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Purpose: Until now molecular biologic techniques have not been easily used in daily clinical practice to stratify patients for therapeutic purposes. Therefore, we have investigated the prognostic relevance of the immunohistochemical (IHC) germinal center B-cell (GCB) versus non-GCB diffuse large B-cell lymphoma (DLBCL) subtypes. Patient; and Methods: We have analyzed tumor samples from patients treated in 2 prospective multicenter phase III trials, ie HOVON 25 (patients >= 65 years, n = 153) and HOVON 26 (patients

Liposarcomas are separated into clinicopathological entities with a characteristic morphological spectrum and mutually exclusive genetic alterations. Therefore, the rare occurrence of cases with combined patterns of well-differentiated liposarcoma and myxoid liposarcoma designated as mixed-type liposarcoma pose a conceptual problem. Moreover, this feature may have consequences for treatment choice and prognosis. Here, we have dissected the molecular relation of tumor components in cases of mixed-type liposarcoma. On the basis of heterogeneous preoperative magnetic resonance image (MRI) features, eight cases of mixed-type liposarcoma were selected. Preoperative biopsy samples and resection specimens were analyzed including molecular and immunohistochemical analysis on all components. As controls, cases with homogeneous MRI features and uniform aspects of myxoid liposarcoma (n = 5), round cell liposarcoma (n = 5) and well-differentiated liposarcoma (n = 5) were studied. All patients with heterogeneous MRI features showed morphological components of myxoid liposarcoma and well-differentiated liposarcoma. Real-time polymerase chain reaction showed FUS-DDIT3 fusion in both components in five of eight cases in the absence (zero of five) of MDM2 and CDK4 amplification. In three of eight patients, MDM2 and/or CDK4 were overexpressed, and amplification was shown by multiplex ligation-dependent probe amplification (MLPA) in the absence of myxoid liposarcoma translocations. All control patients showed a molecular pattern consistent with their morphological features. Therefore, mixed-type liposarcomas should not be regarded as collision tumors, but as an extreme variant of the morphological spectrum within a single biological entity, explaining the biological contradiction of mixed-type liposarcoma. For treatment stratification, detailed classification including molecular support should be performed in tumors with heterogeneous MRI features.

Item does not contain fulltext The Lunenburg Lymphoma Biomarker Consortium (LLBC) evaluated the prognostic value of IHC biomarkers in a large series of patients with diffuse large B-cell lymphoma (DLBCL). Clinical data and tumor samples were retrieved from 12 studies from Europe and North America, with patients treated before or after the rituximab era. Using tissue microarrays from 1514 patients, IHC for BCL2, BCL6, CD5, CD10, MUM1, Ki67, and HLA-DR was performed and scored according to previously validated protocols. Optimal cut points predicting overall survival of patients treated in the rituximab era could only be determined for CD5 (P = .003) and Ki67 (P = .02), whereas such cut points for BCL2, BCL6, HLA-DR, and MUM1 could only be defined in patients not receiving rituximab. A prognostic model for patients treated in the rituximab era identified 4 risk groups using BCL2, Ki67, and International Prognostic Index (IPI) with improved discrimination of low-risk patients. Newly recognized correlations between specific biomarkers and IPI highlight the importance of carefully controlling for clinical and biologic factors in prognostic models. These data demonstrate that the IPI remains the best available index in patients with DLBCL treated with rituximab and chemotherapy.

Background Treatment decisions and prognosis assessment for liposarcoma is based on a classification that depends on morphological and genetic features. Revisions by experienced referral pathologists are often advocated. Methods The process of histopathological classification in referring hospitals and subsequently in a referral center in relation to molecular biological information is evaluated. A total of 331 consecutive liposarcoma patients were evaluated for the added value of histological review at time of referral. Subsequently, cases were reclassified with implementation of present-day molecular information. For all patients, complete data on staging, treatment, and follow-up were available. Results Upon histological revision, 15/54 (28%) diagnoses were reclassified in the first decade, 14/65 (22%) in the second, and 14/53 (26%) in the last decade. Molecular biological analysis enabled well-differentiated liposarcoma with or without dedifferentiated component to be better recognized as such and distinguished from myxoid liposarcoma and pleomorphic liposarcoma. Inclusion of cytogenetic information resulted in reclassification after revision in 4/18 (22%) cases in the first decade, 10/38 (26%) cases in the second decade, and 19/75 (25%) cases in the last decade. Conclusions This study indicates that liposarcomas are heterogeneous tumors. Expert assessment and implementation of molecular biological analysis are valuable for adequate classification as a basis for treatment decisions.

The classification of multifocal myxoid/round cell liposarcoma, which is defined as tumor presentation in at least two separate sites before manifestation in the lungs, as either metastasis or as a second primary tumor, has essential clinical consequences. Genetically, myxoid/round cell liposarcoma is characterized by t(12;16)(q13;p11) or t(12;22)(q13;q12), and various exon fusion transcripts are described with varying incidences, which permits their use as markers for clonality. Moreover, in solid tumors, analysis of loss of heterozygozity is valuable for clonality analysis. Therefore, fifteen multifocal myxoid/round cell liposarcoma patients with two to five metachronous (n = 12) or synchronous (n = 3) localizations were investigated. Using RT-PCR, the detailed molecular characteristics of the FUS-CHOP and EWS-CHOP breakpoints were determined. Loss of heterozygozity analysis at twelve loci was then used to further analyze clonal relationships. In all patients, tumor sites showed identical FUS-CHOP fusion products. In six patients, identical rare fusion transcripts were found, supporting a clonal relationship. Nine patients had the common exon5-FUS/exon2-CHOP fusion transcript, and two of these were identified as clonally related by loss of heterozygozity analysis. In all other patients, loss of heterozygozity analysis was highly suggestive of a clonal relationship, and no evidence for interpretation of a second primary tumor was found. This study supports the metastatic nature of apparent multifocal myxoid/round cell liposarcoma.

Eur J Clin Invest 2010; 40 (5): 433–439 Abstract Background In most cases of renal cell carcinoma there is no family history of renal cancer and no hereditary cause of the disease. Hereditary renal cancer accounts for about 2–4% of cases. Recognition of this subgroup by clinicians is important because of the possibility of severe medical consequences for patients and their relatives. Materials and methods We review the latest data about different genetic conditions characterized by an increased risk of developing renal cancer and we formulate tools to recognize high-risk families. Results In general, a positive family history, young age at diagnosis of renal cancer, multiple and/or bilateral renal tumours and combined occurrence of different histological types of renal tumours should raise suspicion of a hereditary renal tumour syndrome. In addition, the presence of specific extrarenal symptoms in patients could assist in differentiating between tumour syndromes. Conclusions A detailed medical and family history, along with physical examination are key factors to diagnose hereditary renal cancer syndromes. When a genetic predisposition for renal cancer is suspected, referral to a Family Cancer Clinic is warranted to initiate genetic examination and counselling on preventive options.

The term "gray-zone" lymphoma has been used to denote a group of lymphomas with overlapping histological, biological, and clinical features between various types of lymphomas. It has been used in the context of Hodgkin lymphomas (HL) and non-Hodgkin lymphomas (NHL), including classical HL (CHL), and primary mediastinal large B cell lymphoma, cases with overlapping features between nodular lymphocyte predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B cell lymphoma, CHL, and Epstein-Barr-virus-positive lymphoproliferative disorders, and peripheral T cell lymphomas simulating CHL. A second group of gray-zone lymphomas includes B cell NHL with intermediate features between diffuse large B cell lymphoma and classical Burkitt lymphoma. In order to review controversial issues in gray-zone lymphomas, a joint Workshop of the European Association for Hematopathology and the Society for Hematopathology was held in Bordeaux, France, in September 2008. The panel members reviewed and discussed 145 submitted cases and reached consensus diagnoses. This Workshop summary is focused on the most controversial aspects of gray-zone lymphomas and describes the panel's proposals regarding diagnostic criteria, terminology, and new prognostic and diagnostic parameters.

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, II cases of primary testicular and II cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non- germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P

Translocation t(11;18)(q21;q21) is the most frequent chromosomal aberration reported in gastric mucosa-associated lymphoid tissue (MALT) lymphomas. Intriguingly, this translocation has been reported only rarely in diffuse large B-cell lymphomas; it has been proposed that t(11;18)-positive tumors rarely progress to diffuse large B-cell lymphomas. We examined the frequency of chromosomal translocation t(11;18)(q21;q21) in mucosa-associated lymphoid tissue lymphoma and diffuse large B-cell lymphoma of the stomach. Paraffin-embedded tissues from patients with gastric B-cell lymphomas were selected retrospectively. The presence of the t(11;18)(q21;q21) was determined using reverse transcriptase-polymerase chain reaction and/or fluorescence in situ hybridization. beta-Actin transcript was also determined to evaluate the integrity and efficiency of RNA (cDNA) recovery from paraffin-embedded tissues. We analyzed 53 gastric B-cell lymphomas (33 diffuse large B-cell and 20 mucosa-associated lymphoid tissue) obtained from Italy, the USA, or Japan. Beta-actin transcript was amplified in 50 cases (94%), including 19 mucosa-associated lymphoid tissue and 31 diffuse large B-cell lymphomas (five with mucosa-associated lymphoid tissue components). The t(11;18) translocation was detected in 19% (6 of 31) cases with diffuse large B-cell lymphoma versus 26% (five of 19) with mucosa-associated lymphoid tissue lymphoma (P = 0.72). One of five diffuse large B-cell lymphomas with a mucosa-associated lymphoid tissue component showed the t(11;18)(q21;q21). In conclusion, translocation t(11;18)(q21;q21) was found in both mucosa-associated lymphoid tissue lymphomas and diffuse large B-cell lymphomas of the stomach at approximately equivalent frequencies; its presence does not exclude progression to diffuse large B-cell lymphoma.

Context Recently, we identified 2 patients with anaplastic large T-cell lymphoma (ALCL) negative for tyrosine kinase anaplastic lymphoma kinase (ALK-negative) in the fibrous capsule of silicone breast prostheses, placed for cosmetic reasons. Similar cases have been reported in the literature. Although an increased risk of ALCL in patients with breast prostheses has been speculated, no studies have been conducted so far. Objective To determine whether ALCL risk is associated with breast prostheses. Design A search for all patients with lymphoma in the breast diagnosed in the Netherlands between 1990 and 2006 was performed through the population-based nationwide pathology database. Subsequently, we performed an individually matched case-control study. Conditional logistic regression analysis was performed to estimate the relative risk of ALCL associated with breast prostheses. Setting and Patients Eleven patients with breast ALCL were identified in the registry. For each case patient with ALCL in the breast, we selected 1 to 5 controls with other lymphomas in the breast, matched on age and year of diagnosis. For all cases and controls (n = 35), pathological and clinical information was obtained with special emphasis on the presence of a breast prosthesis. Main Outcome Measure Association between breast implants and ALCL of the breast. Results The 11 patients with ALCL of the breast were diagnosed between 1994 and 2006 at a median age of 40 years (range, 24-68 years). In 5 of these patients, bilateral silicone breast prostheses had been placed 1 to 23 years before diagnosis. All received prostheses for cosmetic reasons. Lymphoma classes of 35 eligible control patients were 12 diffuse large B-cell lymphomas, including 1 T-cell rich B-cell lymphoma; 5 Burkitt lymphomas; 10 mucosa-associated lymphoid tissue–type lymphoma; 3 follicular lymphomas; 3 peripheral T-cell lymphomas; and 2 indolent B-cell lymphomas, unclassified. One of 35 control patients had a breast implant placed before diagnosis of lymphoma. The odds ratio for ALCL associated with breast prostheses was 18.2 (95% confidence interval, 2.1-156.8). Conclusions These preliminary findings suggest an association between silicone breast prostheses and ALCL, although the absolute risk is exceedingly low due to the rare occurrence of ALCL of the breast (11 cases in the Netherlands in 17 years). These findings require confirmation in other studies.

Purpose Patients with adenocarcinoma of unknown primary origin (ACUP) constitute approximately 4% of all malignancies. For effective treatment of these patients, it is considered optimal to identify the primary tumor origins. Currently, the success rate of the diagnostic work-up is only 20% to 30%. Our goal was to evaluate the contribution of gene expression profiling for routine clinical practice in patients with ACUP. Patients and Methods Formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 84 patients with a known primary adenocarcinoma and from 38 patients with ACUP. An extensive immunohistochemical panel classified 16 of the patients with ACUP, whereas 22 patients remained unclassified for their histogenetic origin. Information about staging procedures and clinical follow-up were available in all patient cases. The expression data were analyzed in relation to clinicopathologic variables and immunohistochemical results. Results The gene expression–based assay classified the primary site correctly in 70 (83%) of 84 patient cases of primary and metastatic tumors of known origin, with good sensitivity for the majority of the tumor classes and relatively poor sensitivity for primary lung adenocarcinoma. Gene expression profiling identified 15 (94%) of 16 patients with initial ACUP who were classified by immunohistochemistry, and it made a valuable contribution to a potential site of origin in 14 of the 22 patients with ACUP. Conclusion The gene expression platform can classify correctly from FFPE samples the majority of tumors classes both in patients with known primary and in patients with ACUP. Therefore, gene expression profiling represents an additional analytic approach to assist with the histogenetic diagnosis of patients with ACUP.

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

Purpose To evaluate long-term results of stomach-conserving therapy and to assess the value of histological probable minimal residual disease (pMRD) in predicting outcome in patients with gastric MALT lymphoma. Materials and methods We studied 115 patients with stage I–II 2 gastric MALT lymphoma treated between 1975 and 2002. Initially, first-line treatment consisted of radiotherapy only. Since 1994 most patients were primarily treated with Helicobacter pylori eradication; radiotherapy was used in case of eradication failure. To assess the value of pMRD, first follow-up biopsy samples classified as compete remission (CR) according to classical clinico-pathological criteria and biopsy samples 1 year after assessment of histological CR were reviewed; results were related to outcome. Results Following radiotherapy only ( n =56) 96% achieved a clinical CR; 10-year cancer-specific survival rate was 94%. Following H. pylori eradication only ( n =35) CR-rate was 43% and after additional treatment 89%; 5-year cause-specific survival was 93%. There was no difference in relapse rate following initial histological CR or pMRD. Conclusions Patients with early stage gastric MALT lymphoma have a favorable long-term outcome following conservative treatment. Outcome after H. pylori eradication followed by delayed radiotherapy on indication was excellent. In our series pMRD was not associated with increased risk of recurrence.

Background In contrast to gastric extranodal marginal zone mucosa associated lymphoid tissue (MALT) lymphomas, there is little consensus on the value and clinical consequences of extensive staging at diagnosis and during follow-up in non-gastric MALT lymphomas. Design and Methods Complete clinical information at presentation and during follow-up was collected for 72 patients with non-gastric MALT lymphoma treated at the Netherlands Cancer Institute between 1977 and 2005. Dissemination patterns at presentation were studied for nine primary dominant organ groups in our series of 72 patients and in a similar cohort treated at Vienna University (for a total of 106 patients). Results Twenty-three of our patients (32%) had more than one site of extranodal MALT lymphomatous disease, 13 patients (18%) had regional nodal involvement and 7 (10%) had bone marrow involvement. Site-specific dissemination was seen in paired organs (orbit, lung) and in the gastrointestinal tract (stomach, colon) and primary pulmonary MALT lymphoma was specifically related to gastric involvement ( p

Purpose Surgery and adjuvant radiotherapy (RT) have long been the standard treatment for most deep-seated sarcomas; however, since the randomized trial from the National Cancer Institute of Canada, which described similar local control for pre- vs. postoperative RT, both modalities are now widely accepted. As a group, sarcomas are classified as radiation resistant. The subgroup of myxoid liposarcoma (MLS), a sarcoma with a typical vascular crow's feet pattern, is highly radiosensitive, but a mechanism for this phenomenon is unknown. Here we describe our results with preoperative RT and propose a mechanism explaining the high sensitivity based on the distinctive vascularization pattern of MLS. Methods and Materials Between 2002 and 2006, 31 sarcoma patients, including 10 with MLS, underwent preoperative RT at our institute. Resected specimens were histologically evaluated, focusing on classification, grade, and vascularization patterns. Results Twenty sarcomas showed more than 80% pathologic response after preoperative RT. A pathologic complete response was found in all “pure” MLS specimens after preoperative RT (n = 8). There were no pathologic complete responses in the remaining sarcoma patients (n = 23), although 12 showed 80% to 90% pathologic response. In contrast to the remaining RT-resistant sarcomas, the highly responding specimens contained branching vasculature, partial thrombus formation and inflammation of medium sized arterioles, similar to the vascular changes in MLS. Conclusions Both MLS and sarcomas with MLS-like vasculature are highly radiosensitive. Radiation sensitivity may be explained by changes in medium-sized arterioles, obstructing the specific crow's feet vascularization and inducing hypoxia with secondary tumor cell death.

Introduction Primary diffuse large B-cell lymphomas (DLBCL) constitute a heterogeneous group of tumors with a great diversity in both clinical presentation and outcome. Besides nodal DLBCL, about 40% of these tumors present in different extranodal sites, both immune privileged sites such as the testis and the central nervous system (PCNSL) but also non immune privileged sites such as bone, stomach, skin and mediastinum. Remarkably each of these sites show a marked divergence in biological behavior. Recent studies have shown that certain non-coding short RNAs (22nt) called miRNAs may play a role in the pathogenesis of DLBCL. These miRNAs posttrancriptionally down regulate expression of genes. Aim of study To examine the expression levels of selected miRNAs in different DLBCL sites. Methods and Materials 51 cases of Ann Abour stage I and II primary DLBCL were divided into 4 groups; 19 nodal cases, 11 non-immune privileged site-associated (non-IP) extranodal cases, 9 PCNSL, and 12 testicular DLBCL. Each case was classified GCB or non-GCB using the algorithm as described by Hans et al. 13 miRNAs were selected from current literature on the basis of their involvement in B-cell lymphoma (miR-15, -16, -21, -221, -155, - 181a, -18a, -19, -20a, 17-3p, -17-5p and -92). The expression levels of the mature microRNAs were determined by qRT-PCR using Taqman miRNA assays. Results were compared by using 2−ΔCt and 2−ΔΔCt. Differences in expression levels between the four groups was tested using ANOVA. Results Of the selected microRNAs, miR-127, miR-17-5p and miR-221 show a significant difference in expression level between the immune privileged site-associated DLBCL (IP-DLBCL) and nodal DLBCL. Both miR-17-5p (p=0.0003) and miR-221 (p=0.0032) show an increased expression level in PCNSL compared to nodal DLBCL and miR-127 shows a significant increased level in testicular DLBCL compared to nodal (p=0.0011) and non-IP (p=0.0393) DLBCL. Also of interest is the fact that miR-17-5p and miR-127 show a significant difference in the level of expression between both PCNSL and testicular DLBCL. MiR-155 shows no significant difference in expression levels between nodal and extranodal DLBCL. Analysis on the basis of GCB and non-GCB classification is currently being undertaken. Conclusion In PCNSL miR-17-5p and miR-221 are specifically increased whereas miR-127 shows a specifically higher expression in testicular DLBCL when compared to non-IP and nodal DLBCL. Also PCNSL and testicular DLBCL differ in expression of miR-17-5p and miR-127. In contrast to previous reports miR-155 does not show any significant difference between nodal and extranodal groups. This may be caused by the selection of stage I or II primary DLBCL in contrast to previous publications which used a more heterogeneous group of DLBCL.

Contains fulltext : 53318.pdf (Publisher’s version ) (Closed access) Optimal dose and timing of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy for aggressive non-Hodgkin lymphoma (NHL) is still an unresolved issue. We assessed whether dose intensifications with cyclophosphamide and doxorubicin might improve outcome in younger patients with intermediate-risk aggressive NHL. Previously untreated patients were assigned to receive either 8 courses of standard CHOP (n = 239) or 6 courses of intensified (I)-CHOP (n = 238). Although there was a tendency in favor of I-CHOP for overall survival (OS), disease-free survival (DFS), and event-free survival (EFS), the differences were not significant. However, although these analyses were not planned, when the intermediate-risk group was divided into low-intermediate- and high-intermediate-risk patients according to the International Prognostic Index (IPI), low-intermediate-risk patients had improved 6-year OS (67% vs 52%; P = .05), DFS (58% vs 45%; P = .06), and EFS (41% vs 30%; P = .21) when they were treated with I-CHOP compared with standard CHOP. On the other hand, high-intermediate-risk patients seem to have no benefit from I-CHOP. Although clinically relevant side effects occurred more often in the I-CHOP arm, treatment-related mortality was similar. These data suggest that I-CHOP might be preferable to standard CHOP in younger patients with low-intermediate-risk aggressive NHL.

PurposeDespite the generally favorable clinical course in follicular lymphoma (FL), a minority of patients have a poor prognosis—with death within 3 years of diagnosis—most often due to transformation to aggressive disease.Patients and MethodsIn this study, we analyzed the potential of predicting early transformation on the basis of gene expression and immunologic parameters in FL biopsy samples taken at diagnosis.ResultsAt the gene-expression level, FL is a highly uniform disease at the time of diagnosis, precluding the detection of sufficiently validated prognostic gene-expression profiles suitable for a clinical setting. Combinations of differentially expressed genes indicate that immunologic mechanisms play a differential role in the risk of early transformation. Using immunohistochemistry for specific cell populations, the spatial distribution to neoplastic follicles and the activation of CD4–positive T-helper cells (P = .002) and specifically T-helper 1 (P = .004) were shown to be highly discriminatory to predict early transformation. A role for functional modulation of follicular dendritic cells could also be supported (P = .04). Other cell populations, including CD68-positive macrophages and regulatory T cells, were not differentially present.ConclusionThese results support the identification of FL as an immunologically functional disease in which an interaction of the tumor cells and the functional composition of the microenvironment determines the clinical behavior.

Very low-dose irradiation (2 × 2 Gy) is a new, effective, and safe local treatment for follicular lymphoma. To understand the biologic mechanisms of this extremely effective response, we compared by microarray the gene-expression profile of patients' biopsies taken before and after radiation. In all patients, a major and consistent induction of p53 target genes was seen. p53 targets involved in cell-cycle arrest and apoptosis showed the same mode of regulation, indicating that, in vivo, both are activated simultaneously. p53 up-regulation and p53-mediated proliferation arrest and apoptosis were substantiated using immunohistochemistry, with activation of both the intrinsic and the extrinsic apoptotic pathways. The other induced genes revealed a whole set of biologically meaningful genes related to macrophage activation and TH1 immune response. Immunohistochemical analysis suggested a specific activation or differentiation of resident macrophages by apoptotic cells. These biologic insights are important arguments to advocate the use of low-dose radiotherapy as an effective palliative treatment for follicular lymphoma. Moreover, this study is the first in vivo report of the radiation-induced p53 apoptotic response in patients and suggests that this apoptotic response is not immunologically silent.

Introduction. Classical Hodgkin Lymphomas (cHL) include small numbers of malignant Reed-Sternberg (RS) cells within an extensive but ineffective inflammatory/immune cell infiltrate. In cHL, chromosome 9p24.1 alterations increase the abundance of the PD-1 ligands, PD-L1 and PD-L2, and their further induction via JAK2-STAT signaling. PD-1 ligands engage the PD-1 receptor on T-cells and induce PD-1 signaling and T-cell exhaustion. Tumor cells expressing PD-1 ligands on their surface utilize the PD-1 pathway to evade an effective immune response. In recent pilot studies, PD-1 blockade was associated with high response rates and durable remissions in relapsed/refractory cHL. The unique composition of cHL limits its analysis with high throughput genomic assays. Therefore, the precise incidence, nature and prognostic significance of PD-L1 and PD-L2 alterations in cHL remain undefined. Herein, we utilize a recently developed fluorescence in situ hybridization (FISH) assay to characterize 9p24.1/PD-L1/PD-L2 alterations in a cohort of 108 newly diagnosed cHL patients (pts) who were uniformly treated with StanfordV (a combined modality therapy regimen) and have longterm followup. Methods. Pts were characterized as Ann Arbor early stage I/II favorable risk (ES-F), early stage unfavorable risk (bulk ≥ 10cm or ≥ .33 mediastinal dimension and/or B symptoms) (ES-U) or advanced stage III/IV (AS). ES-F pts received 8 weeks of Stanford V and 30 Gy involved field radiation (IFR); ES-U and AS pts received 12 weeks of Stanford V and 36 Gy IFR to initial sites > 5 cm. FISH was performed on formalin-fixed paraffin-embedded diagnostic biopsy specimens using bacterial artificial chromosome probes which covered CD274/PD-L1 (labeled with spectrum orange) and PDCD1LG2/PD-L2 (labeled with spectrum green) and a control centromeric probe (spectrum aqua-labeled CEP9, from 9p11-q11). Malignant RS cells were identified by their nuclear morphologic features and 50 RS cells/case were analyzed. Nuclei with a target:control probe ratio of at least 3:1 were defined as amplified (amp), those with a probe ratio of more than 1:1 but less than 3:1 were classified as relative copy gain, and those with a probe ratio of 1:1 but more than 2 copies of each probe were defined as polysomic for chromosome 9p. In each case, the percent and magnitude of disomy, polysomy, copy gain and amp were noted. In accordance with clinically approved diagnostic criteria, cases were classified by the highest observed level of 9p24.1 alteration. Specifically, cases with polysomy lacked copy gain or amp and cases with copy gain lacked amp. Immunohistochemical staining for PD-L1/PAX5 was performed as previously described and PD-L1 expression in PAX5 dim+ malignant RS cells and PAX5- infiltrating normal cells was assessed separately. Results. Almost all newly diagnosed cHL pts in this series had concordant alterations of the PD-L1 and PD-L2 loci; disomy was found in only 1% (1/108), polysomy in 5% (5/108), copy gain in 56% (61/108) and amp in 36% (39/108) of study pts. There was a correlation between intensity of PD-L1 protein expression and relative genetic alterations in this series. Two additional pts had translocations of PD-L1 or PD-L2 (2%, 2/108). We next assessed the association between specific types of PD-L1/PD-L2 alterations, clinical risk factors and outcome. Overall, the progression-free survival (PFS) was significantly lower for AS pts compared to ES-F/U pts (p=0.017). A model of PFS for the cHL pts by genetic alteration indicated that PFS was also significantly lower for pts with amp (p=0.02). Consistent with these findings, the incidence of 9p24.1 amp increased by clinical risk group: ES-F, 24%; ES-U, 34%; and AS, 50% (p=0.024, Kruskal-Wallis test). Therefore, we fit a full model of clinical and genetic factors including B-symptoms, bulk, stage and amp. Despite the association of amp with increased clinical risk groups, the genetic alteration further delineated PFS in the multivariate model (p=0.075). Conclusions. PD-L1/PD-L2 alterations are a defining feature of cHL with rare polysomy and more frequent copy gain and amp. There is an increased incidence of amp in pts with AS disease and a highly significant association of PD-L1/PD-L2 amp with PFS. These findings underscore the importance of genetically defined PD-1 mediated immune evasion in cHL and provide a rationale for the efficacy of PD-1 blockade in this disease. Disclosures Rodig: Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Shipp:BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Gilead: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees.

2507 Background: AZD1775 (formerly MK-1775) is a potent and selective inhibitor of Wee1, a kinase that phosphorylates CDC2. Phosphorylation of CDC2 inactivates the CDC2/cyclin B complex and is ther...

Cyclin D1 overexpression is believed to be essential in the pathogenesis of mantle cell lymphoma (MCL). Hence, the existence of cyclin D1-negative MCL has been controversial and difficult to substantiate. Our previous gene expression profiling study identified several cases that lacked cyclin D1 expression, but had a gene expression signature typical of MCL. Herein, we report the clinical, pathologic, and genetic features of 6 cases of cyclin D1-negative MCL. All 6 cases exhibited the characteristic morphologic features and the unique gene expression signature of MCL but lacked the t(11;14)(q13; q32) by fluorescence in situ hybridization (FISH) analysis. The tumor cells also failed to express cyclin D1 protein, but instead expressed either cyclin D2 (2 cases) or cyclin D3 (4 cases). There was good correlation between cyclin D protein expression and the corresponding mRNA expression levels by gene expression analysis. Using interphase FISH, we did not detect chromosomal translocations or amplifications involving CCND2 and CCND3 loci in these cases. Patients with cyclin D1-negative MCL were similar clinically to those with cyclin D1-positive MCL. In conclusion, cases of cyclin D1-negative MCL do exist and are part of the spectrum of MCL. Up-regulation of cyclin D2 or D3 may substitute for cyclin D1 in the pathogenesis of MCL.

Follicular lymphoma (FL) is the second most frequent type of non-Hodgkin's lymphoma in adults. The disease is characterized by an indolent course with frequent relapses. Ultimately, resistance to chemotherapy or transformation to a more aggressive phase of the disease in the form of diffuse large B-cell lymphoma develops, and patients die as a result of their disease. Median survival is 8 to 10 years. The range is very wide, however, with patients surviving for more than 15 years and 10% to 15% of the patients who run a rapidly fatal course and die within 3 years after diagnosis. The translocation t(14;18) is the basic molecular defect in FL and results in protection from apoptosis by aberrant overexpression of bcl-2 protein. Accumulation of genomic alterations and clonal selection account for subsequent progression and transformation. Recently, the role of the immunologic microenvironment of FL in determining clinical behavior and prognosis has been substantiated. Combined genetic and immunologic data may now support a model for the development of FL as a disease of functional B cells in which specific molecular alterations infer intrinsic growth properties of the tumor cells as well as dictate a specific functional cross talk with the immunologic regulatory network resulting in extrinsic growth support. These insights may lead to improvement of risk stratification, but most importantly will provide tools for developing new targets and strategies for treatment.

Objectives: In a phase I study the feasibility, toxicity and immunological effects of peri-operative cytokine immunotherapy of renal cell carcinoma were studied. Main goals were to determine the maximal tolerable dose and detailed in situ analysis of tumor infiltrates. Methods: Fifteen patients with renal cell carcinoma, undergoing nephrectomy, received subcutaneous immunotherapy, consisting of low-dose IL-2, IFNα and GM-CSF, from day −3 prior, until day +5 following surgery in a dose escalation study. Infiltrates from resected tumor tissues from patients undergoing immunotherapy or control patients that underwent nephrectomy only, were examined using quantitative immunohistological analysis and 3-color immunofluorescence staining and confocal laser scanning microscope analysis. Results: Toxicity was limited and the maximal tolerable dose was established. In peripheral blood an increase was found in total lymphocytes, (activated) T cells, NK cells and monocytes. Quantitative immunohistological analysis of tumor infiltrates showed enhanced numbers of CD3+ T cells, S100+ DC, CD83+ DC and IL-2 receptor positive cells (4-fold, 2-fold, 10-fold and 20-fold, respectively, compared to controls). In treated patients preferential invasion was observed of TNFα positive CD8+ T cells and DC, positive for DC-SIGN (CD209), CD83, CD80, IL-12 and the DC specific chemokine, DC-CK1 (CCL18). Conclusions: These findings show increased infiltration of activated, mature DC and functionally active CD8+ T cells in renal tumors, which may suggest clinical potential of cytokine immunotherapy.

Follicular lymphoma (FL), except grade 3B, is characterized by the chromosomal translocation t(14;18)(q32;q21), which results in over-expression of the Bcl-2 protein. Ten per-cent of all FLs, however, do not show Bcl-2 protein expression with standard immunohistochemistry using a monoclonal Bcl-2 antibody against residues 41-54 of the Bcl-2 protein. In this study, the biological background of 18 Bcl-2-negative FL cases grade I, II, or IIIa was investigated by immunohistochemical staining and western blot analysis with alternative antibodies. Bcl-2 protein was demonstrated in five of the 18 cases and all of these carried the t(14;18) translocation. Of the 13 cases that were Bcl-2 negative with alternative antibodies, 12 lacked the t(14;18) translocation. PCR and subsequent sequence analysis of cDNA demonstrated that three cases with a t(14;18) contained somatic mutations in the translocated BCL2 gene, resulting in amino acid replacements in the region of the epitope recognized by the antibody. In conclusion, the majority of Bcl-2-negative FL lack a t(14;18) but a significant subset of these tumours are false negative due to mutations in the BCL2 gene. These findings may have consequences for the use of Bcl-2 immunohistochemistry for diagnostic purposes. Copyright (C) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley Sons, Ltd.

In virtually all human tumors, genetic and epigenetic alterations have been found which affect the INK4/-CYCLIN D/RB pathway, which regulates cell cycle entry and exit in normal cells. E2F transcription factors are important downstream components of this pathway, which act by controlling the expression of genes involved in DNA replication and cell cycle progression. To determine whether E2F2 deregulation promotes proliferation and tumorigenesis in vivo, we generated E2F2 transgenic mice, in which the Emu and murine pim1 promoter (pp) direct high expression of E2F2 in thymic epithelial cells. Emu-pp-E2F2 mice start to develop cytokeratin- and ER-TR4-positive cortical thymomas from the age of 20 weeks, and within 1 year, nearly all mice succumb to gross thymic epithelial tumors. General thymic morphology is largely maintained, but T cell development is perturbed in thymomas, with proportionately less CD4(+)CD8(+) double-positive thymocytes. In the first 3 months, E2F2 transgenic thymi exhibit only mild epithelial hyperplasia, and thereafter thymomas arise stochastically, probably following additional mutations. Interestingly, Emu-pp-E2F1 mice do not display cortical thymomas. These data argue that E2F2 promotes unscheduled cell division and oncogenic transformation of thymic epithelial cells.

This report contains the proceedings of the Second International Lunenburg LymphomaWorkshop, held at the Lunenburg Estate in Langbroek, the Netherlands on May 19 and 20, 2003. In this workshop 26 international lymphoma experts from the fields of hematology and pathology were assembled to discuss old and new prognostic factors in follicular non-Hodgkin’s lymphoma (FL) and diffuse large-cell lymphoma (DLCL). The ultimate goal of the meeting was to start integrating current clinical knowledge with novel biological insights to improve on the prognostic stratification of patients with FL and DLCL. The meeting was sponsored by the Ank van Vlissingen Foundation, a private foundation established in 1980 that aims at promoting lymphoma research and treatment. The First Lunenburg Lymphoma Workshop was held in May 2001, and focussed on ongoing clinical trials.

Purpose To evaluate 99m Tc-Annexin-V (TAV) scintigraphy in monitoring radiation-induced apoptotic cell death in follicular lymphoma (FL) patients. Patients and methods Eleven FL patients (7 female and 4 male; median age, 58 years; range, 42–80 years) with recurrent disease underwent TAV imaging before and 24 hours after the last fraction of the 2 × 2 Gy involved field radiotherapy regimen. Fine-needle aspiration cytology was performed on 5 consecutive days to determine the optimal time window for apoptosis detection and to confirm the apoptotic nature of the response. The TAV scintigraphy (total body studies and SPECT of the irradiated sites) was performed 4 hours after the administration of the radiopharmaceutical. Tumor uptake was scored in a semiquantitative manner as absent (−) weak (±), present (+), or intense (++) with corresponding categories for the cytologic slides. Response evaluation was performed after 1 week and 4 weeks both in terms of completeness and speed of remission. Results Baseline TAV uptake was absent in 6 and weak in 5 patients. Sequential cytology indicated that the optimal time period for apoptosis assessment was between 24 and 48 hours after the last fraction of the 2 × 2 Gy regimen. Baseline cytology was concordant with baseline TAV in all patients. Apoptotic feature appearance (nuclear chromatin condensation, margination and apoptotic body formation) after low-dose irradiation matched the irradiation response in all patients. In all but 1 patient the posttreatment TAV uptake matched the posttreatment cytology. In these 10 patients the cytology and TAV results correlated with the type and onset of the clinical response. Conclusion Tumor 99m Tc-Annexin-V uptake can be increased after 2 × 2 Gy involved field radiotherapy. This increase was concordant with the appearance of apoptotic morphology as determined by cytology, and correlated with the clinical outcome. Apoptotic cell death can be observed on Day 4 of this regimen and if so predicts a complete remission within 1 week.

Patients with diffuse large B-cell lymphoma (DLBCL) rarely show relapse after 4 years of complete remission (CR). In this study, we addressed the following questions: (1) Does late-relapsing DLBCL represent clonally related disease or a second malignancy; and (2) is there a characteristic biologic background? In 10 of 13 DLBCL patients with relapse after 4 to 17 years, a clonal relationship was established based on identical IgH-sequences and/or identical bcl2-IgH translocation. Most (77%) showed features of germinal center (GC) cells, as defined by expression of CD10, bcl-2, and bcl-6 protein and ongoing immunoglobulin heavy chain variable region (VH) hypermutation. A GC phenotype was seen in 8 (20%) of 38 control patients matched for age, stage, and (extra)nodal localization with relapse within 2.5 years (P = .005). In conclusion, we have found evidence that late-relapsing DLBCL represents truly clonally related disease episodes in most cases and that this clinical behavior may be related to the biologic features of GC cells. (Blood. 2003;102:324-327)

Introduction. Primary testicular lymphoma (PTL) and primary central nervous system lymphoma (PCNSL) are large B-cell lymphomas (LBCL) that occur in immune privileged (IP) sites and share certain clinical and molecular features. To date, the treatment of these IP lymphomas is largely empiric and more effective targeted therapies are needed. Methods. To define actionable genetic features of IP lymphomas, we performed comprehensive genomic analyses of 21 PCNSLs and 7 PTLs and validated specific alterations in an independent cohort of 43 additional PTLs. Recurrent copy number alterations (CNAs) were detected using high-density single nucleotide polymorphism (SNP) arrays and the GISTIC algorithm and integrated with transcriptional profiles to identify candidate driver genes. Recurrent somatic mutations were identified using a combination of whole exome sequencing (WES) of paired tumor/normal samples and whole transcriptome sequencing (RNA-Seq) of the additional tumors without paired normal samples. Results. In systemic diffuse large B-cell lymphomas (DLBCLs), multiple low-frequency CNAs and associated target genes decrease p53 activity and perturb cell cycle regulation; infrequent somatic mutations of TP53 also deregulate these pathways (Cancer Cell, 2012; 22:359-372). In contrast, PCNSLs and PTLs primarily exhibit bi-allelic deletion of the upstream regulator of p53 activity and cell cycle, CDKN2A (~70% PCNSLs and ~80% of PTLs) and rarely have copy loss or somatic mutations of TP53 or CNAs of additional pathway components. The most commonly mutated genes in PCNSL and PTL, CD79B and MYD88, are also perturbed in a subset of systemic DLBCLs. However, mutations of these two genes are much more frequent in IP lymphomas (70% MYD88 and 61% CD79B of analyzed PCNSLs and PTLs) and these alterations are commonly found in the same cases (57% of cases in this series). These data indicate that concurrent oncogenic activation of the B-cell receptor (BCR) and the Toll-like receptor (TLR) signaling pathways is a characteristic feature of IP lymphomas with implications for targeted therapies. Among the IP lymphomas, ~20% of PCNSLs and ~40% PTLs exhibit 3q12.3/NFKBIZ copy gain and increased expression of the NFKBIZ protein product, IκB-ζ, an atypical IκB family member induced by TLR signaling. In our PTL series, MYD88 wild-type tumors had the highest 3q12.3/NFKBIZ copy gains, and ~90% of all analyzed PTLs had structural bases for NFκB activation via the TLR pathway. Lentiviral-mediated IκB-ζ knockdown decreased expression of the IκB-ζ target genes, IκB-α and BCL-xL, and induced apoptosis of LBCL cell lines with MYD88 L265P mutations, NFKBIZ gain or both alterations. In addition, enforced expression of NFKBIZ enhanced the growth of LBCLs with normal NFKBIZ copy numbers. Taken together, these data suggest that many IP lymphomas depend upon oncogenic MYD88/NFKBIZ signaling. Although the majority of CNAs and somatic mutations were shared by PCNSLs and PTLs, certain alterations were primarily observed in PTL. In both the initial and independent validation series, > 40% of PTLs exhibited copy gain of chromosome 9p24.1/CD274 (PD-L1) / PDCD1LG2 (PD-L2) and associated overexpression of the PD-1 ligands. These observations were of particular interest because 9p24.1 copy gain is a characteristic abnormality in two additional lymphoid malignancies, primary mediastinal LBCL and classical Hodgkin lymphoma, PD-1 signaling promotes tumor immune evasion and the PD-1 pathway is targetable. We also identified one PTL in which a novel translocation juxtaposed the regulatory elements of TBL1XR1 (chromosome 3) to the start codon-bearing exon 2 of PDCD1LG2 (PD-L2) (chromosome 9). This translocation, which was detected by RNA-Seq and confirmed by 5’ RACE and a newly developed split-apart FISH assay, resulted in dramatic overexpression of the PD-L2 protein. These data suggest that PTLs utilize several genetic mechanisms to deregulate the PD-1 ligands and limit anti-tumor immunity. Conclusions. Integrative and comparative genomic studies define PCNSL and PTL as related but unique lymphoid malignancies with targetable genetic alterations, and associated p53 deficiency and cell cycle deregulation, concurrent oncogenic BCR and TLR signaling and PD-1 dependent immune evasion that warrant further clinical investigation. Note: B.C. and M.G.M.R have made equal contributions to this research. Disclosures Feuerhake: Roche Pharma Research and Early Development (pRED) from 2008-2012: Employment. Freeman:Merck: on the PD-1 pathway Patents & Royalties; EMD-Serrono: on the PD-1 pathway Patents & Royalties; Boehringer-Ingelheim: on the PD-1 pathway Patents & Royalties; Amplimmune: on the PD-1 pathway Patents & Royalties; Roche: on the PD-1 pathway Patents & Royalties; Bristol-Myers-Squibb: on the PD-1 pathway Patents & Royalties; Novatis: on the PD-1 pathway, on the PD-1 pathway Patents & Royalties. Shipp:Sanofi: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Membership on an entity's Board of Directors or advisory committees.

Indistinguishable disease states in diffuse large B-cell lymphoma unraveled by microarray expression profiles: A new basis for therapy selection. ( pages 68–74 )

Background. Transformation to diffuse large cell lymphoma (DLCL) is a frequent event in patients with follicular lymphoma (FL), occurring in approximately 30–50% of patients. Upon transformation the prognosis of these patients is dismal, with a median overall survival of 1–2 years. However, a subset of patients does respond to aggressive chemotherapy regimens, including autologous stem cell transplantation and can achieve long-term disease-free survival. Currently, there are no prognostic factors reliably predicting response to treatment. We used gene expression profiling to identify a profile which can predict responsiveness to chemotherapy and long-term survival at transformation. Patients and methods. From the pathology archives of the 4 participating institutions, 46 cases with transformation of FL were identified for whom frozen tissue was available. 11 cases were excluded from the analysis: 4 because of insufficient clinical information and 7 because the patients were not treated with aggressive chemotherapy regimens (defined as containing at least CHOP-like chemotherapy). RNA was extracted from frozen tissue. All samples were hybridized to cDNA microarray slides prepared at the Central Microarray Facility at the Netherlands Cancer Institute. The arrays contain 18336 biological transcripts as well as 864 control probes. Samples were cohybridized with a tonsil reference. Data analysis was performed in the statistical package R as well as with BrB array tools. Results. Of the 35 patients, 62% was male. The median interval between the initial diagnosis of FL and transformation was 27 months (range 0–252; 4 patients had transformed FL at initial diagnosis). Median age at transformation was 52 years (range 32–78). Using hierarchical clustering, no clear separation of the cases was obtained. Of the 35 cases, 13 reached a CR or Cru and had an overall survival of >24 months (range 37–134+, median 60 months). 6 patients achieved a partial remission and had an overall survival of 12–24 months, and 16 patients had stable or progressive disease and died within 13 months (range 3–13, median 5.5 months). Using supervised analysis on patients grouped based on survival, a classifier with an accuracy of up to 88% accuracy could be reached in an LOOCV procedure. An analysis of the most differentially expressed genes related to survival identified mostly genes involved in cell cycle, metabolism and immune related genes. Conclusions. Using gene expression profiling a classifier can be constructed that can separate transformed FL with a long survival from transformed FL with a short survival. This classifier can help to identify patients who benefit from intensive chemotherapy. Functional annotation of the differentially expressed genes identified mostly genes involved in cell cycle control and metabolism, and immune related genes. We plan to validate this approach using immunohistochemistry.

The Dutch Hemato-Oncology Study Group (HOVON) evaluated in a phase II trial the efficacy of the addition of rituximab, high dose Ara-C and autologous stem cell transplantation after BEAM conditioning to conventional CHOP in 88 previously untreated patients with mantle cell lymphoma, with respect to remission rate, failure free survival (FFS) and overall survival (OS). From 2002 to 2005, 86 eligible patients were included and evaluated. The male : female ratio was: 4 and the median age 55 years (32–66). Median follow-up of all patients still alive was 25.5 months. Patients were treated with R-CHOP21 x 3 (rituximab 375 mg/m2, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, vincristine 1.4 mg/m2 day 1 and prednisone 100 mg days 1 – 5). Stem cells were harvested after high dose Ara-C x 1 (2g/m2 BID days 1 – 4), followed by rituximab on day 11 for in vivo purging and G- CSF. After haematological recovery BEAM conditioning (carmustine 300mg/m2 day 1, Ara-C 200mg/m2 and etoposide 200 mg/m2 days 2 – 5 and melphalan 2 x 100 mg/m2 day 6) was given with autologous stem cell support day 8). After 3 x R-CHOP 17 patients did not reach PR and went off study, according to the protocol, and 5 patients were excluded for other reasons. Sixty four patients received HD Ara-C and 63 patients proceeded to BEAM. Haematological toxicity was as follows: the median time of recovery of leukocytes (> 1,0 x 109/l) from start of high dose Ara-C was 17 days (range: 0 – 59 days), from start of BEAM 26 days (range: 17 – 55). For platelets the median time to recovery (> 50 x 109/l) from start of high dose Ara-C was 23 days (range: 0 – 44 days) and from start of BEAM 25 days (range: 16 – 364). Non-hematological toxicity grade 3–4 was seen during CHOP in 13%, after HD Ara-C in 20%, mainly gastro-intestinal, and after BEAM in 51%, mainly gastro-intestinal and liver, of the patients. Grade 3–4 infections were seen during CHOP in 17%, after HD Ara-C in 30% and after BEAM in 59% of the patients. Responses to treatment are summarised in Table 1. At two years FFS was 67%, OS was 81%. An analysis restricted to the 63 patients who completed the protocol treatment showed FFS 90% and OS 98% at two years. We conclude that intensification of first line treatment with rituximab, HD Ara-C and BEAM is beneficial with respect to FFS and OS in younger MCL patients and that this regimen is well tolerated. Table 1. Response to treatment N CR PR NR/PD Not yet known after 3x CHOP 83 13 (16%) 51 (61%) 17 (20%) 2 after HD Ara-C 64 22 (34%) 28 (44%) 14 after BEAM 63 43 (68%) 13 (21%) 7 died early 1

Background : Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived anti-apoptotic Mcl-1 protein, but it is unclear if and how this leads to Bax or Bak activation that is required for most forms of cell death. Aim: To study the effects of seliciclib on apoptosis gene expression and Mcl-1 and Bcl-2 protein interactions in B cell chronic lymphocytic leukemia (B-CLL), a malignancy with known aberrant apoptosis regulation. Methods: Purified B-CLL cells (PBMC consisting of >90% B-CLL cells; n=20) and Ramos cell lines overexpressing different apoptosis regulators were used in this study. The effect of seliciclib on viability, apoptosis gene expression pattern, and protein associations was investigated via RT-Multiplex-Ligation-dependent Probe Amplification (RT-MLPA), Western blotting and co-immunoprecipitation assays. Ramos cells were transduced with retroviral vectors expressing either Noxa siRNA, Bim siRNA or control-GFP virus, and tested for different apoptosis stimuli. Results: We found that although seliciclib resulted in proteasome-dependent Mcl-1 degradation within 4 hrs in B-CLL cells, Bax and Bak activation and apoptosis occurred with a considerable delay, i.e. at 16 hrs. During this period, there was no evidence of transcriptional changes in p53-responsive or apoptosis-related genes. In freshly isolated, viable B-CLL cells, pro-survival Mcl-1 was engaged by the pro-apoptotic proteins Noxa and Bim but not by Bak. The contribution of Noxa [Figure1] and Bim (liberated from McL-1 within 4 hours) as specific mediators of seliciclib-induced apoptosis was demonstrated via RNAi in two model systems. Interestingly, 16 hrs after seliciclib treatment, there was a clear accumulation of Bcl-2, Bim, and Bax in the detergent insoluble (mitochondria containing) fraction of B-CLL cells. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, probably resulting in Bax multimerisation and pore formation in the mitochondria. Conclusions: These data support the ’oncogene-addiction’ model in which malignant cells depend on increased Bcl-2 levels, and extend it to include Mcl-1. Furthermore, since Noxa is elevated in B-CLL, its involvement in p53-independent apoptosis suggests this BH3-only protein may be a therapeutic target. Figure Figure

Seliciclib (cyc202, R-roscovitine) is a novel cyclin dependent kinase inhibitor currently in pre-clinical testing. It indirectly inhibits RNA polymerase II and has been shown to be effective against B-cell malignancies by inducing apoptosis and Mcl-1 degradation. The aim of this study was to dissect the underlying apoptotic pathway(s) of seliciclib in chronic lymphocytic leukemia (B-CLL). Treatment of CLL cells with >10 μM seliciclib in vitro induced apoptosis within 24 hours, irrespective of IgVH mutation status (n=20). Gene profiling by means of RT-MLPA, did not reveal involvement of p53 as indicated by the absence of Puma upregulation. None of the >30 other apoptosis genes tested were induced; instead signals for certain labile mRNAs (Mcl-1, A1/Bfl-1, PI-9) were clearly decreased upon seliciclib. Detailed investigation of B cell lines overexpressing various anti-apoptotic proteins (Bcl-2, caspase-9-DN, Flip, FADD-DN), indicated that seliciclib activated the mitochondrial but not the death receptor pathway. Neither mitochondrial activation nor the rapid degradation of Mcl-1 protein could be prevented by caspase inhibition (zVAD) or overexpression of inactive caspase-9 (DN). This indicated that Mcl-1 decline is an upstream, caspase-independent event. Immuno-precipitation demonstrated that pro-survival Mcl-1 is engaged by pro-apoptotic Noxa and Bim. The specific contribution of Noxa was confirmed by RNAi, resulting in inhibition of apoptosis after seliciclib, but not after staurosporin, CD95- or BCR triggering. These findings demonstrate that seliciclib induces rapid, p53-independent apoptosis via Mcl-1 degradation and mitochondrial activation. The involvement of Noxa, which is highly expressed in CLL, suggests this BH3-only protein may be a novel therapeutic target.

Involved field radiation therapy with 30 to 40 Gy is a valuable local treatment for follicular lymphoma (FL) that is routinely used in clinical practice. We previously showed that very low dose radiation (2x2 Gy, days 1 and 3) is also effective, with rapid and often long lasting remissions in up to 90% of FL patients (Haas et al, JCO, 2003). However, the biological mechanism of this extremely effective response is not known. To study the molecular response to low dose radiation therapy in FL, gene-expression profiling using 35K spotted 60-mer oligo-arrays was performed from lymph node biopsy samples taken before treatment and 24 hours after the second dose of 2 Gy irradiation, in 15 patients. The clinical response was excellent (10 CR, 5 PR). In all patients, a major and consistent induction of p53 and p53 target genes was seen, reflecting both proliferation arrest (e.g., P21, repression of cell-cycle regulated genes) and apoptosis induction (NOXA, PUMA, BAX, TRAIL/DR5 and FAS). P53 upregulation, p53-mediated proliferation arrest and apoptosis were substantiated using immunohistochemistry with dramatic increase of p53 protein levels in B-cells, T-cells and accessory cells. There was also a significant increase in the numbers of cleaved-caspase 8 positive cells (death receptor pathway apoptosis) with a minor increase of cleaved-caspase 3 positive cells and morphological features of apoptosis, suggesting a relatively early stage in the apoptotic process. The other induced genes revealed an ‘immune signature’, with a whole set of biologically meaningful genes related to macrophages (e.g., CD68, TLR4), TH1 immune response (e.g., IL18, CXCL9, 10, 11), clearance of apoptotic cells (e.g., C1Q, lysosomal enzymes), tolerance (ILT-3, IL-4, IDO) and death receptor ligands (FASL, TRAIL). Immunohistochemical analysis did not show an increase in T-cell subsets and macrophages density, rather suggesting an activation or differentiation of resident macrophages by radiation and/or apoptotic cells than recruitment of novel cell populations. This is the first global analysis of the direct molecular effect of radiotherapy and p53 related apoptosis in vivo in human lymphoma. Moreover, the ‘immune signature’ suggests that radiation-induced apoptosis in FL is not an immunologically silent process, but rather an early event that could contribute to the death and clearance of tumor cells. These insights may have important implications for modulation of the cancer-related immune response and for immunotherapeutical approaches in FL.

Follicular lymphoma (FL) is characterized by an indolent clinical course and frequent relapses. Transformation to more aggressive disease is common and one of the main causes of death. The time to transformation may vary widely and some patients show early transformation and a poor prognosis. Main questions in FL center on markers to predict transformation (and prognosis) at the time of diagnosis and to accurately assess the transformed phase to tailor the choice of therapy. Currently, prognostic stratification for actual aggressiveness and overall survival is based on histological grading and clinical criteria; however, in up to 30% of all cases these methods prove to be insufficient. Using 18k cDNA arrays, we analyzed gene-expression patterns of 135 cases of FL addressing both issues. 1) Using supervised classification on a training set of paired samples from the indolent and aggressive disease episodes and on an independent validation set, a 81-gene-expression profile was established that could, with an accuracy of 100% and 93% respectively distinguish indolent from aggressive disease. Importantly, in a third series of FL with ambiguous histological grade, precluding meaningful morphological guidance, the profile showed a 94% classification accuracy supporting the value of this method for practical clinical use. The profile consists of genes involved in cell cycle control, DNA synthesis and metabolism (upregulated in high-grade disease), T-cell related genes (upregulated in low-grade disease) and T-cell and macrophage activation markers, that are significantly upregulated upon transformation. This profile, however, did not predict future transformation in FL samples at diagnosis. 2) A separate analysis was performed in a selection of the biopsy samples of not previously treated patients with grade 1 and 2 FL with transformation to DLBCL within 7–22 months after diagnosis and no transformation with a minimum follow-up of 108 months. Supervised and unsupervised cluster analysis showed no differences between both groups exceeding the threshold of significance to construct a predictor profile with a validated significance, underlining the biological homogeneity of the study cohort. Direct comparison analysis on the basis of signal-to-noise ratio’s suggested a discriminative role of the immune response with enhanced cytokine- and chemokine-mediated T-cell activation and antigen-processing in the extremes of the spectrum. However, differences were slight and the immune-response seemed to play a less distinctive role in patients with transformed disease after more than 3 years. In conclusion, actual indolent and aggressive clinical behavior can be more reliably distinguished using a FL-stratification profile than the currently used histological grading and clinical criteria, and may provide an important alternative to guide the choice of therapy in FL patients both at presentation and at relapse. Although there may be subtle differences between rapidly transforming disease and long-term stable indolent disease, possible inherent variations are insufficient to reliably predict future transformation.

We evaluated the so-called dynamic sentinel node procedure in patients with penile cancer. This new staging technique consists of excisional biopsy of the first lymph node onto which a tumor drains the so-called sentinel node, based on individual mapping of lymphatic drainage.From 1994 to 1998, 55 consecutive patients with stage T2 or greater bilateral or unilateral node negative squamous cell carcinoma of the penis were prospectively entered in this study. Tumor stage was T2N0 in 42, T2N1 in 4 and T3N0 in 9 cases. To locate the sentinel node each patient underwent lymphoscintigraphy with 99mtechnetium nanocolloid injected intradermally around the tumor. The following day the sentinel node was identified intraoperatively using patent blue dye injected intradermally around the tumor and a gamma detection probe. Regional lymph node dissection was restricted to patients with a tumor positive sentinel node only.Scintigraphy revealed 125 sentinel nodes in 107 inguinal regions, including no sentinel node in 2 patients, 1 or more unilateral nodes in 10 and bilateral drainage in 43. At surgery 108 sentinel nodes were removed. In 8 patients with 2 or more sentinel nodes on lymphoscintigraphy only 1 was noted intraoperatively and in 9 an additional sentinel node was removed, which was not identified by scintigraphy. All nodes were identified with the gamma detection probe. In 1 patient a wound abscess developed. Regional lymph node dissection was performed in 11 patients with sentinel node metastasis. Median followup was 22 months (range 4.1 to 61). In 1 patient lymph node metastasis was noted at followup despite prior excision of a tumor-free sentinel node.The dynamic sentinel node procedure is a promising staging technique to detect early metastatic dissemination of penile cancer based on individual mapping of lymphatic drainage, and enables identification of patients with clinically node negative disease requiring regional lymph node dissection.

A 42-year-old man presented with a locally invasive cortical thymoma. Before chemotherapy was commenced 36 months after presentation, an unusual peripheral lymphocytosis of 19 x 10(9)/l had slowly developed over time. After the first course of chemotherapy, the lymphocytosis showed a sharp decline to normal absolute cell numbers and subsequently remained at normal levels. Currently, the patient is in stable partial remission and doing well. Immunophenotypic analysis showed a mature T-cell phenotype with 78% TcR-a beta and 16% TcR-gamma delta in the absence of an immature component. Pretreatment Southern blot analysis of peripheral blood mononuclear cells showed an oligoclonal pattern with 13-20 rearranged fragments of different intensity for the TcR beta-gene. TcR gamma also showed a pattern compatible with an oligoclonal proliferation. After treatment, after normalization of absolute blood counts, the distribution of T-cell subsets still showed a slightly aberrant pattern. Immunophenotypic analysis of a blood sample taken 6 months later, also at normal absolute cell counts, showed an increase of thymocytes as well as of mature T cells with a polyclonal pattern on Southern blot analysis. These findings may be interpreted as the result of aberrant positive and negative selection and development of thymocytes in the microenvironment of neoplastic thymic epithelial cells and clonal selection through continuous peripheral stimulation. Moreover, this case stresses the importance of integrated interpretation of clinical, morphological, immunophenotypical, and molecular data to gain insight in unusual clinical problems.

A case of a patient with bilateral ovarian cancer and a uterine malignant mesodermal mixed tumor with ascites and metastatic disease is presented. Flow cytometry, DNA fingerprinting, and immunohistochemistry were performed to assess the origin of these malignancies. Ploidy analysis showed that both ovarian tumors had different aneuploid stemlines (DNA index [DI] = 1.64, 1.85, right ovary and DI = 1.73, left ovary) indicating independent origins. One of the stemlines in the right ovary (DI = 1.64) was also present in the ascites cells, whereas omentum metastases showed the same stemline (DI = 1.73) as the left ovarian tumor. The uterine malignancy contained three aneuploid stemlines. The highest stemline was associated with epithelial differentiation, but a metastatic origin from the left ovarian tumor seems unlikely. DNA fingerprinting analysis revealed a common change in restriction fragment length pattern in the DNA from all tumor localizations as compared with the patient's constitutional DNA. These results indicate that DNA flow cytometry can be helpful in discriminating intragenital metastatic disease from multiple primary tumors.

In the novel WHO classification 2008, the classification of aggressive B-cell lymphoma has been revised for several categories with the aim to define “clean” entities. Within large B-cell lymphoma, a few distinct clinico-pathological entities have been recognized with more clinically defined entities than pathologically defined ones. The majority of known morphological variations were not considered to merit more than classification as a variant of DLBCL, not otherwise specified. Specifically, a biological subgrouping of DLBCL on the basis of molecular (activated B-cell versus germinal center B-cell) or immunophenotypic (CD5+) features was felt to be too immature to include at this stage. The role of EBV in aggressive B-cell lymphoma has been explored in more depth with the recognition of several novel and re-defined clinico-pathological entities. Also, in these diseases, clinical definitions play a very dominant role in the WHO classification 2008.