Search

The International Network of Cancer Treatment and Research (INCTR) recently organized a workshop on non-Hodgkin lymphomas (NHLs) in selected developing countries with the purpose of examining existing information relating to the pathology and management of these neoplasms, and identifying potential areas for research. This report provides a summary of the information presented and is focused primarily on the pathology of NHLs in children and adults. In most countries, the WHO classification of lymphomas was used and most participating centers included immunohistochemistry using a wide array of lymphoid antibodies as part of routine diagnosis. Some of the series had been reviewed by an external panel of experts. B-cell lymphomas accounted for 82-88% of all NHLs. The proportions of chronic lymphatic leukemia (4-6%), mantle cell lymphoma (MCL, 3-5%), and plasmacytoma (2-4%) were similar in the series presented. However, there was a significant variation in the proportion of follicular lymphoma (FL), which accounted for 15% and 11% in India and Kuwait, but less than 5% in Pakistan and Egypt. All of these frequencies are significantly lower than those reported in Western series. Diffuse large B-cell lymphoma accounted for about 35% of cases in India but for more 50% in other countries, but this difference was not accounted for by an increased incidence in a single lymphoma subtype in India, but rather an apparent paucity of several subtypes (such as mantle cell and marginal zone lymphomas (MZL)) in other series. There were relatively high frequencies of Burkitt lymphoma in Egypt (7%) and precursor T-cell lymphoblastic lymphoma in India (6-7%). Peripheral T-cell lymphomas (PTCLs) (not otherwise specified and angioimmunoblastic subtypes) accounted for 3-5% of NHLs, and extranodal lymphoma of T/NK cell type was rare (

Adhesive interactions between the molecules on cancer cells and the target organ are one of the key determinants of the organ specific metastasis. In this communication we show that b1,6 branched N-oligosaccharides which are expressed in a metastasis-dependent manner on B16-melanoma metastatic cell lines, participate in the adhesion process. We demonstrate that high metastatic cells show significantly increased translocation of one of the major carriers of these oligosaccharides, lysosome associated membrane protein (LAMP1), to the cell surface. LAMP1 on high metastatic cells, carry very high levels of these oligosaccharides, which are further substituted with poly N-acetyl lactosamine (polylacNAc), resulting in the expression of high density of very high affinity ligands for galectin-3 on the cell surface. We show that galectin-3 is expressed in highest amount in the lungs as compared to other representative organs. Blocking galectin-3 by pre-incubating the frozen sections of the lungs with 100 mM lactose, substantially inhibited the adhesion of high metastatic cells, while pre-incubation with sucrose had no effect. Finally, by in situ labeling and immunoprecipitation experiment, we demonstrated that the lung vascular endothelial cells express galectin-3 constitutively on their surface. Galectin-3 on the organ endothelium could thus serve as the first anchor for the circulating cancer cells, expressing high density of very high affinity ligands on their surface, and facilitate organ specific metastasis.

The objectives of this study were to document the oestrogen and progesterone receptor (ER & PR) status of breast cancer in the Indian population (as done by immunohistochemistry on paraffin blocks), and correlate the steroid receptor status of breast cancer with all relevant patient and tumour characteristics. Our current data have been compared with previously published data from other centres. In contrast to the higher rates reported in the Western literature, only 32.6% of our tumours were ER positive and 46.1% were PR positive. Tumours were separated into four categories: ER+PR+ (25%), ER+PR- (7.4%), ER-PR+ (21.1%) and ER-PR- (46.5%). ER and PR immunoreactivity increased with advancing age, and correlated with the presence of elastosis. Infiltrating lobular carcinoma, mucinous carcinoma, and mixed tumours were more frequently ER & PR positive. High-grade infiltrating duct carcinomas, pure comedo ductal carcinoma in situ, and medullary carcinoma were predominantly ER & PR negative. The presence of necrosis and lymphovascular invasion showed an inverse relationship with ER and PR reactivity.

Primary non-Hodgkin’s lymphoma of the bone is an unusual entity. Twenty-five patients with diffuse large cell lymphoma of the bone were registered at the Tata Memorial Hospital (TMH) from August, 1991, to May, 2002. Pain at the local site and soft tissue swelling were the commonest symptoms. Involvement of the bones in the lower half of the body was more frequent than the bones in the upper half. Osteolytic lesions and an associated soft tissue mass were the common radiological findings. Nineteen patients received CHOP chemotherapy and five received COP chemotherapy. Twenty-three patients received involved field radiotherapy. The overall response to therapy was 96%. On follow-up, two patients had a nodal relapse. One patient died of progressive disease, and one patient died of cryptococcal meningitis. There were no deaths due to treatment-related toxicity. The mean progression free survival was 9.39 yr and the overall survival was 11.66 yr. The median overall survival has not been reached. At last follow-up, 21 patients were being following up at TMH and are free of disease. Conclusion: Primary bone lymphoma is a malignancy that is highly curable with a combination of chemotherapy and radiotherapy.

Cytokeratins (CK) are the epithelia specific intermediate filament proteins. We have shown consistent non-expression of CK-5 protein in human oral pre-cancer and cancer, in earlier studies. To investigate whether non-expression of CK-5 protein is the result of transcriptional or translational block and to evaluate the possibility if CK-5 non-expression can be used as a marker for early diagnosis of tobacco related oral cancer, RT-PCR using CK-5 specific primers was conducted. Out of 36 precancerous lesions and 29 squamous cell carcinomas (SCC) of buccal mucosa (BM) samples studied, 11 and 13 samples respectively of precancer and SCC did not show CK-5 product in RT-PCR. Down regulation of CK-5 mRNA expression was also observed in some samples. Thus, in conclusion, our results have shown that CK-5 non-expression is the result of transcriptional block. We proposed CK-5 non-expression as a potential marker for the early diagnosis of tobacco related oral cancer.

Meningiomas represent about one sixth of all primary neoplasms of the central nervous system. They rarely metastasize outside the intracranial compartment. There are no clear criteria to identify the subset of aggressive tumors which recur locally or metastasize. Histological tumor grade is the most important predictor of recurrence or metastases. We report an unusual case of recurrent metastasis in an extrapleural location and review the literature. Our patient developed recurrent thoracic metastases from an intracranial benign meningioma after a disease-free interval of 8 years. She was successfully managed by wide excision of the metastasis and is currently asymptomatic.

Metastases from unknown primary tumours (MUP) are not an uncommon clinical problem. A hypothesis that discusses the plausible role of neoangiogenesis, as a central theme in the development of MUPs, is presented. Invasive cancers, which cannot or do not switch to the angiogenic phenotype, remain subclinical. In situations where a non-angiogenic tumour attempts to grow beyond the volume that can be supported by the vasculature, marked apoptosis and cell turnover result. Tumours with a high cell turnover are biologically advanced. Thus, such subclinical tumours acquire a metastatic phenotype. In the viscera, metastases may remain dormant for varying periods until subclones with an angiogenic phenotype arise due to tumour evolution or they burn out. Visceral MUPs, due to their longer evolution, are biologically advanced and have dismal prognosis. Contrary to this, tumour cells reaching lymph nodes (LN) grow without acquiring any phenotypic or genotypic change, as angiogenesis is redundant for growth within the metastatic LNs.

In patients (pts) with non-Hodgkin's lymphoma (NHL) under 25 years, treatment with MCP-842 protocol, a short duration intense protocol, yields worse survival in pts with lymphoblastic lymphoma (LL) compared to other high grade lymphomas. In order to identify both favourable and unfavourable subgroups in pts with T-cell LL (T-LL) with respect to relapse free survival following treatment with MCP-842 protocol, we analysed the expression of p53 and bcl-2 proteins in 22 pts with T-LL treated at the Tata Memorial Hospital, Mumbai by immunohistochemistry. p53 protein overexpression was noted in 59% cases and bcl-2 overexpression was noted in 29.4% cases. p53 expression correlated with a higher rate of relapse (p = 0.03; RR 7.9). The 5-year relapse free survival (RFS) was better in p53 negative patients compared to positive patients (70 vs 38%) (log-rank sigma = 0.04). In conclusion, in this study, overexpression of p53 protein was common in patients with T-LL. T-LL pts negative for p53 are likely to benefit from the short intense protocol--MCL-842. Bcl-2 protein overexpression was not a prognostic factor in these patients.

The diagnosis of myeloma requires the presence of 10% or more clonal plasma cells on bone marrow examination or of a biopsy-proven plasmacytoma along with evidence of end-organ damage that can be attributed to the underlying plasma cell disorder [1]. The above-mentioned case had >90% neoplastic plasma cells in the bone marrow and a significant anaemia as a result of the bone marrow infiltration. Cytological features of myeloma cells can be highly variable. They include tumors with mature and benign appearing plasma cells; small lymphoid-like cells; atypical but differentiated plasmacytic cells; blastic cells with high nucleus: cytoplasmic ratio and fine chromatin; large cells with immunoblastic features and highly pleomorphic forms. The current case showed immunoblastic features and pleomorphism. Differential diagnosis varies as per the cytological features and includes benign/reactive plasma cell infiltrates, small cell lymphomas, large cell lymphomas, metastatic carcinomas, and other metastatic tumors. Documentation of the expression of immunoglobulin either at protein level or at RNA level confirms the B-cell/plasma cell lineage of these tumors. Expression of CD138, CD79a, and CD56 is helpful in the diagnosis of myeloma, though carcinomas can express CD138 and those with neuroendocrine features can express CD56. Expression of cyclin D1 is noted in 40% of cases and is a very helpful feature in the diagnosis and follow-up of myeloma on bone marrow trephine biopsies [2]. Cases with uniform and strong expression of cyclin D1 are associated CCND1 translocation.

Low-grade B-cell mucosa associated lymphoid tissue (MALT) lymphoma can rarely present primarily in extranodal sites other than stomach, which is the most common site for it. Until now only four cases of primary prostatic MALTomas have been described in the literature and we report the fifth. We describe a case of primary prostatic MALToma in a 67-year-old man and the diagnosis was made on the trans-urethral resection specimen of the prostate. As the disease was limited to prostate (stage IEA), the patient was treated with external beam radiation therapy to a total dose of 4400cGy in 22 fractions. The patient achieved completed remission and has remained free of disease in the following 36 months.

Background Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are the classical types of Philadelphia negative MPN. The JAK2 V617F missense mutation is found in 95% of patients with PV and in 50-60% of patients with PMF or ET. Molecular screening for JAK2 V617F mutation in suspected MPN patients by pyrosequencing has become routine method in diagnostic services. With the introduction of new methods of screening, a growing number of mutations have been described in a range of genes that code for receptor structure proteins (MPL, JAK2 exon 12), intracellular signalling proteins (CBL, SH2B3, NF1, SOCS1/3), proteins involved in leukemic progression (TP53, NRAS/KRAS, NF1, IDH1/2), or epigenetic modifications (EZH2, ASXL1, TET2). Molecular genetic screening for these targets in JAK2 V617F negative cases can be laborious, time-consuming and expensive, but the advent of benchtop-class high-throughput sequencers and custom target enrichment platforms gives the opportunity to test all relevant mutations in multiple genes in a single assay. Aims: To evaluate the Ion Torrent PGM high-throughput sequencer and custom AmpliSeq capture platform for the screening of most relevant MPN associated mutations in JAK2 V617F negative cases and validate candidate mutations using pyrosequencing. Methods 32 DNA samples (30 JAK2 V617F negative by pyrosequencing, 1 positive, 1 not tested) from patients with MPN disorders (thrombocytosis, neutrophilia or polycythemia) were screened (with appropriate ethical consent) for mutations using the Ion Torrent PGM sequencing platform. AmpliSeq multiplex primers were used for the target capture and the panel was designed via the online portal (v1.2), targeting 23 regions of 13 genes associated with MPN. The workflow involved library preparation and multiplex sample pooling following qPCR quantification (16 samples per run), emulsion PCR template preparation, Ion 318 chip loading and sequencing. Alignment and variant calling was via the Torrent Server 3.6.2 plugins, using custom coverage and hot-spot files to define the regions of interest. Variant calls were visualised using IGV and UCSC, and functionally annotated using CONDEL, Mutation Assessor and PROVEAN. Results 31 of the 32 samples were successfully sequenced, with a mean depth of 1049 reads and the FASTQC plugin indicated good quality sequencing metrics. All JAK2 V617F negative samples were called V617F negative by the PGM. The V671F positive control sample was called at a frequency of 18.1% (19% by pyrosequencing). The untested sample was called positive (10%) for JAK2 V617F and this was validated using pyrosequencing (11%). No MPL or JAK2 exon 12 mutations were detected. Novel SNPs were seen in CBL (Chr11:119149062; G>A; intron 8-9), JAK2 (Chr9:5072649; T>C; intron 13-14), SOCS3 (Chr17:76354148; C>G; 3’UTR) and SH2B3 [(Chr12: 111856076; C>T; p.R43C) and (Chr12: 111856105; T>G; p.H52Q); both in exon 2]. Of these, the SH2B3 variants were flagged as deleterious (H52Q) and highly deleterious (R43C: SIFT=0; PPH2=0.998). Five samples (4 het, 1 hom) had a SNP in IDH1 associated with poor outcome in AML (rs11554137; MAF=0.05) and a further 6 samples had variants flagged as deleterious by at least one of the annotation platforms: one in SH2B3 [p.186I (rs183913232 )] and five in TET2 [2 samples with p.G355D (rs61744960); 2 with p.L34F (rs111948941) and one p.Y867H (COSM327337)]. Additionally, one sample with a TET2 L34F also had a COSMIC annotated JAK2splice site variant (c.1864+14C>T; COSM88203). Summary This study shows the ability of the Ion Torrent PGM and AmpliSeq platforms to analyse qualitatively and quantitatively multiple samples for multiple genes with good accuracy and specificity (no false positives for V617F), making this qualitative and quantitative method more time and cost-effective than traditional sequencing techniques. Additionally, several potentially disease-associated variants were detected, which would have been missed by conventional MPN screening strategies and we are proposing to roll this approach out as a diagnostic screening in our MPN clinic in the very near future. Disclosures: Milojkovic: BMS: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria. Apperley:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Pfizer, Ariad: Honoraria (not direct from company), Honoraria (not direct from company) Other.

Background Hepatosplenic T-cell lymphoma (HSTL) is a rare form of lymphoma, comprising less than 1% of the cases. However, HSTL extracts a highly disproportionate toll on patients with a median age of diagnosis of 35 years and an expected median survival of less than two years. The vast majority of HSTL patients eventually succumb to their disease. The genetic basis of the disease is largely unknown. Although abnormalities of chromosome 7, including isochromosome 7q occur commonly in the disease, the role of specific genes and genetic mutations to the disease remains essentially unknown. Methods In this study, we sought to define the genetic features of HSTL through the whole genome sequencing and exome sequencing of 32 HSTL tumors and germline DNA (where available) from the same patients. Exome enrichment of DNA was carried out using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes as well as ∼700 human microRNAs. Both whole genome and exome sequencing were performed using the Illumina platform. Results We identified 28 candidate cancer genes that were recurrently mutated in HSTL. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, KRAS) and chromatin modification (e.g. TET1, SETD2 and MLL3), accounting for 16% and 23% of the total genetic events, respectively. Nearly all of these genes have been implicated in HSTL for the first time and provide new insights into the pathogenesis of the disease and potential targets for therapy. Whole genome sequencing confirmed isochromosome 7q as the most common recurrent chromosomal abnormality in HSTL and additional structural genetic alterations in chromosome 7. Conclusion Our study provides the most comprehensive genetic portrait of HSTL to date, and is a significant step in defining the genetic causes of this disease. Disclosures: No relevant conflicts of interest to declare.

The pathology of immune thrombocytopenia (ITP) is not fully understood. Although antiplatelet antibodies (APAs) are thought to be causative, these are not detected in up to 30% of patients. In addition, T cell directed therapy is frequently successful. Patients with ITP display cytotoxic activity towards platelets, and abnormalities in T cell repertoire. However, there is incomplete understanding of the interactions between T cells and megakaryocytes within the bone marrow. We hypothesized that if T cell or NK cell cytotoxicity against megakaryocytes contributes to thrombocytopenia, then T and/or NK cells should be in direct contact with the megakaryocyte in the bone marrow. We therefore further explored the interactions between T cells and megakaryocytes in bone marrow trephine biopsies - taken at or near to diagnosis - in patients with ITP. To further explore apoptotic pathways, serum TRAIL levels were also assessed at different time points in disease. Methods Bone marrow trephine samples were taken as clinically indicated at or around the time of diagnosis and processed as per department protocol. Samples from patients with known ITP (n=9) were further processed and stained with CD3, CD8, CD4 and CD56. Immunohistochemistry was performed using Leica Bond-max automated immunohistochemistry system. The density of MK cells, CD3, CD8 and CD4 was recorded and the numbers of MK in contact with T or NK cells were recorded on multiple sites on the trephine. Control bone marrows from staging marrows for lymphoma were used for comparison (n=6). For further analysis of the non-antibody component of ITP, serum TRAIL levels were measured in 63 samples from 43 patients at different stages of disease. Patients had all consented for research. Results Results of the bone marrow examination are shown in the table. Bone marrow samples from patients with ITP had higher density of megakaryocytes than control bone marrows (median 41/mm3 compared to 20/mm3 - p=0.03). Megakaryocytes appeared normal in morphology. CD3 density was also higher in patients with ITP, although this was not significant (p=0.17). There was however increased interaction between CD3 and MK cells in patients with ITP compared to controls (median 50% of MK cells in contact with CD3 cells in ITP compared to 32% in controls - p=0.02) – figure 1a. There was no correlation between MK cell density, T cell density and MK-T cell interaction, making this finding unlikely to have occurred by chance. In ITP patient samples further analysis of T cell type was undertaken and shown in the table showing interactions occur between both CD4s and CD8s and the MK cells (figure 1b). Four patients had reverse CD4:8 ratios in the bone marrow. Interaction between MK and CD56+ cells were less frequent (0 to 8%). There was no association with response to treatment and MK or T cell findings, although patients who responded to rituximab appeared to have lower MK density. Patients who responded to thrombopoietic (TPO) agonists - and by virtue of their use, had not responded to other treatments – had a high density of MK cells at diagnosis (bone marrows were taken at diagnosis, and before TPO agonists were started). Serum TRAIL levels were higher in patients with chronic ITP and with platelets 150) (p Conclusion Preliminary evidence suggests increased interactions between T cells and megakaryocytes in the bone marrow of patients with ITP. The demonstration of interactions between CD3 cells and MK cells in both patients and controls shows the importance of the MK cell in the immune system. Further investigation is under way to explore whether CD8 or NK cell interaction is only found in patients with ITP, and whether these interactions result in MK cell death, or reduction in platelet production. High TRAIL levels in patients with chronic disease, refractory to first and second line therapy further suggests that increased apoptosis plays an important role in the ongoing thrombocytopenia in chronic disease. Disclosures: No relevant conflicts of interest to declare.

Non-Hodgkin s lymphomas (NHLs) constitute a heterogeneous group of lymphoid neoplasms and a majority of them in India are of B-cell phenotype. Varying numbers of T lymphocytes and natural killer (NK) cells are consistently present within the lymph nodes (LNs). The role of these reactive cells is becoming understood. TIA-1 is a cytotoxic granule associated RNA binding protein, the expression of which is restricted to cytotoxic T lymphocytes (CTLs) and NK cells. Snap frozen lymph node biopsies obtained from 41 B-cell NHLs were localized for intra-tumoral TIA-1 + cytolytic cells by immunohistochemistry. Distribution of T cell subsets and NK cells were also quantified. Cells expressing TIA-1 antigen was observed in all the cases, seen as a strong granular cytoplasmic signal. Results indicate significantly higher number of TIA-1 cytolytic cells outside (periphery of the follicle and interfollicular areas) than within the neoplastic follicle in follicular lymphomas (p0.001). In small lymphocytic lymphomas, cytolytic cells were mainly seen as uniformly scattered single cells, distributed throughout the tumor environment. In mantle cell and diffuse large B-cell lymphomas these were most often seen as small clusters and less frequently as singly scattered cells. Higher numbers of CD4 + than the CD8 + T cells were observed in most cases. Contrary to the follicles in follicular hyperplasia, CD57 + NK cells were predominantly observed outside the neoplastic follicle in follicular lymphomas (FLs). These results outline specific interactions between the potential anti-tumoral cytolytic and the malignant cells of B-cell NHLs.

The trafficking or homing of different lymphoid subsets to particular microenvironment is mediated by specific cell adhesion molecules (CAMs) expressed on lymphocytes and endothelial cells. B-cell chronic lymphocytic leukaemia (B-CLL) or Non-Hodgkin's lymphoma of small lymphocytic, B-cell type are monoclonal expansions of mature lymphocytes. The relative distribution of the tumor lymphocytes among various lymphoid compartments vary from patient to patient. Very few studies underlying this issue are available. To this effect, we have analysed the expression of LFA-1; VLA-4, ICAM-1; CD44H and CD44v6 (haematopoietic and variant form respectively) on freshly isolated lymphocytes obtained from bone marrow (BM), peripheral blood (PB) and lymph node (LN) by flow cytometry. Overall, we find strong expression of CD44H, low to moderate expression of LFA-1, negative to low expression of VLA-4 and lack of expression of CD44v6. ICAM-1 expression was observed only in patients with prominent lymphadenopathy. Higher expression of CD44H in PB lymphoid cells relative to that of BM lymphoid cells correlated with higher PB lymphocytosis (p < 0.001). Proliferating cell nuclear antigen expression in LN sections correlated inversely with VLA-4 expression on BM and PB lymphoid cells (p < 0.05). There was no significant correlation between expression of CAMs and bcl-2 protein.

Summary Background Though Epstein-Barr virus (EBV) has been linked to classical Hodgkin’s disease (cHD), only a proportion of cHD cases show EBV-association. While there has been evidence to suggest a crucial role for EBV in the early phase of cHD evolution, we are unclear whether continued presence of EBV is essential for the maintenance of the neoplasm. We have addressed this issue by investigating the EBV-association in paired samples of cHD obtained at presentation and relapse. Materials and methods We have analysed lymph node biopsy samples from 23 cases of cHD at presentation and relapse. In situ hybridization with EBER-1 probe and immunostaining with anti latent membrane protein-1 (LMP-1) antibody was performed on the paraffin sections. PCR for EBNA 3C gene was performed for EBV subtype analysis on the DNA extracted from paraffin sections. Results EBV-association was documented in both the presentation and relapse samples of 14 cases. One case showed loss of EBV-association at relapse. PCR analysable DNA was obtained from both presentation and relapse samples in four of the EBV-associated cases. The EBNA 3C amplimer corresponded to type A strain of EBV in all the samples. Conclusion Loss of EBV-association between presentation and relapse seen in one case implies a hit and run mechanism in EBV-induced lymphomagenesis. Though EBV may be essential early in the evolution of cHD, it may not be required for maintenance of the neoplastic clone. Our study also brings out a speculation that a proportion of EBV-negative cHD could represent loss of EBV in the tumour prior to clinical presentation. Alternatively, an unidentified lymphotropic virus other than EBV might play a pathogenetic role in EBV-negative cHD.

Nasal NK/T-cell lymphoma is a unique form of lymphoma highly associated with Epstein-Barr virus (EBV). These lymphomas are rare in Western populations and much more prevalent in some Asian and Latin American countries. Although there are several sizable studies from Asian countries, the same is not true from South America. The aim of this study was to analyze a series of 32 cases of nasal T-cell lymphoma from Peru and to further extend the characterization of this disease. Immunohistochemistry was performed on paraffin sections using the following antibodies: CD20 (L26), CD45RO, CD3, Ki67, CD57, CD56, TIA-1, bcl-2, and p53. The presence of EBV was investigated with immunohistochemical analysis for latent membrane protein (LMP)-1 and in situ hybridization using an antisense riboprobe to EBER 1. The 32 patients included 18 men and 14 women (M:F ratio, 1.2:1), with a median age of 43 years (11 to 72). Three categories were identified: (1) Nasal NK/T cell lymphomas (28 cases): The morphology ranged from small or medium-sized cells to large transformed cells. Necrosis was present in 86% of the cases, and angioinvasion was seen in 36% of the cases. All cases were positive for CD45RO, CD3, and for TIA-1. CD56 was positive in 21 of 27 cases (78%), and CD57 was negative in all cases. EBER 1 positivity was identified in most of the tumor cells in 27 of 28 cases (96%), including the six cases in which CD56 was negative. Overexpression of p53 was detected in 24 cases (86%). (2) Blastic NK cell lymphoma (1 case): The neoplastic cells resembled those of lymphoblastic lymphoma. CD56 and CD45RO were positive; TIA-1, TdT, and EBER-1 were negative. (3) Peripheral T-cell lymphoma (PTCL) unspecified (3 cases): CD56, TIA-1, and EBER-1 were negative. Nasal lymphomas from Peru with a T cell phenotype are predominantly EBV-associated NK/T cell lymphomas, similar to those described in Asian countries. The expression of CD56, TIA-1, and EBER-1, in combination, are very useful markers for the diagnosis of nasal NK/T cell lymphoma in paraffin-embedded tissue. The differential diagnosis of T-cell lymphomas in the nasal region should include rare cases of PTCL unspecified and the blastic variant of NK cell lymphoma. P53 is overexpressed in 86% of the cases. The significance of this finding with regard to clinical behavior and prognosis remains to be determined.

Fas and Fas ligand expression were investigated in twenty two cases of classical Hodgkin's disease (HD) by immunohistochemistry. While Reed-Sternberg (RS) cells in 7/22 (32%) cases expressed Fas ligand, reactive lymphoid cells expressed Fas ligand in only 2 (9%) cases. In 20/22 (91%) cases, the RS cells expressed Fas. A higher proportion of RS cells in the nodular sclerosis subtype expressed Fas as compared to the mixed cellularity subtype. In 18/22 (82%) cases, Fas expression was also noted in the reactive lymphoid cells. In eight cases, the reactive lymphoid cells were also analyzed by flow cytometry and a majority of them were CD4+CD45RO+. Most of these activated T-cells expressed Fas but were negative for Fas Ligand. To investigate the co-expression of Fas and Fas Ligand in the RS cells, six cases were subjected to Fas and Fas ligand immunostaining on consecutive sections. The co-expression was documented in the RS cells in four of six cases. These six cases with expression of both Fas and Fas ligand were investigated for the incidence of apoptosis. There was no statistically significant relationship between expression of Fas on reactive cells, expression of FasL on RS cells and the proportion of apoptotic reactive cells. In all these cases apoptosis was not observed in the RS cells. Thus Fas - FasL interactions may not lead to apoptosis of the RS cells.

Kikuchi-Fujimoto lymphadenitis is a distinctive entity which is easily recognised in its classical histology. However, cases which are biopsied in the early stage of the disease, before entering the necrotic phase may be misdiagnosed as non-Hodgkin's lymphoma. Ten cases of Kikuchi-Fujimoto disease which mimicked non-Hodgkin's lymphoma and in which less than 10% of the lymph node was occupied by necrosis were chosen for this study. The patients included 4 males and 6 females with an age range of 15-40 years. Eight patients presented with cervical lymphadenopathy. The pathologic areas ranged from 70% to 95% of the total nodal area. The features that helped distinguish this lesion from non-Hodgkin's lymphoma were: 1) a sprinkling of karyorrhectic debris throughout the node 2) plasmacytoid monocytes as well as "histiocytes" in the company of transformed lymphocytes (immunoblasts) 3) enlarged mottled T-zone areas, and 4) presence of residual lymphoid follicles. Follow up on six patients revealed no evidence of disease.

Aberrant expression of CD79a has been reported in neoplastic cells in peripheral T cell lymphoma, T-cell acute lymphoblastic leukemia and acute myeloid leukemia (especially those with t(8;21)). In this report, we document the first report of CD79a positivity in erythroid precursor cells in bone marrow. In all, we document this finding in five of 18 re-staging bone marrow trephine samples in patients of lymphoma treated with chemotherapy (one index case and 17 additional validation cases). It is important to appreciate this finding especially in rituximab treated patients where one tends to rely on CD79a to identify minimal marrow disease.

We report four cases of sinonasal teratocarcinosarcoma (SNTCS), a rare malignant tumor that displays combined features of an immature or malignant teratoma and a carcinosarcoma. The patients, three men and one woman, were all adults ranging in age from 21 to 69 years who presented with nasal obstruction and epistaxis. The tumors were typically composed of round cells and short spindle cells with neuroectodermal/rosette-like structures. Also seen were foci of fetal-like squamous epithelium, glandular epithelium, immature mesenchyme, immature cartilage, and neuronal differentiation. Immunohistochemistry performed in three cases showed expression of vimentin, CD99 (MIC2), and neuron-specific enolase in most cells, and focal expression of cytokeratin, epithelial membrane antigen, alpha fetoprotein, glial fibrillary acidic protein, chromogranin, and synaptophysin. The tumors were consistently negative for beta human chorionic gonadotrophin, neurofilament protein, and leukocyte common antigen. The entities considered in the differential diagnosis were poorly differentiated carcinomas, sarcomas, and olfactory neuroblastoma. We suggest that these neoplasms arise from a primitive cell capable of organized divergent differentiation.

Plasma cell dyscrasias are a heterogeneous group of disorders characterised by the expansion of a single clone of immunoglobulin secreting, terminally differentiated, end-stage B cells.1 Many of their clinical and morphological features are the result of the production and accumulation of excessive amounts of monoclonal immunoglobulin. Intracellular inclusions, both intracytoplasmic (Russell bodies) and intranuclear (Dutcher bodies) are occasionally seen in bone marrow biopsy specimens from patients with plasma cell neoplasms, and less frequently in reactive states.2,3 Extracellular protein deposition also occurs, although less frequently. “Myeloma kidney” with tubular casts composed of a heterogeneous mixture of electron dense homogeneous material and Tamm–Horsfall protein, together with elongated crystals, some with a fibrillar or lattice substructure, is an important cause of myeloma-associated morbidity and mortality.4,15 The formation of extracellular crystalline structures within the cornea, skin, thyroid gland, intravascular space and bone marrow along with other sites has been described.5–9 However, this phenomenon is unusual and is limited to isolated reports. In our department, we see approximately 1200 bone marrow trephines per year, including about 300 cases of multiple myeloma, and they have not previously come to our notice. A 72-year-old man presented with back pain. A spinal x ray carried out six months later showed collapse of the first lumbar vertebra. Further investigations included haemoglobin 9.3 g/dl, creatinine 129 μmol/l, calcium 2.5 mmol/l, β2-microglobulin 3.9 mg/l, IgG …

We have generated cytotoxic T-lymphocytes (CTLs) from the peripheral blood (PB) of eight B-cell non-Hodgkin's lymphoma (NHL) patients by in vitro coculture with autologous fresh tumor cells. Their functional activity was assessed in 51Cr release assay and was found to be MHC class I restricted. Our results indicate the presence of T-cells cytotoxic for autologous tumor cells in the PB of these patients but these were relatively small numbers in small lymphocytic lymphomas (SLLs). Treatment of fresh tumor cells with rIFN-gamma and rTNF-alpha alone, or in combination significantly increased their susceptibility in 4/5 cases of SLLs, and a case of diffuse large cell lymphoma and Burkitt lymphoma (BL), while, B-cell lymphoma, rich in T-cells, did not show any appreciable increase. Fresh tumor cells were also analysed for MHC class I and ICAM-1 antigens by flow cytometry, in 5/8 cases before and after cytokine treatment. Significant upregulation of MHC class I antigens but with no detectable change in ICAM-1 observed in a case of SLL and BL, correlated with enhanced susceptibility. These findings suggest the possible role of MHC class I antigens in the cytotoxic susceptibility of autologous tumor cells in B-cell NHL.

A human squamous cell carcinoma (SCC) cell line has been established from the surgical specimen of an untreated, upper aero-digestive tract tumour, diagnosed as a squamous carcinoma, grade III, of the pyriform fossa. The tumour tissue was grown as a xenograft in an athymic nude mouse and was designated as NT-8. Histological examination of the surgical specimen and the nude mouse tumour showed that the two were identical. NT-8 was subsequently passed by subcutaneous injections into nude mice. After the 6th passage in nude mouse, the tumour was cultured in vitro where it grew as an epithelial cell line, with a typical cobblestone appearance. This cell line was designated as NT-8e. Both the primary tumour as well as xenograft and the cells in culture have retained several common morphological and biochemical characteristics. Immunological markers for epithelial cells including epithelial membrane antigen and cytokeratins were seen in all three, confirming the epithelial lineage. Characterization of the NT-8e cell line including growth parameters, anchorage-independent growth and tumorigenicity in nude mice, chromosome counts and DNA content by flow cytometry have been carried out.

About one fourth of patients with Hodgkin's disease relapse after therapy. The mechanisms that lead to resistance to treatment in these patients are poorly understood. The authors describe the differential protein expression of p53, proliferating cell nuclear antigen (PCNA), and p21 at initial presentation and relapse, and discuss their role in disease progression and resistance to therapy. Thirty-four patients with Hodgkin's disease who had relapsed after standard chemotherapy and radiotherapy regimens were assessed for the expression of p53 protein, PCNA, and p21 protein (waf/cip 1). In 14 of these cases, sequential biopsies performed both at presentation and at relapse were available for the study. Seventy-five percent of the cases were positive for the p53 protein. Tumors at relapse had higher p53 and PCNA scores than those at initial presentation. In the paired samples, a significant increase was noted in the number of p53 and PCNA-positive cells and in the intensity of staining with p53 antibody. Six of seven paired samples tested for p21 showed an increased p21 expression at relapse. These results suggest that, at relapse, Reed-Sternberg (RS) cells and their variants positive for p53, PCNA, and p21 are increased in number and individually have an increased expression of p53, PCNA, and p21 proteins. These findings suggest that therapy failure and relapse may, at least in part, be associated with altered p53, p21, and PCNA pathways. HUM PATHOL 28:549-555. This work was carried out during an exchange fellowship program at the National Cancer Institute, Bethesda. There are no restrictions on its use

We studied the expression of CD34 in 169 bone marrow trephines of 53 patients with CML in chronic, accelerated and blastic phases. The bone marrow blast count ranged from 0–82% (median: 4.5%). All were receiving imatinib treatment. We aimed to establish which counting method in bone marrow trephines provided the best correlation with the blast count of the corresponding bone marrow aspirate in all phases of the disease. Paraffin embedded sections were stained with the monoclonal antibody QBEND10. CD34 expression in each marrow trephine was evaluated by three counting methods. The first method, established in our department for several years, expressed the percentage of CD34 positive cells vs. all nucleated cells in a minimum 500-cell count (% CD34+/500 cells). The second recorded the total number of CD34 positive cells in ten high power fields (CD34+/10hpf) using x 40 magnification /numerical aperture 0.65. The third method evaluated the highest number of CD34 positive cells present in a single high power field (CD34+/hpf), this value being taken from the previous 10 hpf count. The two latter methods were employed because they may be more applicable for general use by practicing histopathologists. In addition, counting over 10 hpf provides a much larger sample size than a 500-cell count. CD34 expression in endothelial cells served as a positive internal control. All three methods showed significant positive correlation with blast count (% CD34+/500 cells: Pearson’s Correlation - 0.723, sig

Abstract 898 Burkitt Lymphoma (BL) is a highly proliferative form of non-Hodgkin lymphoma and is characterized by translocation of the C-MYC gene to the immunoglobulin gene loci resulting in deregulation. The role of collaborating gene mutations in BL is largely unknown. We performed whole exome sequencing and gene expression profiling of 57 Burkitt lymphoma and 94 DLBCL exomes. Mutational analysis revealed that ID3 is recurrently mutated in 38% of Burkitt lymphoma samples. ID3 mutations did not occur in any of the 94 DLBCL cases. ID3 gene expression was also found to be a distinguishing feature of Burkitt lymphomas (P We explored the biological significance of ID3 mutations by initially comparing the gene expression profiles of BL cases that had mutated and wild-type ID3. Gene set enrichment analysis showed that those samples with mutated ID3 had higher expression of genes that were involved in cell cycle regulation, specifically those involved in the G1-S transition (P=0.01). In order to experimentally investigate the functional consequences of ID3 mutation, we generated mutant constructs corresponding to six different ID3 mutations observed in BLs. These mutant constructs were cloned into lentiviral vectors and overexpressed in BL cells that were wild type for ID3. We then performed cell cycle analysis for these wild type cells expressing GFP controls or the mutant constructs. We found that BL cells expressing each of the six mutant constructs demonstrated significant cell cycle progression from G1 to S phase compared to wild-type (P=0.01). Separately, we tested the effects of expressing mutant ID3 in cell proliferation assays and found that cells expressing mutant ID3 were considerably more proliferative than those expressing wild type (P=0.03). Conversely, we over-expressed the wild type form of ID3 in BL cells that had mutated ID3. These experiments completely rescued the observed phenotypes of the mutant ID3 constructs, with reduced cell cycle progression through increased G1 phase and decreased S-phase (P=0.04). We also noted decreased cell proliferation in these cells (P=0.03). These experiments support a role for ID3 as a novel tumor suppressor gene in Burkitt lymphoma. ID3 is a basic helix loop helix (bHLH) protein that binds to other E-proteins, blocking their ability to bind DNA. ID3 has been shown to be involved in a variety of biological processes including development and T and B cell differentiation. ID3 knockout mice have been shown to develop T cell as well as B cell lymphomas. Our data implicates this gene for the first time as a tumor suppressor in human cancer. Disclosures: No relevant conflicts of interest to declare.

A case of low grade hemangiopericytoma of three years duration occurring in the upper end of the femur of a 70 year old man is described. Radiologically, the upper third of the right femur showed a destructive expansile osteolytic lesion with soft tissue extension. The tumour was partially encapsulated. Mitoses were 0-1 per 10 high power fields, suggesting the low grade malignant nature of the lesion. It is felt that all hemangiopericytomas occurring in bone should be considered biologically malignant.

A case of ovarian myxoma in a 12-year-old premenarchal girl is reported. This paper details the clinical presentation, gross and microscopic pathology, Immunocytochemical profile, and behavior of this rare tumor. The differential diagnosis and possible histogenesis is briefly discussed.

Eight adult paratesticular sarcomas seen at a Regional Cancer Centre over a 7-year period are described. There were three cases of rhabdomyosarcoma (RMS), three cases of leiomyosarcoma (LMS), and two cases of liposarcoma (LS). The RMS occurred in a younger age group (3rd decade) than the LMS and LS (6th and 7th decades). Most of our patients presented with advanced disease after orchiectomy at other hospitals, three with recurrent or residual disease and four with metastasis. The single patient with RMS who received intensive adjuvant therapy is free of disease at 84 months in spite of the advanced stage at presentation. All three patients with LMS had an unfavourable clinical course. Both the patients with LS had well differentiated (WD) tumours and presented with recurrences, one over several years, following initial local excisions. The necessity for early adequate surgical and adjuvant therapy and the need for a uniform treatment policy are discussed. © 1994 Wiley-Liss, Inc.

Abstract 433 Burkitt lymphoma (BL) is a relatively uncommon lymphoma, but is clinically important because it is curable when diagnosed properly. BL is also an important model disease for studying cancer. Chromosomal translocations of the MYC gene are a defining feature of BL. Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in adults and demonstrates overlapping morphology, immunophenotype and clinical behavior with BL. The genetic causes and the role of specific mutations in BL are largely unknown. Th e decoding of the human genome and the advent of high-throughput sequencing have provided rich opportunities for the comprehensive identification of the genetic causes of cancer. We began by sequencing 2 complete lymphoma tumor genomes (and paired normal tissue) derived from DLBCL and BL respectively. The pattern of somatic base alterations in both DLBCL and BL genomes indicated a predominance of G→A/C→T and A→G/T→C transitions (P In order to comprehensively identify genes that are recurrently mutated in DLBCL and BL, we obtained a total of 95 cases of DLBCLs and 60 cases of BL. The DLBCL cases were divided into a discovery set (N=34) and a prevalence set (N=61). The Burkitt cases were also divided into discovery and prevalence sets (N=15, N=45 respectively). For each of the discovery set cases we also obtained paired normal tissue. We performed whole-exome sequencing for all of these using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 6 GB of sequencing data using high throughput sequencing on the Illumina platform. We identified 525 candidate cancer genes that were recurrently somatically mutated in DLBCL and BL. We found that each tumor had an average of 20 gene alterations, which is fewer than most other solid tumors sequenced to date. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, PDGFRA) and chromatin modification (e.g. MLL3, SETD2), affecting 17.2% and 14.8% of the total genetic events respectively. We found several genes related to cancer that were commonly mutated in both BL and DLBCL, including MYC, BTG1 and SETD2. Mutations in MYC were much more common in BL compared to DLBCL, suggesting that mutation of MYC might serve as an independent oncogenic mechanism in BL, in addition to chromosomal translocations. Many known cancer genes were found to be exclusively mutated in BL including SMARCA4, a gene known to regulate the expression of CD44 which is implicated in tumorigenesis. This study represents one of the first in-depth analyses of a BL genome and one of the largest applications of exome sequencing in cancer. Our data provide the most comprehensive genetic portrait of human BL to date, and provides a significant first step to identifying the genetic causes of the disease. Disclosures: No relevant conflicts of interest to declare.