Johan Linderoth

Evaluation of immunophenotype in diffuse large B-cell lymphoma and its impact on prognosis.

Diffuse large B-cell lymphoma (DLBCL) has been shown to be comprised of at least two prognostic entities, depending on its resemblance to normal germinal center or activated B cells, using global gene expression profiling. Also, the expression patterns of bcl-6, CD10 and IRF-4 (also known as MUM1) have been suggested as alternative means of identifying the germinal- and nongerminal center (activated B-cell like) groups. In the present study, we evaluated by immunohistochemistry the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 in a large material of 161 DLBCL patients. Using two different approaches, patients with germinal center phenotype displayed a significantly better survival than the nongerminal center group. Positive staining for bcl-6 or CD10 predicted for superior survival, while expression of IRF-4 alone showed no association with prognosis. Furthermore, expression of bcl-2 was associated with worse event-free survival and overall survival. In a multivariate analysis, a high international prognostic index score (3–5), non-GC phenotype and bcl-2 were independent adverse prognostic factors. Here we confirm the prognostic importance of determining the germinal- or nongerminal center phenotype in patients with DLBCL.

Protein expression and cellular localization in two prognostic subgroups of diffuse large B-cell lymphoma: Higher expression of ZAP70 and PKC-beta II in the non-germinal center group and poor survival in patients deficient in nuclear PTEN

Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) show varying responses to conventional therapy, and this might be contributed to the differentiation stage of the tumor B-cells. The aim of the current study was to evaluate a panel of kinases (ZAP70, PKC-β I and II and phosphorylated PKB/Akt) and phosphatases (PTEN, SHP1 and SHP2) known to be frequently deregulated in lymphoid malignancies. De novo DLBCL cases were divided into two subgroups, the germinal center (GC) group (14/28) and the non-germinal center (non-GC) or activated B-cell (ABC) group (14/28). ZAP70 and PKC-β II were expressed in a significantly higher percentage of tumor cells in the clinically more aggressive non-GC group compared with the prognostically favourable GC group. Also, the subcellular localization of PKC-β I and II differed in DLBCL cells, with the PKC-β I isoform being expressed in both the cytoplasm and nucleus, while PKC-β II was found exclusively in the cytoplasm. Loss of nuclear PTEN correlated with poor survival in cases from both subgroups. In addition, five cell lines of DLBCL origin were analyzed for protein expression and for mRNA levels of PTEN and SHP1. For the first time, we show that ZAP70 is expressed in a higher percentage of tumor cells in the aggressive non-GC subgroup of DLBCL and that PKC-β I and II are differently distributed in the two prognostic subgroups of de novo DLBCL.

A subset of CD5- diffuse large B-cell lymphomas expresses nuclear cyclin D1 with aberrations at the CCND1 locus.

In 231 diffuse large B-cell lymphomas, the expression of cyclin D1 and CD5 was evaluated. All cases were CD5-. Ten (4.3%) were positive for cyclin D1 and were subjected to fluorescence in situ hybridization at the CCND1 locus. One case showed the t(11;14). In another case, the telomeric probe signal for cyclin D1 was lost in most tumor cells, and in a small proportion of the cells, there were fluorescence signals indicative of the t(11;14). Two other cases displayed additional cyclin D1 signals in the absence of the t(11;14). All cases but 1 were positive for bcl-6 or MUM1, disfavoring the possibility of misdiagnosed blastoid variants of CD5- mantle cell lymphomas. Thus, contrary to the current view, there seems to exist a certain number of cyclin D1+ and CD5- diffuse large B-cell lymphomas, some of which have structural aberrations at the CCND1 locus, including the t(11;14).

Genes associated with the tumour microenvironment are differentially expressed in cured versus primary chemotherapy-refractory diffuse large B-cell lymphoma.

In order to identify genes associated with primary chemotherapy‐resistance, gene expression profiles (GEP) in tumour tissue from 37 patients with de novo diffuse large B‐cell lymphoma (DLBCL), stage II–IV, either in continuous complete remission (n = 24) or with progressive disease during primary treatment (n = 13), were examined using spotted 55K oligonucleotide arrays. Immunohistochemistry was used for confirmation at the protein level. The top 86 genes that best discriminated between the two cohorts were chosen for further analysis. Only seven of 86 genes were overexpressed in the refractory cohort, e.g. RABGGTB and POLE, both potential targets for drug intervention. Seventy‐nine of 86 genes were overexpressed in the cured cohort and mainly coded for proteins expressed in the tumour microenvironment, many of them involved in proteolytic activity and remodelling of extra cellular matrix. Furthermore, major histocompatibility complex class I molecules, CD3D and ICAM1 were overexpressed, indicating an enhanced immunological reaction. Immunohistochemistry confirmed the GEP results. The frequency of tumour infiltrating lymphocytes, macrophages, and reactive cells expressing ICAM‐1, lysozyme, cathepsin D, urokinase plasminogen activator receptor, signal transducer and activator of transcription 1, and galectin‐3 was higher in the cured cohort. These findings indicate that a reactive microenvironment has an impact on the outcome of chemotherapy in DLBCL.

Tissue microarray is inappropriate for analysis of BCL6 expression in diffuse large B-cell lymphoma

Objective:  In this study, our aim was to investigate how different immunohistochemical techniques may influence the result of BCL6 positivity and categorization in germinal center (GC) and non‐GC derived diffuse large B‐cell lymphoma (DLBCL), as it has been proposed that classification of DLBCL according to cell‐of‐origin by immunohistochemistry may be performed as a routine procedure in the diagnostic work‐up. However, a number of technical issues need to be solved before introducing this as a standard technique.

Identification of molecular targets associated with transformed diffuse large B cell lymphoma using highly purified tumor cells.

Follicular lymphoma (FL) frequently transforms into the more aggressive diffuse large B cell lymphoma (DLBCL‐tr), but no protein biomarkers have been identified for predictive or early diagnosis. Gene expression analyses have identified genes changing on transformation but have failed to be reproducible in different studies, reflecting the heterogeneity within the tumor tissue and between tumor samples. Gene expression analyses on Affymetrix Human Genome U133 Plus 2.0 arrays were performed, using flow cytometry sorted tumor cells derived from FL and transformed DLBCL. To identify molecular targets associated with the transformation, subsequent immunohistochemistry (IHC) analyses of the corresponding proteins were performed. Using highly purified cells, this study identified 163 genes, which were significantly deregulated during the transformation in a majority of cases. Among the upregulated transcripts, 13 genes were selected for validation using IHC, based on the availability of commercial antibodies, and galectin‐3 and NEK2 proteins specifically identify DLBCL‐tr, when compared with FL. We demonstrate that by purifying tumor cells through cell sorting, thereby reducing the heterogeneity due to infiltrating cells, it was possible to identify distinct differences between tumor entities rather than variations due to cellular composition. Galectin‐3 and NEK2 both identified a subgroup of DLBCL‐tr, and the function of these protein markers also suggests a biological role in the transformation process. Am. J. Hematol. 2009.

Dominant human herpesvirus type 8 RNA transcripts in classical and AIDS-related Kaposi's sarcoma

Skin biopsy sections of Kaposis sarcoma (KS) from 25 patients (5 AIDS‐related, 20 classical cases) were histologically staged and hybridized in situ with oligonucleotide probes for constitutively transcribed human herpesvirus 8 (HHV‐8) mRNA T0.7 and T1.1 using a colourimetric technique. T1.1 increases during experimental induction of the viral lytic phase in the HHV‐8‐infected lymphocytes of primary effusion lymphoma and its colourimetric detection in KS cells presumably corresponds to virion production. Immunostaining with anti‐CD20, CD45RO, MAC 387, and α‐smooth muscle actin was performed following T1.1 in situ hybridization (ISH). When the amount of T0.7 was above the detection threshold, the signal was made up of multiple coarse intranuclear dots in most spindle cells. Of the six early‐stage lesions, none produced a T1.1 hybridization signal. Two of four AIDS‐related and two of eight classical lesions with incipient spindle cell growth produced rare but distinct dense intranuclear T1.1 signals in endothelial cells lining narrow tubes. In contrast, eight of ten (all classical KS) mature spindle cell lesions displayed a signal, scattered in up to 2 per cent of spindled endothelial cells. Cell types other than endothelium produced no T1.1 hybridization signal in double stains. The results are consistent with other published data indicating latent HHV‐8 infection in endothelium and its tumour cell progeny, with simultaneous virion production in a small subset of cells. Immunodeficiency may not influence the number of cells lytically infected with HHV‐8 in early KS, in contradistinction to other herpesviruses with latent‐lytic cycles. Copyright

No survival benefit associated with routine surveillance imaging for Hodgkin lymphoma in first remission: a Danish-Swedish population-based observational study

The use of routine imaging for patients with classical Hodgkin lymphoma (HL) in complete remission (CR) is controversial. In a population‐based study, we examined the post‐remission survival of Danish and Swedish HL patients for whom follow‐up practices were different. Follow‐up in Denmark included routine imaging, usually for a minimum of 2 years, whereas clinical follow‐up without routine imaging was standard in Sweden. A total of 317 Danish and 454 Swedish comparable HL patients aged 18–65 years, diagnosed in the period 2007–2012 and having achieved CR following ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine)/BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone) therapy, were included in the study. The cumulative progression rates in the first 2 years were 4% (95% confidence interval [CI] 1–7) for patients with stage I–II disease vs. 12% (95% CI 6–18) for patients with stage III–IV disease. An imaging‐based follow‐up practice was not associated with a better post‐remission survival in general (P = 0·2) or in stage‐specific subgroups (P = 0·5 for I–II and P = 0·4 for III–IV). Age ≥45 years was the only independent adverse prognostic factor for survival. In conclusion, relapse of HL patients with CR is infrequent and systematic use of routine imaging in these patients does not improve post‐remission survival. The present study supports clinical follow‐up without routine imaging, as encouraged by the recent Lugano classification.

Family history of cancer as a risk factor for second malignancies after Hodgkin's lymphoma

This study estimated the risk of second primary malignancies after Hodgkins lymphoma (HL) in relation to family history of cancer, age at diagnosis and latency, among 6946 patients treated for HL in Sweden in 1965–1995 identified through the Swedish Cancer Register (SCR). First-degree relatives (FDRs) to the HL patients and their malignancies were then ascertained together with their malignancies through the Multi-Generation Registry and SCR. The HL patient cohort was stratified on the number of FDRs with cancer, and standardised incidence ratios (SIRs) of developing SM were analysed. In the HL cohort, 781 SM were observed 1 year or longer after HL diagnosis. The risk for developing SM increased with the number of FDRs with cancer, SIRs being 2.26, 3.01, and 3.45 with 0, 1, or ⩾2 FDRs with cancer, respectively. Hodgkins lymphoma long-term survivors treated at a young age with a family history of cancer carry an increased risk for developing SM and may represent a subgroup where standardised screening for the most common cancer sites could be offered in a stringent surveillance programme.

Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines.

Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.