Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma.

Follicular lymphoma (FL) is typically an indolent disease, but 30-40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies and identified 45 recurrently mutated genes in the FL-tFL data set and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D (MLL2), CREBBP, EZH2, BCL2 and MEF2B. Many recurrently mutated genes are involved in epigenetic regulation, the Janus-activated kinase-signal transducer and activator of transcription (STAT) or the nuclear factor-κB pathways, immune surveillance and cell cycle regulation or are TFs involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.

Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

Prognostic value of regulatory T cells, lymphoma-associated macrophages, and MUM-1 expression in follicular lymphoma treated before and after the introduction of monoclonal antibody therapy: a Southwest Oncology Group Study.

BACKGROUND: The purpose was to examine the prognostic impact of features of tumor cells and immune microenvironment in patients with follicular lymphoma treated with and without anti-CD20 monoclonal antibody therapy. PATIENTS AND METHODS: Tissue microarrays were constructed from archived tissue obtained from patients on three sequential Southwest Oncology Group (SWOG) trials for FL. All three trials included anthracycline-based chemotherapy. Anti-CD20 monoclonal antibodies were included for patients in the latter two trials. Immunohistochemistry was used to study the number and distribution of cells staining for forkhead box protein P3 (FOXP3) and lymphoma-associated macrophages (LAMs) and the number of lymphoma cells staining for myeloma-associated antigen-1 (MUM-1). Cox proportional hazards regression was used to evaluate the association between marker expression and overall survival (OS). RESULTS: The number or pattern of infiltrating FOXP3 cells and LAMs did not correlate with OS in sequential SWOG studies for FL. The presence of MUM-1 correlated with lower OS for patients who received monoclonal antibody but not for those treated with chemotherapy alone. CONCLUSIONS: Immune cell composition of lymph nodes did not correlate with OS in this analysis of trials in FL. The mechanism of the observed correlation between MUM-1 expression and adverse prognosis in patients receiving monoclonal antibody therapy requires confirmation.

Array-based DNA methylation profiling in follicular lymphoma.

Quantitative methylation profiling was performed using the Illumina GoldenGate Assay in untreated follicular lymphoma (FL) (164), paired pre- and post-transformation FL (20), benign haematopoietic (24) samples and purified B and T cells from two FL cases. Methylation values allowed separation of untreated FL samples from controls with one exception, based primarily on tumour-specific gains of methylation typically occurring within CpG islands. Genes that are targets for epigenetic repression in stem cells by Polycomb Repressor Complex 2 were significantly over-represented among hypermethylated genes. Methylation profiles were conserved in sequential FL and t-FL biopsies, suggesting that widespread methylation represents an early event in lymphomagenesis and may not contribute substantially to transformation. A significant (P<0.05) correlation between FL methylation values and reduced gene expression was shown for up to 28% of loci. Methylation changes occurred predominantly in B cells with variability in the amount of non-malignant tissue between samples preventing conclusive correlation with survival. This represents an important caveat in attributing prognostic relevance to methylation and future studies in cancer will optimally require purified tumour populations to address the impact of methylation on clinical outcome.

Stromal gene signatures in large-B-cell lymphomas.

BACKGROUND: The addition of rituximab to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or R-CHOP, has significantly improved the survival of patients with diffuse large-B-cell lymphoma. Whether gene-expression signatures correlate with survival after treatment of diffuse large-B-cell lymphoma is unclear. METHODS: We profiled gene expression in pretreatment biopsy specimens from 181 patients with diffuse large-B-cell lymphoma who received CHOP and 233 patients with this disease who received R-CHOP. A multivariate gene-expression-based survival-predictor model derived from a training group was tested in a validation group. RESULTS: A multivariate model created from three gene-expression signatures--termed "germinal-center B-cell," "stromal-1," and "stromal-2"--predicted survival both in patients who received CHOP and patients who received R-CHOP. The prognostically favorable stromal-1 signature reflected extracellular-matrix deposition and histiocytic infiltration. By contrast, the prognostically unfavorable stromal-2 signature reflected tumor blood-vessel density. CONCLUSIONS: Survival after treatment of diffuse large-B-cell lymphoma is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment.

FLT3 K663Q is a novel AML-associated oncogenic kinase: Determination of biochemical properties and sensitivity to Sunitinib (SU11248).

Somatic mutations of FLT3 resulting in constitutive kinase activation are the most common acquired genomic abnormality found in acute myeloid leukemia (AML). The majority of these mutations are internal tandem duplications (ITD) of the juxtamembrane region (JM). In addition, a minority of cases of AML are associated with mutation of the FLT3 activation loop (AL), typically involving codons D835 and/or I836. We hypothesized that other novel mutations of FLT3 could also contribute to leukemogenesis. We genotyped 109 cases of AML and identified two novel gain-of-function mutations. The first mutation, N841 H, is similar to previously described mutations involving amino-acid substitutions of codon 841. The other novel mutation, FLT3 K663Q, is the first AML-associated gain-of-function mutation located outside the JM and AL domains. Of note, this mutation was potently inhibited by Sunitinib (SU11248), a previously described FLT3 kinase inhibitor. Sunitinib reduced the proliferation and induced apoptosis of transformed Ba/F3 cells expressing FLT3 K663Q. The potency of Sunitinib against FLT3 K663Q was similar to its potency against FLT3 ITD mutations. We conclude that FLT3 mutations in AML can involve novel regions of the TK1. Future studies are needed to define the incidence and prognostic significance of FLT3 mutations outside the well-established JM and AL regions.

Demonstration of Philadelphia chromosome negative abnormal clones in patients with chronic myelogenous leukemia during major cytogenetic responses induced by imatinib mesylate.

Imatinib mesylate, an Abl-specific kinase inhibitor, produces sustained complete hematologic responses (CHR) and major cytogenetic responses (MCR) in chronic myeloid leukemia (CML) patients, but long-term outcomes in these patients are not yet known. This article reports the identification of clonal abnormalities in cells lacking detectable Philadelphia (Ph) chromosome/BCR-ABL rearrangements from seven patients with chronic- or accelerated-phase CML, who were treated with imatinib. All seven patients were refractory or intolerant to interferon therapy. Six of seven patients demonstrated MCR and one patient, who had a cryptic translocation, achieved low-level positivity (2.5%) for BCR-ABL by fluorescence in situ hybridization. The median duration of imatinib treatment before the identification of cytogenetic abnormalities in BCR-ABL-negative cells was 13 months. The most common cytogenetic abnormality was trisomy 8, documented in three patients. All patients had varying degrees of dysplastic morphologic abnormalities. One patient exhibited increased numbers of marrow blasts, yet consistently demonstrated no Ph-positive metaphases and the absence of morphologic features of CML. The presence of clonal abnormalities in Ph-negative cells of imatinib-treated CML patients with MCR and CHR highlights the importance of routine metaphase cytogenetic testing and long-term follow-up of all imatinib-treated patients.

Evaluation of T cell receptor testing in lymphoid neoplasms: results of a multicenter study of 29 extracted DNA and paraffin-embedded samples.

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

The Burkitt-like lymphomas: a Southwest Oncology Group study delineating phenotypic, genotypic, and clinical features.

The Revised European-American Lymphoma classification gives Burkitt-like lymphoma (BLL) provisional status, leaving unresolved the differential diagnosis with Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). This study compared the biologic features of adult BLL and DLBCL. The phenotypic distinction between BLL and DLBCL was determined by immunohistochemical staining of frozen tissue from 13 patients with BLL and 55 patients with DLBCL by using an extensive antibody panel including Ki-67, CD10, CD11a/lymphocyte function-associated antigen 1alpha (LFA-1alpha), CD18/LFA-1beta, CD58/LFA-3, and CD54/intercellular adhesion molecule, CD8 for tumor-infiltrating cytotoxic T cells (T-TILs), CD44 homing receptor, and p53 and Bcl-2 oncogenic proteins. Compared with DLBCL, BLL had a higher proliferative rate (mean Ki-67, 88% versus 53%), greater expression of CD10 and p53 antigens, and decreased expression of Bcl-2. BLL cases had a consistent absence of one or more cell adhesion molecules (92% versus 27%), low T-TIL numbers, and absence of CD44 homing receptor (92% versus 14%). The t(8;14) translocation was identified in 80% of BLL cases, but no patients with BLL had the t(14;18) translocation. In a 10-year analysis, median survival of patients with BLL was 1.2 years, and that of patients with DLBCL was 2.5 years. Although the proportion of patients cured was similar in the 2 groups, BLL patients had an increased risk of early death. We conclude that BLL can be recognized by its combined morphologic and phenotypic features and that it represents a high-grade lymphoma much closer to BL than DLBCL. Retention of the BLL category or inclusion of BLL as a variant of BL is biologically and clinically more appropriate than absorbing the category of BLL into DLBCL. (Blood. 2001;97:3713-3720)

Nodular sclerosing Hodgkin disease with primary manifestation in the orbit.

Hodgkin disease with primary manifestation in the orbit is extremely rare, and even when suspected can be very difficult to diagnose. Its clinical and histological presentation can be nearly impossible to differentiate from that of a benign inflammatory process, and it is necessary to utilize immunohistochemical techniques to confirm the diagnosis. This article focuses on a case of nodular sclerosing Hodgkin disease with initial manifestation in the orbit. A comparison of the clinical, histological, and immunohistochemical presentations associated with both Hodgkin disease and benign inflammation is discussed. A brief review of the immunohistochemistry specific for Hodgkin disease is also provided.

Mistaken diagnoses of Hodgkin's disease.

This article reviews the frequency of and general types of diagnostic errors in Hodgkin's disease (HD) over the past several decades, discusses the most common diagnostic errors in the four histologic subtypes of HD today, and describes some of the clinical and pathologic features that may aid in avoiding a mistaken diagnosis of HD.

Absence of Epstein-Barr virus in lymphomatoid papulosis. An immunohistochemical and in situ hybridization study.

BACKGROUND AND DESIGN: Lymphomatoid papulosis (LyP) and cutaneous Hodgkin's disease share many clinical, histopathologic, and immunohistochemical features. Epstein-Barr virus (EBV) has been implicated in the pathogenesis of several lymphoid malignancies, including Hodgkin's disease. Given the similarities between LyP and Hodgkin's disease, we asked if EBV could be detected in lesions of LyP. We examined 31 specimens of LyP that were obtained from 24 patients for evidence of EBV by in situ hybridization to EBER1 transcripts and for immunohistochemistry of viral latent membrane protein 1 (LMP1). RESULTS: In no instance there was there any evidence of EBV gene products by either in situ hybridization or immunohistochemistry. CONCLUSIONS: The absence of EBV in LyP suggests that this virus is not operative in the pathogenesis of LyP. Furthermore, it suggests that LyP and Hodgkin's disease may not share the same molecular mechanisms despite their phenotypic similarities.

Detection of myeloperoxidase gene expression in minimally differentiated acute myelogenous leukemia (AML-M0) using in situ hybridization.

Acute leukemias containing > 3% myeloperoxidase (MPO)-positive blast cells, as detected cytochemically, are considered to be myelogenous in origin, regardless of the immunophenotypic markers expressed. Conversely, acute leukemias that express only myeloid antigens are also considered to be acute myelogenous leukemia (AML), even in the absence of MPO. These MPO-negative AMLs, designated AML-M0 in the FAB classification, currently require either immunophenotypic or electron microscopic studies for identification. To examine the association of MPO and myeloid antigen expression in AML, particularly at the early stages of myeloid cell differentiation, we have used in situ hybridization (ISH) to evaluate MPO gene expression in myeloid leukemia cell lines and a variety of well-characterized acute leukemias, including six cases of AML-M0. Strong positivity for MPO mRNA was detected in the myeloid leukemia cell line HL-60 and in 22 of 27 AMLs (three AML-M0, four AML-M1, eight AML-M2, five AML-M4, two AML-M5a). No MPO gene expression was detected in three AML-M0, one AML-M5a, one AML-M7, 5 acute lymphoblastic leukemia, the lymphoid cell lines Molt-4 and Namalwa, or in the early myeloid cell lines KG-1 and KG-1a. Ultrastructural studies for MPO activity were performed on four AML-M0; one leukemia showed both gene expression and cytochemical activity, whereas two others contained neither MPO transcripts nor enzyme. Weak MPO gene expression was evident in one AML-M0 that was negative for enzymatic activity by electron microscopy. These studies show MPO gene expression can be detected by ISH in about half of AML-M0, supporting their presumed myelocytic derivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Prognostic significance of the Ki-67-associated proliferative antigen in aggressive non-Hodgkin's lymphomas: a prospective Southwest Oncology Group trial.

The growth fraction of tumors from patients with non-Hodgkin's lymphomas (NHL) has been shown to correlate with survival in retrospective studies. The growth fraction can be evaluated using immunohistochemical techniques employing the Ki-67 monoclonal antibody (MoAb) that marks a nuclear protein present in cycling cells. The purpose of this study was to evaluate the clinical utility of the Ki-67 MoAb for predicting survival. Using a prospective trial design in a multi-institutional cooperative trials group, the proliferative index, clinical outcome, and statistical correlations were independently assessed for previously untreated patients with advanced stages of intermediate- and high-grade histologies of NHL treated on Southwest Oncology Group study (SWOG 8516, Intergroup 0067). The proportion of Ki-67-positive cells was determined on snap-frozen thin tissue sections. A proliferative index of 80% or greater, as determined from prior retrospective studies, identified a group of patients (18%) who had a poor outcome. Overall survival was significantly reduced in these patients with a high Ki-67-associated proliferative index compared with those with a low proliferative index (P = .001). One-year survival estimates were 82% (low proliferative index) versus 18% (high proliferative index). A multivariate regression analysis incorporating commonly used clinical prognostic features confirmed the independent effect of proliferation on survival (relative risk estimate 5.9; 95% confidence interval, 2.2, 16.1). The Ki-67 MoAb identifies a group of patients with rapidly fatal NHL for whom currently available chemotherapy is inadequate.

Atypical human T-cell lymphotropic virus type-I-associated T-cell lymphoma in a low-prevalence Alaska Native population. Implications for disease surveillance.

An atypical case of adult T-cell leukemia/lymphoma (ATL) associated with human T-cell lymphotropic virus type I (HTLV-I) occurred in a 46-year-old Inupiat Eskimo man with no behavioral risk factors for HTLV-I infection. The case was characterized by lack of atypical circulating lymphocytes, hypercalcemia, and opportunistic infections; and by complete remission of the initial renal parenchymal lymphoma. The lymphoma cells had a helper T-cell (CD4) immunophenotype. Serum antibodies to HTLV I/II, detected by Western immunoblot, were identified in specimens collected 31 months before the onset of illness, at the time of diagnosis, and up to 37 months later, shortly before the patient's death. Polymerase chain reaction was used to identify HTLV-I DNA in peripheral blood mononuclear cells and in lymphoma in involved skin. Clinicians should be alert to sporadic cases of both atypical and classic ATL, even in populations in which the prevalence of HTLV-I infection is low.

Immunophenotypic and genotypic analysis in cutaneous lymphoid hyperplasias.

BACKGROUND: The clinical utility of immunophenotyping and Southern blot analysis in the evaluation of patients with cutaneous lymphoid hyperplasia (CLH) is controversial. OBJECTIVE: Our purpose was to determine whether adjunctive immunophenotyping and Southern blot analysis are of diagnostic and prognostic value in patients with CLH. METHODS: Immunophenotyping was performed on skin biopsy specimens from 26 patients with a routine histologic diagnosis of CLH. Southern blot analysis for immunoglobulin (Ig) gene rearrangements was done on 13 of 26 cases. RESULTS: Twenty-four of 26 patients had polyclonal CLH on immunophenotyping: 2 of 26 had monoclonal lymphoma. Two of 11 patients with polyclonal CLH studied by Southern blot analysis had clonal Ig gene rearrangements. In both, lymphoma developed within 1 to 6 years; comparison of CLH and malignant lymphoma demonstrated overlapping and different clonal bands. Two additional patients with polyclonal CLH developed lymphoma. No clonal gene rearrangements were detected in the CLH or lymphoma from one; the other was not studied. CONCLUSION: Immunophenotyping will identify some patients with lymphoma with nondiagnostic histologic features. Southern blot analysis will predict some patients with polyclonal CLH in whom malignant lymphoma will develop and who may benefit from definitive therapy.

Evolution of B-cell lymphoma from pseudolymphoma. A multidisciplinary approach using histology, immunohistochemistry, and Southern blot analysis.

Cutaneous B-cell lymphomas are rare neoplasms that can present as lesions involving solely the skin or develop in association with a systemic lymphoma. Histologically they are often difficult to differentiate from pseudolymphomas, and the use of immunohistochemistry may be necessary to correctly classify them. We report a study of multiple skin lesions in a patient who initially presented with multiple pseudolymphomas, apparently associated with an immune response to the dye of his tattoos. Over a period of 4 years his skin lesions evolved from histologically benign and immunologically polyclonal pseudolymphomas to a histologically malignant and immunologically monoclonal B-cell large cell lymphoma. Genotypic analysis with a probe for the heavy-chain immunoglobulin gene demonstrated the presence of clonal B-cell populations in all of the pseudolymphoma biopsy samples as well as in the subsequent lymphoma tissue samples, with a pattern of clonal bands suggestive of evolution of the B-cell clones. These findings suggest that the development of B-cell lymphoma in this patient was related to a persistent abnormal immune response to the chronic antigenic stimulus of the dye of the tattoo. The presence of clonal B-cell populations in pseudolymphoma by Southern blot analysis may be useful in predicting those patients who will subsequently develop overt lymphoma.

T-cell lymphoma involving subcutaneous tissue. A clinicopathologic entity commonly associated with hemophagocytic syndrome.

Eight cases of T-cell lymphoma localized primarily to the subcutaneous adipose tissue are described, five of which were referred in consultation with a benign diagnosis having been made or suggested. All patients presented with 1-12-cm-diameter subcutaneous nodules, which preferentially involved the extremities in six individuals. Histologically, the lesions were reminiscent of panniculitis and were composed of a mixture of small and large atypical lymphoid cells (large cells predominated in four cases) infiltrating between adipocytes. Focally, sheets of tumor cells were found. Karyorrhexis, fat necrosis, and benign histiocytes were present in all cases. Involvement of small blood vessels was found in seven cases, but the infiltrates were not primarily angiocentric, and angiodestruction was minimal or absent. Immunophenotypic analysis (paraffin or frozen sections) in all cases showed that the atypical cells were of T-cell phenotype. Frozen-section studies demonstrated a mature T-cell phenotype with evidence of pan-T-cell antigen loss in two of five lesions. Genotypic analysis demonstrated a rearrangement of the T-cell receptor beta-chain gene in one (possibly two) biopsies of three cases studied. All patients had some evidence of hemophagocytosis during their clinical course. Six patients developed a florid hemophagocytic syndrome, fatal in five patients. Autopsies were done in all of the expired patients, and all had residual subcutaneous lymphoma and a hemophagocytic syndrome. Dissemination to nonsubcutaneous sites did not occur. Three patients are currently alive without evidence of lymphoma after aggressive chemotherapy (mean follow-up, 12 months). These results suggest that T-cell lymphomas that are primarily localized to the subcutaneous tissue may represent a distinct clinicopathologic entity. Initial biopsy findings may be misinterpreted as benign. A hemophagocytic syndrome commonly supervenes that may be secondary to lymphokine production by the malignant cells or related to the destruction of normal cells at subcutaneous sites.

Transient myeloproliferative disorder of the Down type in the normal newborn.

Two infants with congenital nonlymphoblastic leukemia were discovered to have mosaicism for trisomy 21. Both infants achieved durable spontaneous remissions. Trisomy was apparently restricted to the leukemic clone and could be detected in neither phytohemagglutinin-stimulated peripheral blood cells or bone marrow in either patient nor in myeloid progenitor cells from the second patient after resolution of the transient myeloproliferative disorder. We conclude that spontaneous remission of congenital leukemia is not confined to infants with partial or complete systemic trisomy 21 but can occur in genetically normal newborns whose leukemic cells contain a third chromosome 21.