Whole-exome sequencing in osteosarcoma reveals important heterogeneity of genetic alterations.

BACKGROUND: Whole-genome sequencing studies have recently shown that osteosarcomas (OSs) display high rates of structural variation, i.e. they contain many somatic mutations and copy number alterations. TP53 and RB1 show recurrent somatic alterations in concordant studies, suggesting that they could be key players in bone oncogenesis. PATIENTS AND METHODS: we carried out whole-genome sequencing of DNA from seven high-grade OS samples matched with normal tissue from the same patients. RESULTS: We confirmed the presence of genetic alterations of the TP53 (including novel unreported mutations) and RB1 genes. Most interestingly, we identified a total of 84 point mutations and 4 deletions related to 82 different genes in OS samples, of which only 15 have been previously reported. Interestingly, the number of mutated genes (ranging from 4 to 8) was lower in TP53mut cases compared with TP53wt cases (ranging from 14 to 45). This was also true for the mutated RB1 case. We also observed that a dedifferentiated OS harboring MDM2 amplification did not carry any other mutations. CONCLUSION: This study suggests that bone oncogenesis driven by TP53 or RB1 mutations occurs on a background of relative genetic stability and that the dedifferentiated OS subtype represents a clinico-pathological entity with distinct oncogenic mechanisms and thus requires different therapeutic management.

Breast implant-associated anaplastic large cell lymphoma: two distinct clinicopathological variants with different outcomes.

BACKGROUND: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Clinical and molecular practice of European thoracic pathology laboratories during the COVID-19 pandemic. The past and the near future.

BACKGROUND: This study evaluated the consequences in Europe of the COVID-19 outbreak on pathology laboratories orientated toward the diagnosis of thoracic diseases. MATERIALS AND METHODS: A survey was sent to 71 pathology laboratories from 21 European countries. The questionnaire requested information concerning the organization of biosafety, the clinical and molecular pathology, the biobanking, the workload, the associated research into COVID-19, and the organization of education and training during the COVID-19 crisis, from 15 March to 31 May 2020, compared with the same period in 2019. RESULTS: Questionnaires were returned from 53/71 (75%) laboratories from 18 European countries. The biosafety procedures were heterogeneous. The workload in clinical and molecular pathology decreased dramatically by 31% (range, 3%-55%) and 26% (range, 7%-62%), respectively. According to the professional category, between 28% and 41% of the staff members were not present in the laboratories but did teleworking. A total of 70% of the laboratories developed virtual meetings for the training of residents and junior pathologists. During the period of study, none of the staff members with confirmed COVID-19 became infected as a result of handling samples. CONCLUSIONS: The COVID-19 pandemic has had a strong impact on most of the European pathology laboratories included in this study. Urgent implementation of several changes to the organization of most of these laboratories, notably to better harmonize biosafety procedures, was noted at the onset of the pandemic and maintained in the event of a new wave of infection occurring in Europe.

[Clear-cell sarcoma of the kidney: about a paediatric case]. / Sarcome à cellules claires du rein : à propos d'un cas pédiatrique.

Clear-cell sarcoma of the kidney (CCSK) is a rare malignant tumor of childhood, known for its aggressiveness, its tendency to recurrence and to metastasis to bone. We report an observation of a child of 48 months carrying a large abdominal mass. The diagnosis of the SCCR was made on biopsy, since imaging remained uncertain as to the renal origin of the mass. Indeed, our observation underlines the difficulty of its diagnosis. Excepting the morphological aspect, there is no criterion for its recognition. Its prognosis has been improved by the new treatments.

Detection of different clonal EBV strains in Hodgkin lymphoma and nasopharyngeal carcinoma tissues from the same patient.

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is linked to the development of several malignancies, including nasopharyngeal carcinoma (UCNT) and Hodgkin lymphoma (HL). Despite the well-known oncogenic properties of the EBV latent membrane protein 1 (LMP-1), the different oncogenic pathways involved in the pathogenesis of each disease remain unclear. This study reported, for the first time, the case of a patient with sequential development of UCNT and HL. Polymerase chain reaction was used to determine the LMP-1 gene sequence and demonstrate that the two tumours contained different clonal viral genomes, suggesting a central and specific role of EBV infection.

Gene expression profiling identifies molecular subgroups among nodal peripheral T-cell lymphomas.

The classification of peripheral T-cell lymphomas (PTCL) is still a matter of debate. To establish a molecular classification of PTCL, we analysed 59 primary nodal T-cell lymphomas using cDNA microarrays, including 56 PTCL and three T-lymphoblastic lymphoma (T-LBL). The expression profiles could discriminate angioimmunoblastic lymphoma, anaplastic large-cell lymphoma and T-LBL. In contrast, cases belonging to the broad category of 'PTCL, unspecified' (PTCL-U) did not share a single molecular profile. Using a multiclass predictor, we could separate PTCL-U into three molecular subgroups called U1, U2 and U3. The U1 gene expression signature included genes known to be associated with poor outcome in other tumors, such as CCND2. The U2 subgroup was associated with overexpression of genes involved in T-cell activation and apoptosis, including NFKB1 and BCL-2. The U3 subgroup was mainly defined by overexpression of genes involved in the IFN/JAK/STAT pathway. It comprised a majority of histiocyte-rich PTCL samples. Gene Ontology annotations revealed different functional profile for each subgroup. These results suggest the existence of distinct subtypes of PTCL-U with specific molecular profiles, and thus provide a basis to improve their classification and to develop new therapeutic targets.

[Psycho-pathological risk factor of arterial disease (prevalence, superposition or vinculation with other factors)]. / Estudio oncativo: factores de riesgo psicopatológicos de enfermedad arterial (prevalencia, superposición o vínculo con otros factores).

Alexitimia and depression may or not coexist with others risk factors (comportment o physical). Frecuently they have relation with socio-echonomic status and with ethnia. Sometimes are determinants of the atherosclerotic process by increasing the vascular reactivity by the alteration of the evolution. There is no information in our country about this problem in general population. The present study result of the investigation of these aspects and the comportamental and physical factors of arterial disease, in a population of Cordoba province (Argentina Republic).

"In situ-like" mantle cell lymphoma: a report of two cases.

Mantle cell lymphoma (MCL) is a B cell neoplasm that most often shows a diffuse growth pattern. Two cases of MCL are reported here, both with a previous diagnosis of lymphoid hyperplasia. Morphologically, germinal centres are hyperplasic with a normal or discretely enlarged mantle zone, where foci of irregularly shaped small lymphocytes are seen. These are positive for CD20, CD5 and cyclin D1, confirming a diagnosis of in situ-like MCL. This type differs from the mantle zone pattern in that the neoplastic mantle zone is very thin and there is very little or no spread of tumour cells into interfollicular areas. To the best of our knowledge, this is the first report on such a pattern of MCL, which is important to recognise, as it can be confused with lymphoid hyperplasia.

Mutational hot spots within the carboxy terminal region of the LMP1 oncogene of Epstein-Barr virus are frequent in lymphoproliferative disorders.

We have recently identified in Epstein-Barr virus (EBV) positive Hodgkin's disease (HD) a variant of the latent membrane protein 1 (LMP1) oncogene characterized by four point mutations and a 30 base pair deletion. These findings led us to test whether such mutants were also present in other lymphoproliferative disorders (LPD). We analysed 98 EBV DNA positive cases (67 LPD, 15 benign conditions, 16 lymphoblastoid cell lines) by PCR for deletions within the LMP1 gene. DNA sequencing of the region coding for the carboxy terminal protein domain was performed on 24 cases. In 13 cases the same combination of 4 point mutations at positions 168,320, 168,308, 168,295 and 168,225 was identified. Of these cases, 12 had an additional point mutation at position 168,357 and eight at position 168,355, and nine had a 30 base pair deletion including nucleotides 168,285 to 168,256. These deletion mutants were identified in HD, angioimmunoblastic lymphadenopathy, B-immunoblastic lymphoma, peripheral T-cell lymphoma, and two lymphoblastoid cell lines. Our findings reveal a high frequency of non-random point mutations at preferential sites within the 3' (carboxy terminal) region of the LMP1 oncogene. The association of these mutational hot spots with LPD suggests that they are involved in EBV related lymphomagenesis and that they define a clinically relevant EBV strain.

Immunodetection of SV40 large T antigen in human central nervous system tumours.

BACKGROUND/AIMS: DNA sequences from Simian virus 40 (SV40) have been previously isolated from various human tumours of the central nervous system (CNS). This study aimed to investigate a series of tumours of the CNS for the expression of the SV40 large T antigen (Tag), which is an oncogenic protein of the virus. METHODS: A French series of 82 CNS tumours was investigated for Tag expression using a monoclonal antibody and immunohistochemistry. A Tag positive hepatocellular carcinoma cell line from transgenic mice and a kidney biopsy from a patient infected by SV40 were used as positive controls. RESULTS: None of the tumours (20 ependymomas, 20 glioblastomas, 12 oligodendrogliomas, three plexus choroid adenomas, two plexus choroid carcinomas, 15 meningiomas, and 10 medulloblastomas) contained SV40 Tag positive cells. CONCLUSIONS: The lack of SV40 Tag in 82 CNS tumours of various types is at variance with previous studies from different countries, and suggests that the virus may not be an important factor in CNS tumorigenesis, at least in French cases.

B-cell lymphoma following polycythemia vera: evidence for the involvement of two different clones.

We report here two patients with polycythemia vera (PV) who developed secondary non-Hodgkin's lymphoma (NHL). Both cases were high grade B-cell NHL. Cytogenetic analysis of bone marrow and lymph node was performed in each case and showed numerous chromosomal abnormalities. Of interest, chromosomal abnormalities of the PV and of the NHL clones were different, suggesting the possible involvement of two different clones. A 11q23 breakpoint was common between the two cases and the putative role of this breakpoint in the pathogenesis of the NHLs is discussed.

Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL.

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.

Epstein-Barr virus (EBV) replicative gene expression in tumour cells of AIDS-related non-Hodgkin's lymphoma in relation to CD4 cell number and antibody titres to EBV.

OBJECTIVE: To determine whether activation of Epstein-Barr virus (EBV) replication in tumour cells of AIDS-related non-Hodgkin's lymphoma (ARNHL) is correlated with CD4+ cell counts and influences antibody response to EBV [anti-Z Epstein-Barr replicative activator (ZEBRA), anti-early antigen (EA), anti-viral capsid antigen (VCA)]. DESIGN: Retrospective study based on immunohistochemistry and in situ hybridization to detect EBV replicative gene products in tissue samples from patients affected by ARNHL and correlation with CD4+ cell counts and results of EBV serology (including anti-ZEBRA activity) in sera from the same patients. METHODS: Seventeen out of 22 cases of ARNHL were selected for the presence of EBV [Epstein-Barr early region (EBER) RNA-positive]. Immunohistochemistry was performed with anti-ZEBRA, anti-EA-restricted, anti-VCA antibodies and in situ hybridization with BHLF1/NotI oligoprobes on tumour samples. Results were statistically correlated with those of CD4+ cell counts (17 out of 17) and with anti-EBV antibody titres (13 out of 17) assessed using standard immunofluorescence method and enzyme-linked immunosorbent assay procedure using recombinant ZEBRA protein and synthetic peptides as antigens. RESULTS: BZLF1 (ZEBRA) or early gene products (EA-R and EA-D/BHLF1/NotI) were detected in a small proportion (< 0.01-5%) of tumour cells in eight of these 17 cases by immunohistochemistry and in situ hybridization. Demonstration of replicative gene expression did not correlate with either low CD4+ cell counts (P > 0.05) or anti-EBV antibody titres (P > 0.05). Anti-ZEBRA activity was not significantly increased in patients affected with ARNHL, the cells of which expressed replicative gene products (P > 0.05). CONCLUSION: The degree of immunodeficiency does not clearly enhance replicative gene expression in tumour cells of ARNHL. EBV serology, including anti-ZEBRA activity, is not a reliable tool for predicting the occurrence of such proliferations.

Expression of the cell death-inducing gene bax in carcinomas developed from the follicular cells of the thyroid gland.

The expression of apoptosis-regulating proteins, Bcl-2, Bax, Mcl-1, and Bcl-X, was evaluated by immunohistochemical methods in 39 cases of thyroid carcinomas. Normal thyroid tissues showed a consistent expression of Bcl-2 and Mcl-1 whereas Bax and Bcl-X proteins were essentially absent from most follicular thyroid cells. Bax expression was observed in all papillary carcinomas (n = 23) and in 8 of 10 follicular carcinomas. The intensity of Bcl-2 immunostaining was generally higher in follicular tumors (n = 10) than in papillary carcinomas (n = 21 of 23). However, in undifferentiated tumors, both Bax and Bcl-2 were weakly expressed. Mcl-1 protein expression was similar to that of Bax in papillary and follicular tumors, but was also frequently detectable in undifferentiated tumors. Bcl-X immunostaining was seen in all undifferentiated tumors (n = 6), in 22 of 23 papillary tumors, and in 5 of 10 follicular tumors. Our findings show that the regulation of bcl-2 family gene expression is different in normal thyroid tissue compared to that of its neoplastic counterpart and varies with the tumor subtype. In particular, unlike normal thyroid epithelium, the apoptosis-blocking gene bcl-X and the apoptosis-inducing gene bax are frequently expressed in thyroid carcinomas derived from the follicular cells. Thus, alterations in the expression of these bcl-2 family genes may contribute to the pathogenesis of thyroid carcinomas.

Telomerase activity in human thyroid carcinomas originating from the follicular cells.

Telomerase activity is known to be absent from most normal and well-differentiated tissues, although being detectable in the vast majority of malignant tumors. An increasing number of reports demonstrate that telomerase may be activated in benign tumors, such as adenomas. We have investigated a series of normal and neoplastic thyroid tissues for the presence of telomerase activity. As expected, all normal thyroid tissues (n = 20) had no display of telomerase activity. Amongst cancers, the incidence of telomerase activity varied with the histological subtypes. Telomerase activity was present in only 3/15 cases (20%) of papillary carcinomas. Telomerase activity was more frequently detected in follicular (4/6) and in undifferentiated (2/3) carcinomas. Unexpectedly, one case (1/12) of adenoma contained high levels of telomerase activity. Taken together, these results indicate that telomerase may play some role in the pathogenesis of thyroid tumors, in particular in follicular and undifferentiated carcinomas that are known to have the most aggressive behavior.

Inflammatory pseudotumor of the liver. Evidence for follicular dendritic reticulum cell proliferation associated with clonal Epstein-Barr virus.

We describe an "inflammatory pseudotumor" of the liver that, which on detailed investigation, proved that the spindle-cell component of this lesion is derived from follicular dendritic reticulum cells (FDRC). This contention is supported by morphologic observations and by immunophenotype. The FDRC population contain Epstein-Barr virus (EBV). It is known that FDRC express the EBV receptor CD21. In this particular case, the FDRC contained clonal EBV genomes, EBV RNA (EBER) transcripts, and expressed EBV latent membrane protein (LMP1). DNA sequencing of PCR products showed three point mutations compared with the standard LMP1 sequence of the EBV strain B95-8. The findings in this case corroborate those of other investigators concerning the possible role of EBV in the development of some inflammatory pseudotumors, including the recent production of functionally active EBV-transformed FDRC-like cell lines. This association could prove instructive in delineating the histogenesis of these tumors and further assist in making prognostic and therapeutic decisions.

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

Acquisition of a stimulatory activity for Vgamma9/Vdelta2 T cells by a Burkitt's lymphoma cell line without loss of HLA class I expression.

Daudi Burkitt's lymphoma cells activate Vgamma9/Vdelta2 T cells through TCR ligation by an unknown antigen. This activity is for a large part revealed by their lack of HLA class I antigen expression, allowing their escape from KIR downregulation. We characterize here a culture variant of the Burkitt's lymphoma line Raji, RJ-A3, which is able to promote as efficiently as Daudi cells the outgrowth of Vgamma9/Vdelta2 T cells in cocultures in spite of unchanged HLA class Ia/Ib antigen expression. RJ-A3 is resistant to lysis by most Vgamma9/Vdelta2 lines and clones, even those lacking CD9-4/NKG2 and p58, p70 p140 KIR molecules. However, one Vgamma9/Vdelta2 line which can efficiently kill RJ-A3 do so in a TCR-dependent manner since killing is modulated by anti-TCR antibodies. The CDR3 sequences of the T cell clones amplified with Daudi and RJ-A3 reveal that some clones can be expanded with both lines while others are expanded preferentially with one or the other but not both. This indicates differences in the antigenic determinants of the two Burkitt's lines. The occurrence of this Raji variant line demonstrates that the stimulatory phenotype for Vgamma9/Vdelta2 cells can be acquired by some tumors independently of the loss of class I antigens and comforts the hypothesis of an anti-tumoral function for the Vgamma9/Vdelta2 T cell population.

Colocalization of the viral interleukin-6 with latent nuclear antigen-1 of human herpesvirus-8 in endothelial spindle cells of Kaposi's sarcoma and lymphoid cells of multicentric Castleman's disease.

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.

Characterization of Epstein-Barr virus-infected cells in benign lymphadenopathy of patients seropositive for human immunodeficiency virus.

The authors investigated 25 benign lymph nodes in patients infected with the human immunodeficiency virus (HIV) by in situ hybridization (ISH) and immunohistochemistry (IHC) to detect and characterize the Epstein-Barr virus (EBV)-infected cells. After ISH, 22 lymph nodes were found to contain various numbers of Epstein-Barr-encoded RNA (EBER)-positive cells. Most of these cells were B cells. In six lymph nodes with numerous EBV-infected cells, EBNA2-positive/LMP1-positive lymphoblastoid cells were detected by IHC. Exceptional cells (in two specimens) were positively labeled with antigen-Z Epstein-Barr replicative activator (ZEBRA) antibody or BamHI Left Frame 1/Not I (BHLF1/Not I) probes, indicating that EBV replication is not enhanced in the lymphocytes. In normal conditions (healthy individuals), small lymphocytes that express a restricted pattern of viral genes do escape immune response, whereas lymphoblastoid cells do not. Thus, impaired immune system may account for the late proliferation of lymphoblastoid cells (Epstein-Barr nuclear antigen [EBNA]2positive/latent membrane protein [LMP]1 positive) in HIV-infected patients, and could explain why EBV-driven, acquired immunodeficiency syndrome (AIDS)-related, non-Hodgkin's lymphoma occur more frequently in patients with low CD4-positive T cells.