150 and high BIC/miR-155 expression.

Several miRNAs have been reported to be associated with immunoglobulin heavy chain (IgH) mutation and ZAP-70 expression status in blood samples of B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma (B-CLL/SLL). In the bone marrow and lymphoid tissues, proliferation centres (PCs) represent an important site of activation and proliferation of the neoplastic cells, suggesting that these tissues better reflect the biology of CLL than circulating blood cells. We collected 33 lymph nodes and 37 blood CLL samples and analysed IgH mutation status and ZAP-70 expression status. Expression of 15 miRNAs was analysed by qRT-PCR and RNA-ISH. Sixty-three per cent of the lymph node cases contained mutated IgH genes and 49% of the lymph node cases were ZAP-70-positive, and a significant correlation was observed between ZAP-70 expression and IgH mutation status. Of the blood CLL samples, 49% contained mutated IgH sequences. The miRNA expression pattern in CLL lymph node and blood samples was very similar. Three of 15 miRNAs (miR-16, miR-21, and miR-150) showed a high expression level in both blood and lymph node samples. No difference was observed between ZAP-70-positive or -negative and between IgH-mutated or unmutated cases. No correlation was found between miR-15a and miR-16 expression levels and 13q14 deletion in the blood CLL samples. RNA in situ hybridization (ISH) revealed strong homogeneous staining of miR-150 in the tumour cells outside the PCs. In reverse BIC/pri-miR-155 expression was observed mainly in individual cells including prolymphocytes of the PCs. This reciprocal pattern likely reflects the different functions and targets of miR-150 and miR-155.

Primary testicular diffuse large B-cell lymphomas have activated B-cell-like subtype characteristics.

Diffuse large B-cell lymphomas (DLBCLs) constitute a heterogeneous group of lymphomas in which germinal centre B-cell-like and activated B-cell-like subtypes can be discerned based on pathology, clinical presentation, and gene expression patterns. Testicular DLBCLs form an immune-privileged site-related subgroup of DLBCLs with an unfavourable prognosis. In the present study, cDNA microarray analysis, immunohistochemistry for CD10, Bcl6 and MUM1, and somatic hypermutation analysis of the immunoglobulin heavy chain gene rearrangements were used to determine the subtype of primary testicular DLBCL. Immunohistochemistry revealed 14/22 testicular DLBCLs with an activated B-cell-like immunophenotype and 8/22 with an ambiguous immunophenotype co-expressing CD10 and high levels of MUM1. cDNA microarray analysis of these 22 and four additional cases showed a uniform activated B-cell-like gene expression pattern for both immunophenotypes. Somatic hypermutation analysis of immunoglobulin heavy chain genes showed a very high mutation load in seven cases tested, but intraclonal heterogeneity was found at low level in only one of these cases. It is concluded that primary testicular DLBCLs have uniform activated B-cell-like subtype characteristics despite a number of cases showing an ambiguous immunophenotype.

Short intensive sequential therapy followed by autologous stem cell transplantation in adult Burkitt, Burkitt-like and lymphoblastic lymphoma.

The feasibility and efficacy of up-front high-dose sequential chemotherapy followed by autologous stem cell transplantation (ASCT) in previously untreated adults (median age 33 years; range 15-64) with Burkitt lymphoma (BL), Burkitt-like lymphoma (BLL) or lymphoblastic lymphoma (LyLy), both without central nervous system or extensive bone marrow involvement was investigated in a multicenter phase II study. Treatment consisted of two sequential high-dose chemotherapy induction courses incorporating prednisone, cyclophosphamide, doxorubicin, etoposide and mitoxantrone, without high-dose methotrexate or high-dose cytarabine. Patients with at least PR went on with BEAM and ASCT. Protocol treatment was completed by 23/27 (85%) BL/BLL and 13/15 (87%) LyLy patients. Median treatment duration until BEAM was 70 (range: 50-116) days. No toxic deaths occurred. Response to treatment was complete response (CR) 81% and partial response (PR) 11% for BL/BLL, CR 73% and PR 20% for LyLy. At a median follow-up of 61 months of patients still alive, six BL/BLL and eight LyLy patients have died. The actuarial 5-year overall and event-free survival estimates are 81 and 73% for BL/BLL vs 46 and 40% for LyLy patients. In conclusion, this short up-front high-dose sequential chemotherapy regimen, followed by ASCT is highly effective in adults with BL/BLL with limited bone marrow involvement, but less so in patients with LyLy.

Changes in salivary gland immunohistology and function after rituximab monotherapy in a patient with Sjogren's syndrome and associated MALT lymphoma.

OBJECTIVES: To report the successful use of rituximab on salivary gland immunohistology and function in a patient with Sjogren's syndrome (SS) and associated MALT lymphoma. CASE REPORT: The patient was a 42 year old woman with primary SS and associated MALT lymphoma located in the parotid gland and the hard palate. Four infusions of rituximab (375 mg/m(2)) weekly resulted in complete remission of the lymphoma. An incision biopsy of the parotid gland before and after treatment showed improvement of the (immuno)histopathological characteristics of SS, with possible regeneration of salivary gland tissue. Furthermore, salivary analysis showed decreased inflammatory characteristics and increased stimulated salivary flow. DISCUSSION: Rituximab is a promising agent in the treatment of SS associated MALT lymphoma. In addition to the effect on MALT lymphoma, B cell depletion by rituximab may also attenuate the activity of SS. This case report is the first to describe the effect of rituximab on histological and sialometric/chemical characteristics of SS. The efficacy of rituximab in the treatment of SS warrants further investigation.

Lack of prognostic significance of BCL2 and p53 protein overexpression in elderly patients with diffuse large B-cell non-Hodgkin's lymphoma: results from a population-based non-Hodgkin's lymphoma registry.

The prognostic significance of age was studied in 372 patients with diffuse large B-cell non-Hodgkin's lymphoma, in relation to the prognostic factors of overexpressed BCL2 and p53 oncoprotein. Overexpression of BCL2 and p53 oncoprotein was defined when more than 50% of the tumor cells showed positive staining. The data were analyzed with respect to the age groups < 65 and > or = 65 years of age. There was a trend for BCL2 overexpression to occur significantly more often among patients older than 65 years of age (P = 0.065). Patients with BCL2 overexpression showed significantly inferior disease free survival rate, but only for patients younger than 65 years (log-rank test P = 0.0002), and the overexpression showed also an independent prognostic significance (P < 0.001). With respect to overexpressed p53 a significant difference was reached for complete remission rate (P = 0.01) and 5-year survival rate (log-rank test P = 0.04), again only for the younger age group. When the analyses were repeated for the older patients who had been treated adequately, the same lack of significance was found, both for BCL2 and p53. This study demonstrates that the negative prognostic value of overexpressed BCL2 and p53 protein is not of concern for patients older than 65 years of age. Among elderly patients the International Prognostic Index score seems the predominant risk factor for inferior prognosis.

Primary extranodal non-Hodgkin's lymphoma (NHL): the impact of alternative definitions tested in the Comprehensive Cancer Centre West population-based NHL registry.

BACKGROUND: The definition of primary extranodal non-Hodgkin's lymphoma (NHL) is a controversial issue, especially in patients where both nodal and extranodal sites are involved. PATIENTS AND METHODS: The impact of different definitions of primary extranodal NHL on incidence and prognosis is explored using data from a population-based NHL registry. RESULTS: Using liberal criteria, 389 (34%) cases were classified as primary extranodal NHL. Overall survival (OS) rates of nodal and extranodal NHL patients defined this way were comparable; however, extranodal NHL patients had a better disease-free survival (DFS). When strict criteria were applied, 231 cases (20%) were classified as primary extranodal NHL. OS and DFS rates of extranodal NHL patients defined this way were superior to nodal NHL patients; however, the difference in OS was reversed after correction for differences in International Prognostic Index and malignancy grade. CONCLUSION: This study illustrates the selection bias that is introduced when a strict definition of primary extranodal NHL, that excludes cases with disseminated disease, is used. Patients with primary extranodal NHL were found to have a superior DFS, irrespective of which definition of primary extranodal NHL was used.