Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK(-) and cutaneous ALK(-) ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK(-) ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK(+) and four ALK(-) systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4(+), CD8(+) or CD30(+) T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFkappaB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK(-) ALCL and cHL despite their different cellular origin. ALK(+) ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

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.

p53 immunostaining is correlated with reduced survival and is not correlated with gene mutations in resected pulmonary large cell carcinomas

Malignancy of pulmonary large cell carcinomas (LCC) increases from classic LCC through LCC with neuroendocrine morphology (LCCNM) to large cell neuroendocrine carcinomas (LCNEC). However, the histological classification has sometimes proved to be difficult. Because the malignancy of LCC is highly dependent on proteins with functions in the cell cycle, DNA repair, and apoptosis, p53 has been targeted as a potentially useful biological marker. p53 mutations in lung cancers have been shown to result in expression and protein expression also occurs in the absence of mutations. To validate the importance of both p53 protein expression (by immunostaining) and p53 gene mutations in lung LCC (by PCR-single strand conformational polymorphism analysis of exons 5, 6, 7, and 8) and to study their relationships with clinical factors and sub-classification we investigated the correlation of p53 abnormalities in 15 patients with LCC (5 classic LCC, 5 LCNEC, and 5 LCCNM) who had undergone resection with curative intent. Of these patients, 5/15 expressed p53 and none had mutant p53 sequences. There was a negative survival correlation with positive p53 immunostaining (P = 0.05). After adjustment for stage, age, gender, chemotherapy, radiotherapy, and histological subtypes by multivariate analysis, p53 expression had an independent impact on survival. The present study indicates that p53 assessment may provide an objective marker for the prognosis of LCC irrespective of morphological variants and suggests that p53 expression is important for outcome prediction in patients with the early stages of LCC. The results reported here should be considered to be initial results because tumors from only 15 patients were studied: 5 each from LCC, LCNEC and LCCNM. This was due to the rarity of these specific diseases.

Chronic reparative changes in medium-sized vessels in a case of primary cutaneous anaplastic large-cell lymphoma with angioinvasive features and cytotoxic phenotype: new histopathological findings in line with indolent clinical behavior

Angioinvasion/angiodestruction has been reported in a small subset of primary cutaneous anaplastic large-cell lymphomas (PCALCL). Recently, PCALCL with angioinvasive features and cytotoxic phenotype has been characterized as a variant associated with good clinical outcomes despite worrisome histopathologic features. We report a case of PCALCL with angioinvasive features and cytotoxic phenotype associated with reparative changes on the wall of medium-sized vessels involved by the neoplasm, including intimal fibroblastic proliferation and luminal obliteration. This vascular pattern, although previously unreported in PCALCL, is in accordance with the indolent behavior observed in this entity and provides a further link with lymphomatoid papulosis type E.

Hypereosinophilia driven by GM-CSF in large-cell carcinoma of the lung

In contrast to leukocytosis, paraneoplastic hypereosinophilia is uncommon in lung cancer. We present a patient with large-cell carcinoma of the lung, in which cancer cells generate large amounts of GM-CSF leading to a leukemoid reaction with prominent hypereosinophilia and potentially involved in autocrine tumor stimulation.

Radiation therapy for HIV-associated diffuse large cell non-Hodgkin lymphoma

PURPOSE: To report the clinical experience with external beam radiotherapy (RT) for AIDS-related lymphoma (ARL) with or without the involvement of the central nervous system (CNS) in HIV-infected patients. PATIENTS AND METHODS: Clinical outcome of 24 HIV-seropositive patients with ARL treated with RT from 1995 to 2004 was reviewed, testing factors associated with outcome. RESULTS: After 1 and 5 years, the overall survival was 65% and 35%, respectively. The mean RT dose was 31 Gy after normalization to fractions of daily 2 Gy (range, 7.8-47.2 Gy). Radiotherapy dose was associated with survival in univariate (P = .04) and multivariate analysis (P = .01). Other factors in univariate analysis associated with outcome were viral load (VL), highly active antiretroviral therapy (HAART), ARL stage, and CNS involvement. Patients with CNS involvement achieved complete response in 46% and improved clinical performance was seen in 73%. CONCLUSIONS: After chemotherapy, RT in combination with HAART is highly active, and RT should be encouraged especially after suboptimal responses to induction treatment.

Studies on the adhesion properties of the liver-metastatic murine RAW117 large-cell lymphoma cells: Roles of $\beta\sb3$ integrin

Metastasis is the major cause of death in cancer patients. Since many cancers show organ-preference of metastasis, elucidation of the underlying mechanisms of metastasis will benefit diagnosis or treatment of metastatic diseases. Adhesion mechanisms are thought to be involved in organ-preference of metastasis, because metastatic cells show organ preference in adhering to organ-derived microvascular endothelial cells. The adhesion molecules in this process remain largely unidentified. I have examined a series of murine RAW117 large-cell lymphoma cells variants selected in vivo for liver-colonizing properties ($\rm{H10{>>}L17>P}$). The highly liver-metastatic H10 cells were found to differentially express much higher levels of integrin $\alpha\rm\sb{v}\beta\sb3$ than L17 or P cells. H10 cells also adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin and type I collagen, and adhered at significantly higher rates to (GRGDS)$\sb4$ than to monomeric RGD-peptides. In contrast, P and L17 cells did not adhere well to the above substrates. H10 cells also spread well on vitronectin and migrated toward vitronectin concentration gradients. Pretreament of H10 cells with anti-$\beta\sb3$ monoclonal antibodies resulted in significant decreases in adhesion of H10 cells to vitronectin and immobilized (GRGDS)$\sb4$, and reduced the formation of experimental liver metastases in syngeneic Balb/c mice.^ Adhesion of RAW117 cells under hydrodynamic shear stresses was also studied because tumor cell adhesion occurs under fluid shear stresses in target organ microvessels. Similar to their properties found with static adhesion assays, H10 cells stabilized their hydrodynamic adhesion to vitronectin, fibronectin and (GRGDS)$\sb4$ much more quickly than P or L17 cells. Unlike their static adhesion properties, RAW117 cells showed differential adhesion stabilization to liver-sinusoidal endothelial cell-derived extracellular matrix ($\rm{H10{>>}L17>P}$). Although not supporting static adhesion of RAW117 cells, monomeric RGD-peptides mediated adhesion stabilization of H10 cells but not L17 or P cells. Integrin $\rm\alpha\sb{v}\beta\sb3$ was found to be involved in stabilizing H10 cell adhesion to vitronectin, (GRGDS)$\sb4$, monomeric RGD-peptide R1, and liver sinusoidal endothelial cell-derived extracellular matrix.^ This study is the first to provide evidence that integrin $\rm\alpha\sb{v}\beta\sb3$ is differentially expressed in liver-metastatic lymphoma cells and involved in differential adhesion of these cells. The results indicate that strong static adhesion and especially the unique hydrodynamic adhesion of RAW117 cells to the RGD-containing substrates correlate with liver-metastatic potentials. Thus, integrin $\rm\alpha\sb{v}\beta\sb3$ may play an important role in liver-preferential metastasis of RAW117 large-cell lymphoma cells. ^

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (VH) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma.

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.

Nonsmall cell lung cancer: evaluation of 737 consecutive patients in a single institution

OBJECTIVE: To analyze surgical and pathological parameters and outcome and prognostic factors of patients with nonsmall cell lung cancer (NSCLC) who were admitted to a single institution, as well as to correlate these findings to the current staging system. METHOD: Seven hundred and thirty seven patients were diagnosed with NSCLC and admitted to Hospital do Cancer A. C. Camargo from 1990 to 2000. All patients were included in a continuous prospective database, and their data was analyzed. Following staging, a multidisciplinary team decision on adequate management was established. Variables included in this analysis were age, gender, histology, Karnofsky index, weight loss, clinical stage, surgical stage, chemotherapy, radiotherapy, and survival rates. RESULTS: 75.5% of patients were males. The distribution of histologic type was squamous cell carcinoma 51.8%, adenocarcinoma 43.1%, and undifferentiated large cell carcinoma 5.1%. Most patients (73%) presented significant weight loss and a Karnofsky index of 80%. Clinical staging was IA 3.8%, IB 9.2%, IIA 1.4%, IIB 8.1%, IIIA 20.9%, IIIB 22.4%, IV 30.9%. Complete tumor resection was performed in 24.6% of all patients. Surgical stage distribution was IA 25.3%, IB 1.4%, IIB 17.1%, IIIA 16.1%, IIIB 20.3%, IV 11.5%. Chemotherapy and radiotherapy were considered therapeutic options in 43% and 72%, respectively. The overall 5-year survival rate of nonsmall cell lung cancer patients in our study was 28%. Median survival was 18.9 months. CONCLUSIONS: Patients with NSCLC who were admitted to our institution presented with histopathologic and clinical characteristics that were similar to previously published series in cancer hospitals. The best prognosis was associated with complete tumor resection with lymph node dissection, which is only achievable in earlier clinical stages.

Peripheral T-cell lymphoma with t(6;14)(p25;q11.2) translocation presenting with massive splenomegaly.

Recurrent chromosomal translocations associated to peripheral T-cell lymphomas (PTCL) are rare. Here, we report a case of PTCL, not otherwise specified (NOS) with the karyotype 46,Y,add(X)(p22),t(6;14)(p25;q11) and FISH-proved breakpoints in the IRF4 and TCRAD loci, leading to juxtaposition of both genes. A 64-year-old male patient presented with mild cytopenias and massive splenomegaly. Splenectomy showed diffuse red pulp involvement by a pleomorphic medium- to large-cell T-cell lymphoma with a CD2+ CD3+ CD5- CD7- CD4+ CD8+/- CD30- TCRbeta-F1+ immunophenotype, an activated cytotoxic profile, and strong MUM1 expression. The clinical course was marked by disease progression in the bone marrow under treatment and death at 4 months. In contrast with two t(6;14)(p25;q11.2)-positive lymphomas previously reported to be cytotoxic PTCL, NOS with bone marrow and skin involvement, this case was manifested by massive splenomegaly, expanding the clinical spectrum of PTCLs harboring t(6;14)(p25;q11.2) and supporting consideration of this translocation as a marker of biological aggressiveness.

Small nucleolar RNA expression profiling identifies potential prognostic markers in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a rare, heterogeneous type of non-Hodgkin lymphoma (NHL) that, in general, is associated with a poor clinical outcome. Therefore, a current major challenge is the discovery of new prognostic tools for this disease. In the present study, a cohort of 122 patients with PTCL was collected from a multicentric T-cell lymphoma consortium (TENOMIC). We analyzed the expression of 80 small nucleolar RNAs (snoRNAs) using high-throughput quantitative PCR. We demonstrate that snoRNA expression analysis may be useful in both the diagnosis of some subtypes of PTCL and the prognostication of both PTCL-not otherwise specified (PTCL-NOS; n = 26) and angio-immunoblastic T-cell lymphoma (AITL; n = 46) patients treated with chemotherapy. Like miRNAs, snoRNAs are globally down-regulated in tumor cells compared with their normal counterparts. In the present study, the snoRNA signature was robust enough to differentiate anaplastic large cell lymphoma (n = 32) from other PTCLs. For PTCL-NOS and AITL, we obtained 2 distinct prognostic signatures with a reduced set of 3 genes. Of particular interest was the prognostic value of HBII-239 snoRNA, which was significantly over-expressed in cases of AITL and PTCL-NOS that had favorable outcomes. Our results suggest that snoRNA expression profiles may have a diagnostic and prognostic significance for PTCL, offering new tools for patient care and follow-up.

CD30-positive peripheral T-cell lymphomas share molecular and phenotypic features.

Peripheral T-cell lymphoma, not otherwise specified is a heterogeneous group of aggressive neoplasms with indistinct borders. By gene expression profiling we previously reported unsupervised clusters of peripheral T-cell lymphomas, not otherwise specified correlating with CD30 expression. In this work we extended the analysis of peripheral T-cell lymphoma molecular profiles to prototypical CD30(+) peripheral T-cell lymphomas (anaplastic large cell lymphomas), and validated mRNA expression profiles at the protein level. Existing transcriptomic datasets from peripheral T-cell lymphomas, not otherwise specified and anaplastic large cell lymphomas were reanalyzed. Twenty-one markers were selected for immunohistochemical validation on 80 peripheral T-cell lymphoma samples (not otherwise specified, CD30(+) and CD30(-); anaplastic large cell lymphomas, ALK(+) and ALK(-)), and differences between subgroups were assessed. Clinical follow-up was recorded. Compared to CD30(-) tumors, CD30(+) peripheral T-cell lymphomas, not otherwise specified were significantly enriched in ALK(-) anaplastic large cell lymphoma-related genes. By immunohistochemistry, CD30(+) peripheral T-cell lymphomas, not otherwise specified differed significantly from CD30(-) samples [down-regulated expression of T-cell receptor-associated proximal tyrosine kinases (Lck, Fyn, Itk) and of proteins involved in T-cell differentiation/activation (CD69, ICOS, CD52, NFATc2); upregulation of JunB and MUM1], while overlapping with anaplastic large cell lymphomas. CD30(-) peripheral T-cell lymphomas, not otherwise specified tended to have an inferior clinical outcome compared to the CD30(+) subgroups. In conclusion, we show molecular and phenotypic features common to CD30(+) peripheral T-cell lymphomas, and significant differences between CD30(-) and CD30(+) peripheral T-cell lymphomas, not otherwise specified, suggesting that CD30 expression might delineate two biologically distinct subgroups.

Tricky and terrible T-cell tumors: these are thrilling times for testing: molecular pathology of peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) encompass a group of rare and usually clinically aggressive diseases. The classification and diagnosis of these diseases are compounded by their marked pathological heterogeneity and complex clinical features. With the exception of ALK-positive anaplastic large cell lymphoma (ALCL), which is defined on the basis of ALK rearrangements, genetic features play little role in the definition of other disease entities. In recent years, hitherto unrecognized chromosomal translocations have been reported in small subsets of PTCLs, and genome-wide array-based profiling investigations have provided novel insights into their molecular characteristics. This article summarizes the current knowledge on the best-characterized genetic and molecular alterations underlying the pathogenesis of PTCLs, with a focus on recent discoveries, their relevance to disease classification, and their management implications from a diagnostical and therapeutical perspective.