Search

International audience; Although follicular lymphoma (FL) is usually graded as FL1-2, FL3A, and FL3B, some borderline cases can be observed and led us to investigate the clinicopathologic diversity of grade 3 FL (FL3). Among 2449 FL patients enrolled in Lymphoma Study Association (LYSA) trials, 1921 cases with sufficient material underwent a central pathologic review. The resulting diagnoses comprised 89.6% FL1-2 (n=1723), 7.2% FL3A (n=138), and 0.5% purely follicular FL3B (n=9). The remaining 51 unclassifiable cases (2.7%) exhibited high-grade features but did not meet WHO criteria for either FL3A or FL3B; and were considered as “unconventional” high-grade FL (FL3U). FL3U morphological pattern consisted of nodular proliferation of large cleaved cells or small-sized to medium-sized blast cells. Compared with FL3A, FL3U exhibited higher MUM1 and Ki67 expression, less BCL2 breaks and more BCL6 rearrangements, together with a higher number of cases without any BCL2, BCL6 or MYC rearrangement. FL3U harbored less frequent mutations in BCL2, KMT2D, KMT2B, and CREBBP than FL3A. MYC and BCL2 were less frequently mutated in FL3U than FL3B. Rituximab cyclophosphamide, doxorubicin, vincristine, and prednisone treated FL3U patients had a worse survival than FL1-2 patients with similar follicular lymphoma international prognostic index and treatment. These results suggest that high-grade FLs encompass a heterogeneous spectrum of tumors with variable morphology and genomic alterations, including FL3U cases that do not strictly fit WHO criteria for either FL3A or FL3B, and display a worse outcome than FL1-2. The distinction of FL3U may be useful to allow a better comprehension of high-grade FLs and to design clinical trials.

AimsPrimary mediastinal large B-cell lymphoma (PMBL) diagnosis can be challenging on needle biopsies. Robust techniques are needed to ensure diagnosis of this lymphoma which is highly sensitive to recently developed therapy protocols.MethodsIn this study, we sought to determine precise PMBL phenotype, compared with diffuse large B-cell lymphoma not otherwise specified, by combining immunohistochemistry with anti-MAL antibody and RNA in situ hybridisation (RNAscope) with specific MAL probes.ResultsThe overall MAL positivity level reached 93% (14/15) of cases of PMBL. Among the 15 cases enrolled in the study, 11 were undoubtedly positive for MAL immunostaining whereas 13 were positive by RNA in situ hybridisation. Interestingly, one case that was negative by in situ hybridisation turned out to be positive by immunohistochemistry.ConclusionsTaken together, our results demonstrate that in situ detection of both MAL transcripts and protein are complementary and increase the sensitivity and specificity of PMBL diagnosis.

BACKGROUND Congo red-positive material was described in normal and diseased parathyroids (adenoma and hyperplasia) 50 years ago. However, the incidence and the clinical significance of such observation are unknown, and the causal fibril protein has never been convincingly demonstrated. METHODS We conducted the present study including an exceptional case report accompanied with a retrospective study of 105 parathyroid adenomas. We used histopathological, immunohistochemical, ultrastructural, mass spectrometry-based proteomic analysis of parathyroid adenoma tissue samples, and genetic analysis. RESULTS We describe a 57-year-old man with mild hypercalcemia and elevated parathyroid hormone (PTH) level for whom histopathological analysis revealed a parathyroid adenoma associated with nodular typical amyloid deposits. Tandem mass spectrometry after laser microdissection (LMD-MS) of amyloid adenoma identified PTH as the fibril protein, and no germline mutation in the PTH gene was detected. Congo red-positive PTH-deposits were further observed in 6.6% of the parathyroid adenomas analyzed, and were associated with complete/incomplete or absent universal amyloid signature, but with fibrillar morphology at ultrastructural level. CONCLUSIONS Inappropriate PTH production leads to progressive disease-amyloid aggregation of PTH in a subset of parathyroid adenomas, providing new insights into the pathophysiology of this condition and adding PTH to the list of amyloid protein derived from hormones.

Breast implant-associated anaplastic large cell lymphoma (BI-ALCL) is a rare form of lymphoma arising adjacent to a breast implant. We aim to review the pathogenesis and clinico-biological features of BI-ALCL.BI-ALCL is a new provisional entity in the 2017 updated WHO classification. Among several hypotheses, BI-ALCL development seems to be determined by the interaction of immune response related to implant products and additional genetic events.BI-ALCL is an uncommon T-cell lymphoma which is increasingly diagnosed since its first description in 1997 with 500 estimated cases worldwide. Two BI-ALCL subtypes correlating with clinical presentation have been described. Although most BI-ALCL patients with tumor cell proliferation restricted to the periprosthetic effusion and capsule have excellent outcomes, other patients presenting with a tumor mass, may have a more aggressive disease. The pathogenesis of BI-ALCL remains elusive. It is postulated that local chronic inflammation elicitated by bacterial infection or implant products may promote the activation and proliferation of T cells. Additional genetic events resulting in the activation JAK/STAT pathway are also incriminated. Further investigations are needed to better characterize the pathogenesis of this disease in order to determine the potential risk to develop BI-ALCL after surgical implants.

Classical Hodgkin's Lymphoma (cHL) has been well studied over the past few decades. Newly diagnosed patients are treated with combinations of chemotherapeutics, the most common of which includes adriamycin, bleomycin, vinblastine, dacarbazine (ABVD). Approximately 75-85% of newly diagnosed cHL patients show favourable outcomes in response to ABVD treatment resulting in long lasting remission. The remaining 15-25% of diagnosed cHL patients will either show primary resistance to ABVD or relapse during or after the completion of therapy. Despite the fact that cHL biology has been extensively investigated, a robust biomarker to predict initial anthracycline based treatment failure remains an unresolved challenge in the management of the disease. Refractory and relapsing HL patients can be treated with several other treatment modalities including the recently developed brentuximab vedotin, and the PD-1 inhibitors nivolumab and pembrolizumab. The improvement in the outcomes of refractory and relapsed cHL patients treated with such advanced therapeutics presents a rational to advance such therapeutics to first line therapy for all newly diagnosed cHL patients. However, these therapeutics are accompanied with more adverse side effects compared to ABVD, and they present a significantly higher cost burden on the healthcare systems. In addition, clinical studies have shown that not all patients do respond favourably to these therapeutics. In spite of the development of the advanced therapies, the absence of a tool to stratify cHL patients at point of diagnosis remains to be a critical unmet clinical need. In previous reports, the 3-dimensional analysis of telomeres using TeloView® technology conducted on a proof of concept cohort of 27 HL patients revealed that TeloView® analysis, on the group patient level, showed distinct telomere profiles for HL patients who relapsed versus patients who remained stable. In this report, we conducted a retrospective clinical study including a multi-centre cohort of 156 HL patients, 125 remained stable for over 5 years, and 31 patients relapsed within 12 months. We targeted to identify, on the individual patient's level, significant telomeres parameters suitable to predict individual patient outcome at point of diagnosis. General univariate analysis including t-test and Anova identified derived telomeres parameters suitable for predictive modelling analysis including: total and average telomeres length, telomeric aggregates, nuclear volume, quartile nuclear volume and very short telomeres (t-stumps). Multivariate analysis using logistic regression procedures allowed for developing of significant predictive models using combinations of 2, 3 & 4 predictors. The highest predictive power was attained by a 4-predictors model with a predictive power of 0.76 presented as area under the curve (AUC) in receiver operating characteristic (ROC) curve illustration, achieving 75% positive predictive value, and 71% negative predictive value. The results of this study offer a long awaited path to a precision medicine approach for the treatment of cHL patients. The ready-to-use predictive model will further be validated retrospectively on an independent patient cohort, and can further be validated prospectively in longitudinal studies once the test is adopted in the clinic. Disclosures Louis: Telo Genomics Corp.: Consultancy, Current equity holder in publicly-traded company. Johnson:Roche/Genentech, Merck, Bristol-Myers Squibb, AbbVie: Consultancy; AbbVie: Research Funding; Roche/Genentech, Merck: Honoraria. Ludkovski:Telo Genomics Corp.: Current Employment. Shifrin:Telo Genomics Corp.: Current Employment. Mai:Telo Genomics Corp.: Consultancy, Current equity holder in publicly-traded company. Knecht:Telo Genomics Corp.: Consultancy, Current equity holder in publicly-traded company.

Aims Primary prostatic lymphomas (PPL) is exceedingly rare. The aim of this study was to investigate the largest series of PPL obtained from a nationwide expert pathologist network, and thus try to understand the pathophysiology of these tumours. Methods and results Up to 66 000 lymphoma cases have been collected and submitted for central expert review by the French Lymphopath network. We confirm the low frequency of PPL (n = 77; 0.12%), all cases being of B-cell origin. Diffuse large B-cell lymphoma and small lymphocytic lymphoma were the most frequent subtypes, comprising 31% and 26% of cases respectively, followed by mucosa-associated lymphoid tissue (MALT)/lymphoplasmacytic lymphoma (19%), follicular lymphoma (12%), mantle cell lymphoma (6%), Burkitt lymphoma (4%), and unclassified lymphoma (1%). Clinical data obtained in 25 cases suggests that PPLs are rather indolent tumours. Our hypothesis for B-cell recruitment in the prostatic tissue was derived from the observation in chronic inflammation (prostatitis) of frequent heterotopic proliferation of high endothelial venules (HEVs). The latter are dedicated to lymphocyte entry into secondary lymphoid organs, here putatively driving circulating clonal B-lymphocytes from the blood into the inflamed prostate. This may account for the relatively high incidence of small lymphocytic lymphoma consistently reported in series of primary or secondary prostatic lymphoma. As in other organs or glands, chronic inflammation may promote antigen-dependent intraprostatic MALT lymphoma and diffuse large B-cell lymphoma development. Conclusions PPLs are exclusively of B-cell origin, and chronic inflammation resulting from the proliferation of high endothelial venules could play some role in their development.

Background Immunotherapy is currently under investigation in B3 Thymoma (TB3) and Thymic Carcinoma (TC). PD-L1 expression has been evaluated on a limited number of patients with selected antibodies. We aimed to analyze cohort of TB3 and TC with a panel of antibodies to assess the prevalence of PD-L1 expression, its prognostic value and to set up a reproducible test. Methods We retrospectively studied 103 patients samples of FFPE histologically confirmed TB3 (n = 53) and TC (n = 50) by expert pathologists within the RYTHMIC national network. We compared PD-L1, PD1, CD8 and PD-L2 expression and performed correlation with tumor types and patients outcomes. Four PD-L1 antibodies were tested, three of them validated as companion tests in lung cancer, one tested on two automates on whole section of tumors. We evaluated the percentage and intensity of both epithelial and immune stained cells. Results TB3 epithelial cells had a higher and more diffuse expression of PD-L1 than TC regardless the antibodies tested (p Conclusion Frequent PD-L1 expression, particularly in TB3, paves the way for immunotherapy in TET (Thymic Epithelial Tumor). Otherwise, we have set up three reproducible LDT (laboratory-developed test) for four PD-L1 antibodies.

Purpose: The detection of preexisting EGFRT790M subclones and the assessment of their clinical significance in the pretreatment of patients with EGFRT790M non–small cell lung cancer (NSCLC) remain unclear. Experimental Design: A total of 179 tumor samples from patients treated or not with a first-generation tyrosine kinase inhibitor (TKI) was analyzed. The presence of ultra-low levels of preexisting EGFRT790M mutation was evaluated using ultra-sensitive droplet digital PCR (ddPCR) and the clinical implication of these mutations on first-generation TKI efficiency assessed. Results: With a ddPCR linear performance of 0.999 and an analytical sensitivity of approximately 0.001%, we observed a 66% (99/150) overall incidence of ultra-low EGFRT790M mutation. Among 82 patients harboring EGFRactivating mutations, the presence of a preexisting EGFRT790M mutation prior to any treatment was significantly associated with a longer progression-free survival (PFS; P = 0.009; log-rank test). Interestingly, longer PFS was linked to concomitant EGFRdel19 and ultra-low EGFRT790M mutations. Moreover, the presence of both EGFRdel19 and ultra-low EGFRT790M mutations was identified as the best fit for predicting the clinical outcome of patients treated with TKI compared with an ultra-low EGFRT790M mutation status or an activating mutation alone (P = 0.042 and P = 0.0071, respectively). Conclusions: We demonstrate that the detection of the ultra-low EGFRT790M mutation in TKI-naïve patients is not a rare event. We suggest that ddPCR should be used in clinical practice to distinguish patients who may respond to first- or third-generation TKIs.

We previously reported a new form of light chain deposition disease (LCDD) presenting as diffuse cystic lung disorder that differs from the usual systemic form with respect to patient age, the male/female ratio, the involved organs, and the hematologic characteristics. We also demonstrated that the light chains were produced by an intrapulmonary B-cell clone and that this clone shared a stereotyped antigen receptor IGHV4-34/IGKV1. However, we only analyzed 3 patients. We conducted a retrospective study including lung tissue samples from 24 patients with pulmonary LCDD (pLCDD) matched with samples from 13 patients with pulmonary κ light chain amyloidosis (pAL amyloidosis) used as controls. Mass spectrometry–based proteomics identified immunoglobulin κ peptides as the main protein component of the tissue deposits in all patients. Interestingly, in pLCDD, IGKV1 was the most common κ family detected (86.4%), and IGKV1-8 was overrepresented compared with pAL amyloidosis (75% vs 11.1%, P = .0033). Furthermore, IGKV1-8 was predominantly associated with a diffuse cystic pattern (94%) in pLCDD. In conclusion, the high frequency of IGKV1-8 usage in cystic pLCDD constitutes an additional feature arguing for a specific entity distinct from the systemic form that preferentially uses IGKV4-1.

International audience; Amyloidoses are rare life-threatening diseases caused by protein misfolding of normally soluble proteins. The fatal outcome is predominantly due to renal failure and/or cardiac dysfunction. Because amyloid fibrils formed by all amyloidogenic proteins share structural similarity, amyloidoses may be studied in transgenic models expressing any amyloidogenic protein. Here we generated transgenic mice expressing an amyloidogenic variant of human apolipoprotein AII, a major protein of high density lipoprotein. According to amyloid nomenclature this variant was termed STOP78SERApoAII. STOP78SER-APOA2 expression at the physiological level spontaneously induced systemic amyloidosis in all mice with full-length mature STOP78SERApoAII identified as the amyloidogenic protein. Amyloid deposits stained with Congo red, were extracellular, and consisted of fibrils of approximately 10 nm diameter. Renal glomerular amyloidosis was a major feature with onset of renal insufficiency occurring in mice older than six months of age. The liver, heart and spleen were also greatly affected. Expression of STOP78SERAPOA2 in liver and intestine in mice of the K line but not in other amyloid-laden organs showed they present systemic amyloidosis. The amyloid burden was a function of STOP78SER-APOA2expression and age of the mice with amyloid deposition starting in two-month old highexpressing mice that died from six months onwards. Because STOP78SER-ApoAII conserved adequate lipid binding capacity as shown by high STOP78SER-ApoAII amounts in high density lipoprotein of young mice, its decrease in circulation with age suggests preferential deposition into preformed fibrils. Thus, our mouse model faithfully reproduces early-onset hereditary systemic amyloidosis and is ideally suited to devise and test novel therapie

ALK-negative anaplastic large cell lymphoma associated with breast implant (i-ALCL) has been recently recognized as a distinct entity. Among 43 830 lymphomas registered in the French Lymphopath network since 2010, 300 breast lymphomas comprising 25 peripheral T-cell lymphomas (PTCL) were reviewed. Among PTCL, ALK-negative ALCL was the most frequent and all of them were associated with breast implants.Since 2010, all i-ALCL cases were collected from different institutions through Lymphopath. Immuno-morphologic features, molecular data and clinical outcome of 19 i-ALCLs have been retrospectively analyzed.The median age of the patients was 61 years and the median length between breast implant and i-ALCL was 9 years. Most implants were silicone-filled and textured. Implant removal was performed in 17 out of 19 patients with additional treatment based on mostly CHOP or CHOP-like chemotherapy regimens (n = 10/19) or irradiation (n = 1/19). CHOP alone or ABVD following radiation without implant removal have been given in two patients. The two clinical presentations, i.e. effusion and less frequently tumor mass correlated with distinct histopathologic features: in situ i-ALCL (anaplastic cell proliferation confined to the fibrous capsule) and infiltrative i-ALCL (pleomorphic cells massively infiltrating adjacent tissue with eosinophils and sometimes Reed-Sternberg-like cells mimicking Hodgkin lymphoma). Malignant cells were CD30-positive, showed a variable staining for EMA and were ALK negative. Most cases had a cytotoxic T-cell immunophenotype with variable T-cell antigen loss and pSTAT3 nuclear expression. T-cell receptor genes were clonally rearranged in 13 out of 13 tested cases. After 18 months of median follow-up, the 2-year overall survival for in situ and infiltrative i-ALCL was 100% and 52.5%, respectively.In situ i-ALCLs have an indolent clinical course and generally remain free of disease after implant removal. However, infiltrative i-ALCLs could have a more aggressive clinical course that might require additional therapy to implant removal.

Mastocytosis is a rare disease characterized by clonal neoplastic proliferation of mast cells (MCs). It ranges from skin lesions as cutaneous mastocytosis (CM) which may spontaneously regress to highly aggressive neoplasms with multiorgan involvement corresponding to some aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), and/or mast cell sarcoma (MCS). There is increasing evidence of CD30 expression in neoplastic MCs of the bone marrow. This expression has been described almost exclusively in aggressive forms of systemic mastocytosis (SM). The aim of the present study is to evaluate CD30 expression both in cutaneous and systemic forms of mastocytosis. Forty-two mastocytosis cases were reviewed, including cutaneous (n = 29) and systemic (n = 13) forms to assess the prevalence of CD30 expression. Thirty-nine out of 42 (92.8%) cases were CD30 positive. In cases of CM, 28/29 (96.5%) cases were CD30 positive, 11/13 cases of SM (84.6%) were positive for CD30. MCs in normal skin biopsies and in urticaria lesions were CD30-negative. This study found that CD30 is also frequently expressed in CM as well as in systemic forms. This finding is a major departure from the prevailing concept that CD30 expression is often related to aggressive systemic forms of mastocytosis.

Rearrangements of the anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) represent a novel molecular target in a small subset of tumors. Although ALK rearrangements are usually assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), molecular approaches have recently emerged as relevant alternatives in routine laboratories. Here, we evaluated the use of two different amplicon-based next-generation sequencing (NGS) methods (AmpliSeq and Archer®FusionPlex®) to detect ALK rearrangements, and compared these with IHC and FISH. A total of 1128 NSCLC specimens were screened using conventional analyses, and a subset of 37 (15 ALK-positive, and 22 ALK-negative) samples were selected for NGS assays. Although AmpliSeq correctly detected 25/37 (67.6%) samples, 1/37 (2.7%) and 11/37 (29.7%) specimens were discordant and uncertain, respectively, requiring further validation. In contrast, Archer®FusionPlex® accurately classified all samples and allowed the correct identification of one rare DCTN1-ALK fusion, one novel CLIP1-ALK fusion, and one novel GCC2-ALK transcript. Of particular interest, two out of three patients harboring these singular rearrangements were treated with and sensitive to crizotinib. These data show that Archer®FusionPlex® may provide an effective and accurate alternative to FISH testing for the detection of known and novel ALK rearrangements in clinical diagnostic settings.

Purpose To prospectively assess the clinical impact of expert review of lymphoma diagnosis in France. Materials and Methods From January 2010 to December 2013, 42,145 samples from patients with newly diagnosed or suspected lymphomas were reviewed, according to the 2008 WHO classification, in real time by experts through the Lymphopath Network. Changes in diagnosis between referral and expert review were classified as major or minor according to their potential impact on patient care. Results The 42,145 reviewed samples comprised 36,920 newly diagnosed mature lymphomas, 321 precursor lymphoid neoplasms, 314 myeloid disorders, and 200 nonhematopoietic neoplasms, with 4,390 benign lesions. There were 4,352 cutaneous and 32,568 noncutaneous lymphomas. The most common mature noncutaneous lymphomas were diffuse large B-cell lymphomas (32.4%), follicular lymphomas (15.3%), classic Hodgkin lymphomas (13%), peripheral T-cell lymphomas (6.3%) of which angioimmunoblastic T-cell lymphomas (2.3%) were the most frequent, and mucosa-associated lymphoid tissue lymphomas (5.8%). A diagnostic change between referral and expert review occurred in 19.7% of patients, with an estimated impact on patient care for 17.4% of patients. This rate was significantly higher for patients sent with a provisional diagnosis seeking expert second opinion (37.8%) than for patients sent with a formal diagnosis (3.7%). The most frequent discrepancies were misclassifications in lymphoma subtype (41.3%), with 12.3% being misclassifications among small B-cell lymphoma entities. Fewer than 2% of changes were between benign and malignant lymphoid conditions. Minor changes (2.3%) mostly consisted of follicular lymphoma misgrading and diffuse large B-cell lymphoma subtype misclassification. Conclusion To our knowledge, this study provides the largest ever description of the distribution of lymphoma entities in a western country and highlights how expert review significantly contributes to a precise lymphoma diagnosis and optimal clinical management in a proportion of patients.

Purpose: Randomized, multicenter, open-label, phase 2/3 trial investigating lenalidomide versus investigator's choice (IC) in relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Experimental Design: Patients with DLBCL who received ≥2 prior therapies were stratified by DLBCL subtype [germinal center B-cell (GCB) vs. non-GCB; determined by immunohistochemistry (IHC)] and then randomized 1:1 to lenalidomide (25 mg/day, 21 days of 28-day cycle) or IC (gemcitabine, rituximab, etoposide, or oxaliplatin). Crossover to lenalidomide was permitted for IC-treated patients with radiologically confirmed progressive disease. The primary endpoint was overall response rate (ORR). Progression-free survival (PFS), overall survival, and subtype analysis [GCB vs. activated B-cell (ABC)] using gene expression profiling (GEP) were exploratory endpoints. Results: Stage 1: 102 DLBCL patients (by IHC: non-GCB, n = 54; GCB, n = 48) received ≥1 dose of lenalidomide or IC. Hematologic treatment-emergent adverse events with lenalidomide versus IC included neutropenia (42.6%; 36.4%), anemia (33.3%; 47.3%), thrombocytopenia (24.1%; 43.6%), and leukopenia (5.6%; 12.7%), respectively. Overall, lenalidomide-treated patients had an ORR of 27.5% versus 11.8% in IC (ORRs were similar regardless of IHC-defined DLBCL subtype). Median PFS was increased in patients receiving lenalidomide (13.6 weeks) versus IC (7.9 weeks; P = 0.041), with greater improvements in non-GCB patients (15.1 vs. 7.1 weeks, respectively; P = 0.021) compared with GCB (10.1 vs. 9.0 weeks, respectively; P = 0.550). Conclusions: The clinical benefit of lenalidomide monotherapy in DLBCL patients was more evident in the non-GCB subtype. Exploratory analyses suggest that this preferential benefit was more pronounced in the GEP-defined ABC population, demonstrating a need for additional studies of lenalidomide in DLBCL using GEP subtyping. Clin Cancer Res; 23(15); 4127–37. ©2017 AACR.

Mycosis fungoides (MF) represents the most common epidermotropic cutaneous T-cell lymphoma (CTCL), and tumor cells typically express a mature T-helper memory phenotype. A minority of MF patients display an unusual phenotype, which may be either CD4(-)/CD8(+) or double negative. Herein, we report a case of biopsy-proven MF in a 31-year-old woman who presented with infiltrated plaques involving photoprotected areas of the skin. Immunohistochemical study combined with confocal microscopy revealed co-expression of CD4 and CD8 in a subset of atypical T lymphocytes. To our knowledge, this is the second report of a CD4/CD8 dual-positive MF.

Fluorescence in situ hybridization is an indispensable technique used in routine pathology and for theranostic purposes. Because fluorescence in situ hybridization techniques require sophisticated microscopic workstations and long procedures of image acquisition with sometimes subjective and poorly reproducible results, we decided to test a whole-slide imaging system as an alternative approach. In this study, we used the latest generation of Pannoramic 250 Flash digital microscopes (P250 Flash digital microscopes; 3DHISTECH, Budapest, Hungary) to digitize fluorescence in situ hybridization slides of diffuse large B cells lymphoma cases for detecting MYC rearrangement. The P250 Flash digital microscope was found to be precise with better definition of split signals in cells containing MYC rearrangement with fewer truncated signals as compared to traditional fluorescence microscopy. This digital technique is easier thanks to the preview function, which allows almost immediate identification of the tumor area, and the panning and zooming functionalities as well as a shorter acquisition time. Moreover, fluorescence in situ hybridization analyses using the digital technique appeared to be more reproducible between pathologists. Finally, the digital technique also allowed prolonged conservation of photos. In conclusion, whole-slide imaging technologies represent rapid, robust, and highly sensitive methods for interpreting fluorescence in situ hybridization slides with break-apart probes. In addition, these techniques offer an easier way to interpret the signals and allow definitive storage of the images for pathology expert networks or e-learning databases.

Purpose To evaluate safety of medium-chain triglycerides used as a possible intraocular tamponading agent. Methods A 20-gauge pars plana vitrectomy was performed in the right eye of 28 rabbits. An ophthalmologic examination was performed every week until rabbits were killed. At days 7, 30, 60, and 90, rabbits were killed and the treated eyes were examined macroscopically and prepared for histologic examination. Principal outcome was retinal toxicity evaluated by light and electron microscopy, and secondary outcomes were the presence of medium-chain triglyceride emulsification, inflammatory reactions, and the development of cataract. Results Histologic examination did not reveal any retinal toxicity. Two cases of moderate emulsification were observed, but in these cases, emulsification was caused by the perioperative injection of the agent and did not increase during the postoperative period. We noted 13 cases of inflammatory reaction in vitreous cavity and no case of inflammatory reaction in anterior chamber. Two eyes developed cataract as a result of perioperative trauma to the lens with the vitreous cutter and not secondary to the presence of medium-chain triglycerides in the vitreous cavity. Conclusion Medium-chain triglycerides did not induce morphologic evidence of retinal toxicity. The results suggest that medium-chain triglycerides could be a promising alternative intraocular tamponading agent for the treatment of retinal detachments.

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.

International audience; Background: Visceral leishmaniasis (VL) is endemic in large areas of the tropics, the subtropics, and the Mediterranean basin. Besides classical VL presentation, exceptional cases of a limited form of VL have been reported. Here we describe the challenges of diagnosis and management of this intriguing entity.Case summary: A 25-year-old French Caucasian man presented with marked asthenia that had lasted 6 months and was strictly isolated except for a 2-cm left cervical lymphadenopathy. The rest of the clinical examination and extensive biological exploration were unremarkable.Histological examination of the cervical lymphadenopathy showed a reactive lymphoid hyperplasia with granulomatous organization associated with small particles in the cytoplasm of epithelioid histiocytes and giant cells evocative of Leishman-Donovan bodies. Polymerase chain reaction (PCR) performed on the tissue confirmed the presence of Leishmania donovani/infantum DNA. Direct examination of a bone marrow aspiration, together with blood and bone marrow PCR, did not find other evidence for VL. Serology for leishmaniasis was unreactive. Extensive work-up for other causes of granulomatous lymphadenitis was negative. A diagnosis of localized leishmanial lymphadenopathy was made. Intravenous liposomal amphotericin B (20 mg/kg in five infusions) was initiated and well tolerated. Asthenia disappeared promptly and the patient fully recovered.Conclusion: Localized lymph node enlargement because of leishmanial infection should be included in the differential diagnosis of lymphadenopathy of unknown origin in patients who stayed or visited, even a long time ago and for a short period, endemic areas for leishmaniasis such as the Mediterranean basin. Fine-needle aspiration cytology and/or PCR for Leishmania sp of the lymphadenopathy might contribute to the diagnosis. A low-dose liposomal amphotericin B treatment might be effective, and deserves further study.

Background Cyclin D1-positive B cells are occasionally found in the mantle zones of reactive lymphoid follicles, a condition that has been called 'in situ mantle cell lymphoma'. The clinical significance of this lesion remains uncertain. Design and Methods The clinical and pathological characteristics, including SOX11 expression, of 23 cases initially diagnosed as in situ mantle cell lymphoma were studied. Results Seventeen of the 23 cases fulfilled the criteria for in situ mantle cell lymphoma. In most cases, the lesions were incidental findings in reactive lymph nodes. The t(11; 14) was detected in all eight cases examined. SOX11 was positive in seven of 16 cases (44%). Five cases were associated with other small B-cell lymphomas. In two cases, both SOX11-positive, the in situ mantle cell lymphoma lesions were discovered after the diagnosis of overt lymphoma; one 4 years earlier, and one 3 years later. Twelve of the remaining 15 patients had a follow-up of at least 1 year (median 2 years; range, 1-19.5), of whom 11 showed no evidence of progression, including seven who were not treated. Only one of 12 patients with an in situ mantle cell lymphoma lesion and no diagnosis of mantle cell lymphoma at the time developed an overt lymphoma, 4 years later; this case was also SOX11-positive. The six remaining cases were diagnosed as mantle cell lymphoma with a mantle zone pattern. Five were SOX11-positive and four of them were associated with lymphoma without a mantle zone pattern. Conclusions In situ mantle cell lymphoma lesions are usually an incidental finding with a very indolent behavior. These cases must be distinguished from mantle cell lymphoma with a mantle zone pattern and overt mantle cell lymphoma because they may not require therapeutic intervention.

Background In susceptible individuals, multiple events may trigger pulmonary vascular remodeling and pulmonary arterial hypertension (PAH). Human herpesvirus-8 (HHV-8), a γ-herpesvirus homologous with Epstein-Barr virus (EBV), was suggested to act as a “second hit” in the development of PAH in susceptible patients. Although there is indirect evidence from in vitro and animal studies in favor of a link between γ-herpesviruses and the pathophysiology of PAH, results remain controversial. Therefore, we investigated the presence of EBV and HHV-8 in the lungs of patients with PAH. Methods Thirty-four lungs explanted from French patients with end-stage PAH (mean age, 38 ± 14 years; 19 women) were studied. Tissue samples were incorporated into tissue microarrays. Normal lung tissues served as negative controls. Kaposi sarcoma tissue served as a positive control for HHV-8, and EBV-associated lymphoma served as a positive control for EBV. The presence of HHV-8 was investigated with immunohistochemistry and polymerase chain reaction. The presence of EBV was investigated with immunohistochemistry and in situ hybridization. Results For HHV-8, none of PAH lung samples showed a “stippling” nuclear pattern classically observed in HHV-8-positive Kaposi sarcoma lesions. When studied by polymerase chain reaction, all cases remained negative. For EBV, none of the PAH lung samples showed positive staining, whatever the technique applied. Conclusions HHV-8 and EBV cannot be detected in the lungs of patients with end-stage PAH. The role of these γ-herpesviruses in the pathophysiology of PAH is, therefore, unlikely.

International audience; The clinical management of osteosarcoma differs significantly from that of chondrosarcoma; therefore, it is extremely important to diagnose these 2 types of bone tumor accurately. In the absence of a specific marker, differential diagnosis by histochemistry is sometimes impossible, especially between chondroblastic osteosarcoma and conventional chondrosarcoma. We analyzed 165 bone sarcomas by immunohistochemical staining of tissue microarrays for expression of the galectin-1 (GAL1) lectin and by Western blot experiments. We found that GAL1 was abundant in normal human osteoblasts from benign proliferations and in osteosarcomas, including chondroblastic osteosarcomas, but not in chondrosarcomas. There was a highly significant statistical difference in the percentage of stained cells (P < 10(-4)) and in the staining intensity (P < 10(-3)) of chondroblastic osteosarcomas compared to conventional chondrosarcomas. This discriminatory potential of GAL1 staining for osteosarcoma-derived tumors was confirmed by Western blotting. We propose a diagnostic test for bone tumors that takes into account the optimal discriminative values for the percentage of cells stained and the intensity of staining. The positive and negative predictive values were 85.7% (trust interval of 63.7%-97%) and 90% (trust interval of 80%-95.9%), respectively, demonstrating the pertinence of the test. Altogether, our data indicate that GAL1 is a powerful diagnostic marker that distinguishes chondroblastic osteosarcomas from conventional chondrosarcomas.

Survivin is a member of the inhibitor of apoptosis gene family, which is also implicated in mitosis regulation. Most reports in the literature impute poor prognosis to neoplasms with overexpression of this protein. The purpose of the present study is to validate and compare the immunohistochemical reactivity of malignant lymphomas and reactive lymphoid tissue using a new mouse monoclonal antibody to Survivin produced in our laboratory, 6-78. Survivin was detected by immunohistochemistry on tissue microarrays. It was shown that the antibody anti-Survivin 6-78 reliably stains formalin-fixed, paraffin-embedded reactive and neoplastic lymphoid tissues, mostly in a nuclear pattern. We confirmed using this novel antibody that Survivin immunostaining has a tendency to be lower in reactive lymphoid tissues and low-grade B cell lymphomas than in aggressive lymphomas. This antibody may represent a useful tool for standardizing the study of the immunoexpression of Survivin in neoplasms.

Purpose Anaplastic lymphoma kinase (ALK) –positive diffuse large B-cell lymphoma (DLBCL) is a rare variant of DLBCL that has been described only in small case reports. To shed more light on the clinical and pathologic features and outcome of these tumors, we reviewed data from 38 patients. Patients and Methods We retrospectively analyzed 38 patients with ALK-positive DLBCL treated with cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) or CHOP-like regimens from different institutions to better define the presenting features, clinical course, and response to treatment. Results The histologic findings in all patients were similar. All patients expressed ALK fusion proteins, but virtually all were CD30 and CD20 negative. The median age was 43 years with a 5:1 ratio of males to females. Most patients (60%) followed an aggressive clinical course with advanced stage at diagnosis, frequent marrow infiltration, and poor outcome. Overall survival was 20.3 months (95% CI, 12.2 to 42.6 months). Of note, the median survival was only 12.2 months (95% CI, 9.1 to 32.5 months) in patients with advanced-stage disease. Conclusion ALK-positive DLBCLs display clinicopathologic features that distinguish them from common DLBCL. Conventional therapy, as used for typical DLBCL, is of limited efficacy. Recognition of this new entity and the characteristic lack of CD20 expression are paramount. Novel front-line intensive chemotherapy regimens should be evaluated in this group of patients.

The retrospective study of 34 patients with Hodgkin lymphoma-associated hemophagocytic syndrome led us to define this association as a specific disorder. Its characteristics are male predominance (male-to-female sex ratio, 3.3:1), immunodeficiency-like histological features (lymphocyte depletion, 45% of cases; mixed cellularity Hodgkin lymphoma subtype, 40%), and strong association with Epstein-Barr virus (94%).

Over the last 10 years, 240 cases of hyperplasic lymphadenitis have been systematically tested in our institution for the presence of the human immunodeficiency virus (HIV). This series comprised patients between 15 and 90 years (median of age: 38.51) without a past history of HIV infection. The technical approach consisted in an immunohistochemical procedure with a monoclonal antibody against the p24-gag protein of HIV. Among the 240 cases, 105 had a true follicular hyperplasia. Overall, this survey found that 4 cases (3 males and 1 female) were positive for p24-gag without previous knowledge of HIV infection (4/240=1.66%). HIV infection was further confirmed by serologic and molecular investigations in all cases. These results were seen exclusively in those cases with prominent follicular hyperplasia (4/105=3.80%). Staining with the anti-p24 antibody was intense and restricted to the follicular dendritic cell networks. In one case, beside hyperplasic germinal centers, one could see a regressed onion bulblike structure. One important conclusion can be drawn from this study. A systematic research of HIV proteins should be performed in all lymph node biopsies with marked follicular hyperplasia, in a context of polyadenopathy, fever, and general status alteration. Besides giving an accurate diagnosis, this approach may be helpful in cases of recent infection in which anti-p24 antibodies are not yet detectable in the serum.

Background: Mycosis fungoides (MF) is the most common skin lymphoid neoplasm. In initial stages, differential diagnosis of MF from other benign dermal lymphoid infiltrates (BDLI) may be impossible on morphological basis alone. In previous studies, only deletion of CD7 in MF proved to be of diagnostic help, but not the ratio between immunoexpression of CD4 and CD8. Methods: 30 cases of MF and 11 cases of BDLI were analysed, in order to compare morphometric parameters, which could be of diagnostic aid. As CD7 is frequently deleted in MF, immunohistochemical detection of T-cells was made using an antibody to CD3. Images of 100 CD3-positive cells per case in both groups were captured and analysed using a simple computer program for nuclear perimeter, area, diameter and nuclear contour index. Results: All parameters showed statistically significant higher values for MF. Area was the variable with the strongest discriminating power between the two groups of patients. Thus even if morphological evaluation is not accurate to distinguish benign versus malignant dermal lymphoid infiltrates, due to the variability of size and shape of these cells, a more sensitive method promptly shows this difference. Conclusion: Results suggest that morphometry of CD3-positive lymphoid cells may add valuable information in the differential diagnosis of MF and benign dermatoses.

Summary Objective Human herpesvirus 8 (HHV-8) and Epstein–Barr virus (EBV) are human pathogens associated to a number of neoplasms, including tumors of the Waldeyer's ring. Both viruses have been previously detected by in situ methods in tonsils and adenoids from children. HHV-8 was found in 6.8% of the cases and EBV in about one third of the cases. As they belong to the same γ-herpesvirus subfamily and share some biological characteristics, it is of medical interest to further explore their possible relationship in the Waldeyer's ring, an issue not yet addressed in the specialized literature. The purpose of the present study is to compare the presence of EBV by in situ hybridization (ISH) in tonsils and adenoids from children up to 14 years of age in cases previously shown to be positive and negative for HHV-8. Methods Paraffin wax-embedded sections consisting of 38 tonsils and two adenoids from 40 patients were analyzed. HHV-8 was detected by ISH, using the T1-1 probe for the viral mRNA. EBV was also detected by ISH, using the EBER probe. Both probes and the detection systems were provided by Novocastra. Results HHV-8 was detected in 19 tonsils and one adenoid. The other 19 tonsils and one adenoid taken from the HHV-8-negative group were selected by pairing age and gender of patients with the HHV-8-positive group. In both groups EBV was detected in 13 cases and was negative in other 7. Conclusion Although both viruses are related in many aspects, some biological and epidemiological features differ. This is reflected in the present results, as EBV is similarly detected in the groups negative and positive for HHV-8, favoring different mechanisms of spread.

A retrospective investigation of the JAK2 V617F mutation was carried out in DNA samples from 131 bone marrow (BM) core biopsy specimens corresponding to patients with polycythemia vera (PV) (n = 31), essential thrombocythemia (ET) (n = 31), chronic idiopathic myelofibrosis (CIM) (n = 18), as well as patients with normal BM and secondary reactive hyperplasia. We used the TaqMan polymerase chain reaction single nucleotide polymorphism genotyping assay to detect the specific JAK2 mutation. This technique allowed us to detect the JAK2 V617F mutation in a population containing at least 5% of homozygous mutants. Overall, the incidence of the JAK2 V617F mutation was 87% in PV, 67% in ET, and 66% in CIM. This approach proved to be reliable and more sensitive in detecting the mutation compared with that of initial studies on different materials but similar to that of recent work with various polymerase chain reaction-based techniques. Two essential findings arose from our study. First, this technique could be carried out with DNA samples, even partially degraded, from routinely processed BM core biopsy specimens. Second, after correlation with morphological features, it turned out that the characteristics of the megakaryocytes were more specific than the mutational status of JAK2 in characterizing ET and CIM. Concerning PV, as expected, the incidence of the JAK2 mutation was higher, but the morphological criteria were misleading in some cases, strongly suggesting that the combination of both histologic and molecular data would enable the characterization of virtually all cases.

Anaplastic large-cell lymphoma (ALCL) is a distinct biological and cytogenetic entity with a broad spectrum of morphological features (common type, small-cell variant and lymphohistiocytic variant). Few cell lines of ALCL are available and they all originate from primary tumors demonstrating the common type morphology (ie large-sized lymphoma cells). We established a new ALCL cell line (COST) from the peripheral blood of a patient with a small-cell variant of ALCL, at diagnosis. Cells growing in vitro and in SCID mice consisted of two populations, that is, small- and large-sized cells as seen in the patient's tumor. Both large and small malignant cells were positive for CD43/MT1 T-cell associated antigen, perforin, granzyme B and TIA-1, but negative for CD2, CD3, CD5, CD7, CD4 and CD8 antigens. Standard cytogenetic studies as well as multiplex FISH confirmed the presence of the canonical t(2;5)(p23;q35) translocation, but also revealed additional numerical and structural abnormalities. The COST cell line is the first ALCL small-cell variant cell line, and thus provides a potentially useful tool for further functional and molecular studies that should improve our understanding of the small-cell variant of ALCL, which is more frequently complicated by a leukemic phase.

Biopsies from 319 haematopoietic neoplasms were immunostained for intracellular leucocyte-specific protein 1 (LSP1) to assess its distribution and to compare its diagnostic value with that of CD45 (leucocyte common antigen: LCA). Most small B-cell neoplasms expressed LSP1, but one third of diffuse large B-cell lymphomas (DLBCL) were LSP1 negative. Among the cases with DLBCL (76 samples) tested for both LSP1 and CD45, one fifth expressed only CD45, but five samples were LSP1-positive and negative for CD45. The latter pattern was also seen in four of nine myelomas. Five out of 14 T-lymphoblastic lymphomas co-expressed LSP1 and CD45, and three cases were LSP1 negative and CD45-positive. Most peripheral T-cell lymphomas co-expressed LSP1 and CD45. All anaplastic lymphoma kinase (ALK)-negative lymphomas of anaplastic large cell morphology (T and null phenotype) expressed LSP1 although the percentage of LSP1-positive tumour cells was variable, however, only seven out of 30 cases with ALK-positive lymphoma were LSP1 positive. LSP1 was expressed on Reed-Sternberg cells in 60 out of 66 cases with classic Hodgkin's disease but neoplastic cells were usually negative in lymphocyte predominant Hodgkin's. This study confirms the wide expression of LSP1 within haematopoietic neoplasms and its diagnostic value for a minority of lymphoid tumours that have lost CD45 expression. Furthermore, the strong expression of LSP1 in classic Hodgkin's disease, contrasting with its heterogeneous expression in ALK-negative anaplastic lymphomas, may help to distinguish the latter lymphomas from patients with tumour cell-rich Hodgkin's disease.

Epstein-Barr virus (EBV) has been closely associated with undifferentiated nasopharyngeal carcinoma (NPC) and T/NK nasal non Hodgkin lymphoma. Nevertheless, the presence of EBV in non neoplastic lymphoid tissue of the nasopharynx has been rarely investigated. In a previous study by our group, using in situ hybridization to detect EBV in adenoids of children (2-13 years old) resected because of nasal obstruction due to hypertrophy, we found EBV genome in 72% of the cases. It was now intended to study the frequency of EBV expression in adenoids from children that underwent surgical removal, belonging to a lower age group (1-2 years old). It was also intended to establish which lymphoid subsets are involved in this infection. Adenoidal paraffin sections from 21 patients aged 1-2 years old (mean 1.6 years), 15 males and six females were submitted to double labeling: in situ hybridization with EBER 1/2 probes to detect EBV and immunohistochemistry to determine the lymphocyte typing of EBV-positive cells (CD20 for B-lymphocytes, CD3 for T-lymphocytes and CD56 and CD57 for NK-cells). Among 21 patients, seven showed positive lymphoid cells for EBV (33%). In almost all cases, EBV-positive cells were also CD20-positive. Some EBV-positive cells showed no labeling with any of the lymphoid markers, but in no instance they were positive for CD3, CD56 or CD57. This study confirms the preferential infection of B-lymphocytes by EBV, which in some instances can down regulate the expression of CD20.

We report a retrospective immunohistochemical study on bone marrow biopsies of 43 patients with different types of lymphomas showing unusual intrasinusoidal infiltration. Most of these patients presented with splenomegaly (74.4%) and peripheral lymphocytosis (83%). In 20/43 patients, lymphoid infiltrates were not detectable on haematoxylin-eosin sections. After immunohistochemistry on bone marrow biopsies and blood and bone marrow smear examinations, the following diagnoses were made: splenic marginal zone lymphoma with villous lymphocytes (SLVL) in 24 patients, large granular lymphocyte (LGL) leukaemia in 14 patients, hepatosplenic T-cell lymphoma in two patients, anaplastic large cell lymphoma in two patients and intravascular large B-cell lymphoma in one patient. In the presence of intrasinusoidal infiltrates of small lymphocytes, a B-cell phenotype (CD20+, CD76/DBA44+/-) was associated with splenic marginal zone lymphoma whereas intrasinusoidal CD3/CD45RA-positive T-cell infiltrates were strongly suggestive of LGL leukaemia. Intrasinusoidal bone marrow infiltration appears to be a common feature of distinct lymphoma subtypes. Immunohistochemical analysis is essential to detect intrasinusoidal medullary infiltrates (which may be minimal) and should be systematically performed in patients with splenomegaly and peripheral lymphocytosis.

In the thymus, the relationship between lymphofollicular hyperplasia and mucosa-associated lymphoid tissue (MALT)-type lymphoma is uncertain. We analyzed 14 cases with a diagnosis of thymic follicular hyperplasia in patients with connective tissue disease (n = 2), myasthenia gravis (n = 11), or both (n = 1). In 11 cases, well-defined reactive lymphoid follicles were surrounded by a continuous layer of medullary epithelial cells. A polyclonal rearrangement of the immunoglobulin heavy chain gene (IgH) was observed. In 3 cases, ill-defined lymphoid follicles with sheets of centrocytic-like B cells disrupting the medullary cytokeratin epithelial network were observed on certain sections. These cells expressed the phenotypic features of memory B cells with CD20, CD79a, and bcl-2 positivity and CD5, CD10, CD23, and bcl-6 negativity, and a monoclonal rearrangement of the IgH gene was detected. Appropriate sampling, cytokeratin staining, and molecular analyses may help to identify early MALT-type lymphoma developing in the setting of thymic lymphofollicular hyperplasia.

Post-transplantation lymphoproliferative disorders (PTLDs) and Kaposi's sarcoma (KS) are immunosuppression-related tumours developing in solid organ transplant patients. Although the Epstein-Barr virus (EBV) is detected in the majority of the PTLDs during the first year after transplantation, the proportion of EBV-negative PTLDs has increased in recent years. We report a case of a 17-year-old man who developed severe immune haemolytic anaemia, KS and human herpesvirus 8 (HHV-8)-associated, polymorphic-type PTLD 9 months after allogeneic renal transplantation from his HHV-8-seropositive father. It is suggested that: (i) HHV-8 may be associated with EBV-negative, polymorphous-type PTLD occurring less than 1 year after transplantation, and (ii) PTLD may be listed among other tumours, including KS, Castleman's disease and primary effusion lymphoma (PEL), that are related to HHV-8 infection.

Human herpesvirus-8 (HHV-8) also called Kaposi's sarcoma-associated herpesvirus infects spindle cells in Kaposi's sarcoma (KS) and lymphoid cells in multicentric Castleman's disease (MCD). In KS cells, HHV-8 is mainly latent with the expression of latent nuclear antigen-1 (LNA-1), whereas in MCD both lytic and latent antigens are produced by lymphoid cells. We show by immunohistochemical labeling that in KS viral interleukin-6 (vIL-6) is expressed in rare spindle cells, whereas in MCD, vIL-6 is detectable in lymphoid cells around lymphoid follicles but also within the follicular dendritic reticulum cell network. The staining of apoptotic bodies with anti IL-6 antibody suggests the achievement of a complete lytic cycle in a subset of lymphoid cells. Interestingly, in MCD, some areas contained vascular spindle cells latently infected by HHV-8 on the basis of LNA-1 expression. This finding might imply that in MCD, both vascular and lymphoid cells proliferate in response to the viral infection. Double immunostaining with anti LNA-1 and anti vIL-6 in MCD and KS identifies 2 subsets of HHV-8 infected (vascular and lymphoid) cells, some with exclusive expression of LNA-1 and some with coexpression of vIL-6 and LNA-1. This suggests that in vivo the regulation of the expression vIL-6 and LNA-1 protein varies with the cell type. In addition, the detection of infected endothelial cells in MCD may indicate that these cells belong to the reservoir for HHV-8. H UM P ATHOL 32:95-100. Copyright © 2001 by W.B. Saunders Company

A new recurrent translocation t(11;14)(q24;q32) involving IGH@ and miR-125b-1 in B-cell progenitor acute lymphoblastic leukemia

This report summarizes a cumulative 4-year experience in polymerase chain reaction (PCR) analysis of immunoglobin heavy chain (IgH) and TcR-gamma chain gene rearrangements in 525 cases of lymphoproliferative disorders. Because the sensitivity of the PCR methodology was found to be tissue dependent, in the study of the presence of clonal cell population in tissues containing a small number of polyclonal lymphocytes, such as skin and gastrointestinal biopsy specimens, we used the multiple-PCR run approach. In this latter methodology, we repeat the PCR reaction from the same sample at least three times to confirm the reproducibility of the results. In the study of 273 cases of B- or T-cell lymphomas with characteristic immunomorphological and clinical features, a clonal IgH or TcR-gamma chain gene rearrangement was detected in approximately 80% of cases. A clonal rearrangement involving both IgH and TcR-gamma chain genes was found in 10% of cases of both B-cell and T-cell lymphomas. The study of 167 cases of nonneoplastic lymphoid tissue samples showed the presence of clonally rearranged cell populations for IgH or TcR-gamma genes in 3 and 9% of cases, respectively. We also applied PCR for the study of 85 cases of lymphoproliferations with no definite diagnosis (i.e., benign versus malignant) after immunomorphological analysis. In 65 cases (76%), the correlation of immunomorphological features with the presence (48 cases) or the absence (17 cases) of clonal lymphoid cell populations led to a definite diagnosis. In almost all these cases, the final diagnosis was found to be in agreement with the clinical course. In the 20 remaining cases (24%), no definite diagnosis could be made. We also assessed the value of PCR in detecting bcl-2/J(H) gene rearrangement as an additional clonal marker in the diagnosis of follicular lymphoma. Bcl-2/J(H) rearrangement and/or IgH gene rearrangement was found in approximately 85% (71/85) of follicular lymphoma cases studied.

Immunoblotting and immunohistochemical analysis were performed to identify the cells expressing and secreting annexin 1 during experimental rat colitis induced by trinitrobenzene sulfonic acid. Annexin 1 expression was increased during the inflammation. Likewise, annexin 1 secretion was induced in inflamed colons at one, three, six, and nine days after trinitrobenzene sulfonic acid treatment but was not detected in colons from controls and rats at 12 days. Immunohistochemistry showed that the rise in annexin 1 expression resulted from the infiltration of two types of leukocytes highly positive for annexin 1: neutrophils (the most abundant) and macrophages. At day 1 after treatment, neutrophils of the inflammatory site, in mucosa and submucosa, are the only cells expressing annexin 1. Immunoblotting showed that they secreted annexin 1 whereas neutrophils from blood or tunica muscularis did not. This indicates that, during this colitis, annexin 1 can be secreted by neutrophils located in the inflammatory site.

Follicular lymphoma (FL) is a neoplasm originating from germinal centre cells, corresponding to 25-40% of non-Hodgkin's lymphomas. Transformation into diffuse large B cell lymphoma (DLBCL) occurs in about one-third of cases. CD5 is expressed in B-chronic lymphoid leukaemia/small lymphocytic lymphoma and mantle cell lymphoma, but can rarely be expressed in conjunction with CD10 in well-documented cases of FL. In this report one case of grade 1 FL is described, which transformed into a DLBCL 6 months after initial diagnosis, with both tumours expressing CD5. In both specimens, neoplastic cells were strongly positive for CD20, CD79a, bcl-2, bcl-6 and CD5 in virtually all cells. CD10 was strongly positive in initial specimens and weakly positive in the DLBCL. Investigation using the PCR confirmed the derivation of the DLBCL from the FL as they presented the same immunoglobulin heavy chain gene rearrangement and the same BCL2-J(H) break point.

Epstein-Barr virus (EBV) has been frequently documented in the putative neoplastic Hodgkin-Reed-Sternberg (HRS) cells, in lymph nodes from patients with Hodgkin's disease (HD). This association varies in different geographic areas and between industrialized and developing countries, as does the epidemiological pattern of the disease. In the present study of 106 cases of HD from the Soroka Medical Center in Beer-Sheva, which serves as the only hospital for most of the southern part of Israel, we found an association with EBV expression in only 30% of the patients; 45% of mixed cellularity (MC) cases compared with 21% of nodular sclerosis (NS) cases were positive for EBV. The number of patients in the 0-14-year-old age group was limited; however, 8 of these 11 children were EBV positive. This low association rate of HD with the presence of EBV sequences is probably related to the small number of children in our series. A low proportion of EBV-associated disease in older adults may be contributory. Other factors may be involved. Int. J. Cancer, 71:138–141, 1997. © 1997 Wiley-Liss, Inc.

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.

Fifty-one cases of acquired immunodeficiency syndrome (AIDS)—related primary brain lymphomas (AR-PBL) were investigated for clinical characteristics; human immunodeficiency virus (HIV)—associated disorders; histopathologic features; immunophenotype; Epstein-Barr virus (EBV) infection; and, when frozen tissue was available, oncogene rearrangements. AR-PBL occurred late in the course of AIDS and were usually associated with other systemic or cerebral disorders and with a low level of CD4 lymphocytes. All cases were high grade lymphomas according to the Working Formulation or updated Kiel classification, and often displayed a multifocal pattern. Thirty cases were classified as immunoblastic with plasmacytic differentiation, 18 cases were large cell lymphomas with an immunoblastic component or centroblastic polymorphic lymphomas, and 2 were small noncleaved non-Burkitt lymphomas (Working Formulation). This latter category is classified as Burkitt's-like lymphoma in the REAL nomenclature. One case could not be classified because of necrosis. AR-PBL showed a high level expression of activation and adhesion molecules. The presence of EBV was detected in most cases, and, when PCR was used, this was a constant finding. bcl-2 oncoprotein and latent membrane protein-1 (LMP-1) were strongly expressed. None of the tested cases expressed p53, or were rearranged for bcl-2 or c- myc oncogenes. This study confirms the immunophenotypic specificity of AR-PBL, which may reflect the special immune status of the brain.

We used the recently described sensitive and rapid detection assay called telomeric repeat amplification protocol (TRAP) to detect telomerase activity in lymphoblastoid (n = 5) and lymphoma cell lines (n = 7), hyperplastic lymph nodes (n = 6) and tonsils (n = 5), and tissues involved by non-Hodgkin's lymphoma (NHL) (n = 43) and Hodgkin's disease (HD) (n = 14). Clearly evident telomerase activity was found in all lymphoblastoid and lymphoma cell lines, and in 34 of 43 cases (80%) of NHL. These results were expected because of the proliferative and immortal nature of the cell lines and most malignant cells. However, positive results were obtained with the TRAP assay in all hyperplastic lymph nodes and tonsils, which raises the issue of derepression of telomerase activity during an immune response. Telomerase activity alone therefore does not distinguish malignant lymphoid proliferations from reactive states. Telomerase activity was detected in only 1 of 14 cases (7%) of lymphoid tissues involved by HD. Eight of the 13 negative cases were considered to be interpretable because of the lack (3 of 13 cases) or low level (5 of 13 cases) of telomerase inhibition. The five remaining cases could not be evaluated because of their telomerase inhibitor content. The findings imply either transient or very low levels of telomerase activity in HD or that HD for the greater part is a telomerase-independent neoplasm. Microdissection studies are needed to identify subsets of cells carrying telomerase activity in both reactive and neoplastic lymphoid tissues.