ALBERT

Description: Key Points IHC is a valuable clinical tool for assessing CD30+ PTCL patients who may respond to CD30-targeting treatment. CD30 mRNA and protein expression are highly correlated.

Description: Anaplastic lymphoma kinase (ALK)-positive lymphomas are characterized by expression of a hybrid protein, comprising the cytoplasmic portion of the ALK tyrosine kinase fused to a partner protein. This hybrid kinase is often encoded by the nucleophosmin (NPM)NPM-ALK fusion gene resulting from the (2;5)(p23;q35) chromosomal translocation. However, the ALK gene at 2p23 may also be involved in 2 variant translocations, namely t(1;2)(q25;p23) and t(2;3)(p23;q21), which create the TPM3-ALK andTFG-ALK fusion genes, respectively. We report here 2 lymphomas with an unusual finely granular cytoplasmic ALK staining pattern, clearly different from the pattern observed in ALK-positive lymphomas carrying NPM-ALK or its variants. A cloned complementary DNA sequence from 1 of these 2 lymphomas contained the ALK gene fused to the second clathrin heavy chain gene (also referred to as clathrin heavy polypeptide-like gene) (CLTCL). The distinctive granular cytoplasmic staining pattern for ALK was likely to be due to binding of the fusion protein to clathrin-coated vesicles. TheCLTCL gene is constitutively expressed in lymphoid cells and therefore presumably contributes an active promoter for theCLTCL-ALK gene. The fusion protein had a molecular weight (250 kd) that differs from all known ALK products, and it was autophosphorylated in an in vitro kinase assay, confirming that it is constitutively active and hence capable of contributing to malignant transformation. These 2 cases, therefore, represent a hitherto undescribed mechanism of ALK activation in lymphoma and further illustrate the diversity of fusion partners for the ALKgene.

Description: Anaplastic large cell lymphomas (ALCL) are frequently associated with the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene at 5q35, which encodes a nucleolar protein involved in shuttling ribonucleoproteins from the cytoplasm to the nucleus, to the anaplastic lymphoma kinase (ALK) gene at 2p23, encoding a tyrosine kinase receptor. In this report, we describe a typical case of ALCL whose malignant cells exhibited a novel (1;2)(q25;p23) translocation. These cells expressed ALK protein, but, in contrast to t(2;5)-positive ALCL (which show cytoplasmic, nuclear, and nucleolar staining), labeling was restricted to the malignant cell cytoplasm. Using a polymerase chain reaction (PCR)-based technique to walk on chromosome 2 from the known ALK gene across the breakpoint, we showed that the gene involved at 1q25 is TPM3, encoding a nonmuscular tropomyosin. We subsequently identified, using reverse transcription-PCR analysis of cases showing similar ALK cytoplasm-restricted staining, fusion of the ALK andTPM3 genes in 2 other cases of ALCL. The TPM3 gene has been previously found in papillary thyroid carcinomas as a fusion partner with the TRK kinase gene. We showed that TPM3 is constitutively expressed in lymphoid cell lines, suggesting that, in these t(1;2)-bearing ALCL cases, the TPM3 gene contributes an active promoter for ALK expression. Activation of the ALK catalytic domain probably results from homodimerization of the hybrid protein TPM3-ALK, through the TPM3 protein-protein interaction domain. The present cases of ALCL associated with a novel t(1;2)(q25;p23) demonstrate that at least one fusion partner other than NPM can activate the intracytoplasmic domain of the ALK kinase.

Description: Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK+ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase–positive (ALK+) lymphomas and human ALK+ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK+ cells, and demonstrate extracellular HMGB-1–stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK+ cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK+ cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK+ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK+ cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK+ lymphoma epidermotropism.

Description: Seven cases of large B-cell lymphoma which define a previously unrecognized subgroup are reported. Morphologically they are comprised of monomorphic large immunoblast-like cells, containing large central nucleoli, which tend to invade lymphatic sinuses. Superficially they resemble anaplastic large cell lymphoma (ALCL) but they lack CD30. These lymphomas express epithelial membrane antigen (as do ALCL), but also contain intracytoplasmic IgA of a single light chain type (five cases) and an endoplasmic reticulum–associated marker detected by antibody VS38. They lack lineage-associated leukocyte antigens with the exception of CD4 (5 of 5 cases) and CD57 (5 of 7 cases). They are labeled by antibodies detecting both the intracytoplasmic and extracellular regions of the ALK receptor kinase, suggesting that they express the full-length form of this molecule. This was confirmed by Western blotting (in the one case tested) which showed a band of 200 kD in tumor cell lysates, and by polymerase chain reaction (PCR) amplification of mRNA encoding intracellular and extracellular ALK sequences (in the two cases tested). There was no evidence by cytogenetics (one case analyzed) or reverse transcriptase-PCR (three cases tested) of the 2; 5 translocation or the resultant NPM-ALK gene, as is commonly found in ALCL. All but one of the patients were male and all but one were adults, and in all but the latter case the disease followed an aggressive course.

Description: With the use of microarray gene-expression profiling, we analyzed a homogeneous series of 32 patients with systemic anaplastic large-cell lymphoma (ALCL) and 5 ALCL cell lines. Unsupervised analysis classified ALCL in 2 clusters, corresponding essentially to morphologic subgroups (ie, common type vs small cell and “mixed” variants) and clinical variables. Patients with a morphologic variant of ALCL had advanced-stage disease. This group included a significant number of patients who experienced early relapse. Supervised analysis showed that ALK+ALCL and ALK− ALCL have different gene-expression profiles, further confirming that they are different entities. Among the most significantly differentially expressed genes between ALK+ and ALK− samples, we found BCL6, PTPN12, CEBPB, and SERPINA1 genes to be overexpressed in ALK+ ALCL. This result was confirmed at the protein level for BCL-6, C/EBPβ and serpinA1 through tissue microarrays. The molecular signature of ALK− ALCL included overexpression of CCR7, CNTFR, IL22, and IL21 genes but did not provide any obvious clues to the molecular mechanism underlying this tumor subtype. Once confirmed on a larger number of patients, the results of the present study could be used for clinical and therapeutic management of patients at the time of diagnosis.

Description: Abstract 2678 Introduction: Peripheral T-cell Lymphomas (PTCL) are types of rare and heterogeneous Non-Hodgkin's Lymphoma (NHL) that, in general, are associated with a poor clinical outcome. Discovery of new prognostic tools is thus a current and major challenge. In a cohort of 122 cases of PTCL collected from a multicentric T-cell lymphoma consortium (TENOMIC), we analyzed the expression of 80 non-coding small nucleolar RNAs (snoRNA) using high-throughput quantitative PCR (Fluidigm). SnoRNAs belong to the non-coding RNA family, and participate to diverse biological processes, most importantly ribosomal RNA maturation. Patients and methods: PTCL samples (n=122, 32 ALCL (22 ALK+, 10 ALK-) and 90 non-ALCL cases) were collected from TENOMIC. For each case, a consensus diagnosis was made by a panel of expert hemopathologists (some of the patients participated in a GELA-sponsored clinical trial, the others benefited from a national review protocol for T-cell lymphomas). Total RNA from sorted healthy donor-derived T-lymphocytes (n=35) and tumor samples were extracted (Trizol), integrity was verified using Agilent NanoChip, and reverse transcribed and assessed for snoRNA expression levels using The Fluidigm high-throughput quantitative PCR method. Medical cases were used to calculate progression-free and overall survival (PFS and OS) in 78 non-ALCL patients (26 PTCL-NOS, 46 AITL, 6 other rare entities) who received CHOP or CHOP-like regimen first-line. Results: First, we used unsupervised hierarchical clustering to show that, irrespectively from cell of the origin of the T cells, PTCL cells had a significant down-regulation of snoRNA expression in 63% of the snoRNA tested. In particular, there was a clear delineation between AITL, a tumor of T-follicular Helper (TFH) origin, and normal TFH cells from healthy donors. Among PTCL entities, ALCL had a specific snoRNA profile, but unsupervised clustering was not able to distinguish ALK- from ALK+ patients. However, a supervised comparison identified snoRNA U3 as a discriminant marker that sorted ALK+ from ALK- ALCL samples. Unsupervised or supervised snoRNA clustering failed to distinguish AITL from the other subgroups of PTCL. Second, we assessed prognostic impact of snoRNA expression in 78 non-ALCL patients. Characteristics of the cohorts were as follows: median age 65y/74y, Ann-Arbor stage III-IV in 94%/88%, elevated LDH in 76.7%/73.1%, IPI score 3–5 in 70%/61%, respectively. Although the snoRNA expression profiles of AITL and other PTCL subtypes appeared very similar, unsupervised clustering revealed the over-expression of 8 snoRNA in a subgroup of 31 patients with 45% OS at 5y (vs 47 patients with 18% OS at 5y). In AITL and PTCL-NOS cases, median OS was 26mo and 13mo, respectively. These thresholds were used in a supervised analysis to try to identify a specific prognostic snoRNA signature. A three snoRNA set was found significantly over-expressed in patients with the best prognosis, both in AITL patients (HBII-239, U59B, U90 impacted both on OS and PFS), and in PTCL-NOS patients (HBII-239, HBII-438A, U80 impacted on OS). Very interestingly, these signatures were not correlated neither with IPI or IPI-T scores, nor with overall response rates after CHOP. Lastly, we investigated the prognostic impact of HBII-239 snoRNA as a single marker. HBII-239 over-expression was statistically associated with prolonged PFS and OS in the entire cohort of 78 patients. Third, we investigated the impact of HBII-239 over-expression on cellular pathways in the FEPD cell line. This snoRNA is a precursor for microRNA-768, which is processed from the 3p strand of HBII-239. The FEPD cell line had a weaker expression of miR-768-3p as compared to patients' samples. Transfection of a miR-768-3p microRNAmimic induced a significant reduction of proliferation rate (as assessed by an MTS assay), due to a block at the G1/S checkpoint. In patients with low HBII-239 level, it is therefore expected that proliferation rate of lymphomatous cells would be increased, translating into decreased OS. Conclusions: Our study showed that snoRNAs are differentially regulated in normal compared to malignant T-cell populations. Besides a global down-regulation of these molecules, specific signatures may have a prognostic significance in PTCL. The different snoRNAs that can be regarded as potential biomarkers in these tumors may play a direct role in different cellular pathways. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1607 Background: Lymphoid malignancies derived from T and NK cells (PTCLs) constitute a heterogeneous group of uncommon disease entities, with marked geographic variation in their distribution. The most recent data from the International T-cell Lymphoma Project based on a retrospective analysis of PTCLs diagnosed between 1990 and 2002 (Blood. 2011;117(25): 6756–67) indicate that PTCL, not otherwise specified (PTCL,NOS) represents the most common PTCL worldwide (25,9%), followed by angioimmunoblastic T-cell lymphoma (AITL) (18,5%). Over the last few years, a better characterization of the cellular origin and pathophysiology of PTCLs has led to the development of new diagnostic markers. Aim of the study: To characterize the epidemiology of mature T/NK-cell malignancies in Western Europe (France and bordering countries) and to examine whether the availability of new tools/antibodies and changing concepts over the past years might have translated into an apparent change in the relative distribution of PTCL entities. Materials and methods: The histopathological diagnosis of PTCL entities according to the 2008 WHO classification were collected through two independent sets of PTCLs in France and bordering countries. Results: Over the past two years (2010–2011), 933 newly diagnosed non-cutaneous PTCLs were reviewed in reference centers through the prospective network “Lymphopath” aiming to review any newly diagnosed lymphoma in France. According to the 2008 WHO classification, the 933 PTCLs comprised: 314 AITL (33,6%), 239 PTCL,NOS (25,6%), 78 ALK-positive anaplastic large cell lymphoma (ALCL) (8,3%), 72 ALK-negative ALCL (7,7%), 59 extranodal NK/T-cell lymphomas (ENKTL 6%), 33 enteropathy-associated T-cell lymphoma (4%), 32 HTLV1+ adult T leukemia/lymphoma (3%), 7 hepatosplenic T-cell lymphoma (1%) and 99 cases of other entities or unclassifiable (11%). A high prevalence of AITL was also found in an independent set of PTCLs retrospectively collected in the framework of a multicentric T-cell lymphoma research consortium “Tenomic” where non-cutaneous PTCL with frozen material available (n=623) from 1999 to 2009 in France and Belgium were retrieved and collegially reviewed for consensus diagnosis. In this collection, AITL (n=288, 46%) also outnumbered PTCL, NOS (n=215, 35%). Of the 196 AITL cases extensively investigated for CD10, TFH markers (PD1, CXCL13), CD21/CD23 follicular dendritic cells (FDC) and EBV, the initial diagnosis was recorded in 178 cases as: AITL in 155 cases (87%), PTCL, NOS in 21 cases (12%), and intermediate between PTCL,NOS and AITL (PTCL,NOS/AITL) in 2 cases, indicating the impact of additional stainings for the diagnosis of AITL. The 107 PTCL,NOS cases also extensively immunostained included 9 follicular variant of PTCL,NOS, 8 PTCL,NOS/AITL cases, 5 cases intermediate between PTCL,NOS and ALK-negative ALCL (of which 2 had been diagnosed as such and two as PTCL,NOS), and 85 remaining cases truly unspecified. Of these, 60 had been initially diagnosed as PTCL,NOS; 2 as PTCL,NOS/AITL; 4 as ALK-negative ALCLs and 1 as ENKTL. Conclusion: This study based on two independent large cohorts of non-cutaneous PTCLs highlights AITL as the most prevalent entity in Western Europe. It shows that extensive studies including investigation for CD10, TFH markers, FDC and EBV can at least partly contribute to the reclassification of some PTCL, NOS into the AITL spectrum category. Disclosures: No relevant conflicts of interest to declare.

Description: AITL and PTCL-U, the two most common forms of T-cell lymphomas in western countries, usually present as nodal disease and pursue an aggressive clinical course. AITL is commonly associated with a constellation of clinical symptoms and distinct pathological features. Conversely, PTCL-U lacks precise diagnostic criteria, and by default comprises cases not fulfilling criteria for other entities, including tumors with borderline features to ALCL and AITL. The genetic alterations and pathogenic mechanisms underlying AITL and PTCL-U are largely unknown. To determine whether the molecular signature of AITL and PTCL-U could help in distinguishing both entities and in understanding ther ontogeny, we performed gene expression profile (GEP) analysis of 15 PTCL-U tissue samples (6 CD30+ and 9 CD30−) and 19 AITL samples (including 2 sorted tumor cell suspensions) using Affymetrix HG-U133A Plus2.0 pan-genomic oligonucleotide microarrays, with comparison to that of previously published normal T-cell subsets (J Immunol173:68; J Immunol175: 7837; Blood 104: 1952). Principle component analysis (PCA, accumulated variance 95%) of all 33 tissue samples yielded three groups of tumors: one group of 12 AITLs, one group of 10 PTCLs-U and one mixed group comprising 5 AITLs (some with features borderline to PTCL-U) and 6 PTCLs-U (including 5 of 6 CD30+ tumors). The AITL molecular signature consisted of 442 genes with increased levels of expression in AITL compared to PTCL-U (t test, p

Description: Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK+) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK+ ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK+ ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.