Eva Geissinger

Autologous Stem-Cell Transplantation As First-Line Therapy in Peripheral T-Cell Lymphomas: Results of a Prospective Multicenter Study

PURPOSE Peripheral T-cell lymphomas (PTCLs) are rare malignancies with poor outcome after conventional chemotherapy. The role of myeloablative therapy and autologous stem-cell transplantation (autoSCT) is still unclear. Therefore, we initiated the first prospective multicenter study on upfront autoSCT in PTCL and recently reported good feasibility and efficacy of this approach. Here, we present the final analysis of the study. PATIENTS AND METHODS The treatment regimen consisted of four to six cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone followed by mobilizing therapy with either the dexamethasone, carmustine, melphalan, etoposide, and cytarabine protocol or the etoposide, methylprednisolone, cytarabine, and cisplatin protocol and stem-cell collection. Patients in complete remission (CR) or partial remission (PR) underwent myeloablative chemoradiotherapy (fractionated total-body irradiation and high-dose cyclophosphamide) and autoSCT. RESULTS From June 2000 to April 2006, 83 patients were enrolled onto the study. Main subgroups were PTCL not specified (n = 32) and angioimmunoblastic T-cell lymphoma (n = 27). Fifty-five (66%) of the 83 patients received transplantation. The main reason for not receiving autoSCT was progressive disease. In an intent-to-treat analysis, the overall response rate after myeloablative therapy was 66% (56% CR and 8% PR). With a median follow-up time of 33 months, 43 patients are alive; the estimated 3-year overall and disease-free survival rates for patients in CR (calculated from CR to the date of relapse) and 3-year progression-free survival rate were 48%, 53%, and 36%, respectively. CONCLUSION The results of this prospective study suggest a substantial impact on outcome for upfront autoSCT in PTCL and should be further evaluated in randomized trials. Pretransplantation treatment needs to be improved to increase the transplantation rate.

Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of neoplasms with generally poor clinical outcome. Currently 50% of PTCL cases are not classifiable: PTCL-not otherwise specified (NOS). Gene-expression profiles on 372 PTCL cases were analyzed and robust molecular classifiers and oncogenic pathways that reflect the pathobiology of tumor cells and their microenvironment were identified for major PTCL-entities, including 114 angioimmunoblastic T-cell lymphoma (AITL), 31 anaplastic lymphoma kinase (ALK)-positive and 48 ALK-negative anaplastic large cell lymphoma, 14 adult T-cell leukemia/lymphoma and 44 extranodal NK/T-cell lymphoma that were further separated into NK-cell and gdT-cell lymphomas. Thirty-seven percent of morphologically diagnosed PTCL-NOS cases were reclassified into other specific subtypes by molecular signatures. Reexamination, immunohistochemistry, and IDH2 mutation analysis in reclassified cases supported the validity of the reclassification. Two major molecular subgroups can be identified in the remaining PTCL-NOS cases characterized by high expression of either GATA3 (33%; 40/121) or TBX21 (49%; 59/121). The GATA3 subgroup was significantly associated with poor overall survival (P = .01). High expression of cytotoxic gene-signature within the TBX21 subgroup also showed poor clinical outcome (P = .05). In AITL, high expression of several signatures associated with the tumor microenvironment was significantly associated with outcome. A combined prognostic score was predictive of survival in an independent cohort (P = .004).

Gene Expression Profiling Uncovers Molecular Classifiers for the Recognition of Anaplastic Large-Cell Lymphoma Within Peripheral T-Cell Neoplasms

PURPOSE To unravel the regulatory network underlying nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) -mediated lymphomagenesis of anaplastic large-cell lymphoma (ALCL) and to discover diagnostic genomic classifiers for the recognition of patients with ALK-positive and ALK-negative ALCL among T-cell non-Hodgkins lymphoma (T-NHL). PATIENTS AND METHODS The transcriptome of NPM-ALK-positive ALCL cell lines was characterized by silencing the expression of ALK or STAT3, a major effector of ALK oncogenic activity. Gene expression profiling (GEP) was performed in a series of systemic primary T-NHL (n = 70), including a set of ALK-positive and ALK-negative ALCL (n = 36). Genomic classifiers for ALK-positive and ALK-negative ALCL were generated by prediction analyses and validated by quantitative reverse-transcriptase polymerase chain reaction and/or immunohistochemistry. RESULTS In ALCL cell lines, two thirds of ALK-regulated genes were concordantly dependent on STAT3 expression. GEP of systemic primary T-NHL significantly clustered ALK-positive ALCL samples in a separate subgroup, underscoring the relevance of in vitro ALK/STAT3 signatures. A set of genomic classifiers for ALK-positive ALCL and for ALCL were identified by prediction analyses. These gene clusters were instrumental for the distinction of ALK-negative ALCL from peripheral T-cell lymphomas not otherwise specified (PTCLs-NOS) and angioimmunoblastic lymphomas. CONCLUSION We proved that experimentally controlled GEP in ALCL cell lines represents a powerful tool to identify meaningful signaling networks for the recognition of systemic primary T-NHL. The identification of a molecular signature specific for ALCL suggests that these T-NHLs may represent a unique entity discernible from other PTCLs, and that a restricted number of genes can be instrumental for clinical stratification and, possibly, therapy of T-NHL.

Primary cutaneous marginal zone B-cell lymphoma: a molecular and clinicopathological study of cases from Asia, Germany, and the United States

Primary cutaneous marginal zone B-cell lymphoma is considered the cutaneous counterpart of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Although its molecular pathogenesis is currently unknown, an etiological link with Borrelia burgdorferi infection has been identified in European, but not in American or Asian cases. To better understand the pathogenesis and the geographical differences of cutaneous marginal zone B-cell lymphoma, 60 cases from the East Asia, Germany, and the United States at their initial presentation were subjected to the following analyses; (1) clinicopathological comparison between the geographical regions, (2) detection of B. burgdorferi DNA, (3) detection of the API2–MALT1 fusion transcript, a gene alteration specific to mucosa-associated lymphoid tissue lymphoma, and (4) inactivation of tumor suppressor genes (death-associated protein kinase (DAPK), p16INK4a, p14ARF, MGMT, TIMP3, CDH1, and RARB) by hypermethylation of the CpG islands. Cases from the three geographical regions showed similar clinicopathological features. However, moderate/marked tissue eosinophilia was found in 9/25 Asian cases, but only 1/23 German cases (P=0.011) and 0/12 American cases (P=0.015). All 60 cases were negative for either Borrelia DNA or API2–MALT1 fusion. Tumors from the three regions were highly methylated for DAPK (38–50% of the cases, mean 43%) and p16INK4a (42–70%, mean 49%), and the positivities were significantly higher than those of nonneoplastic skin (8%, P=0.0010 and 14%, P=0.0032, respectively). Methylation of these genes had no significant association with progressive features of the tumor. Primary cutaneous marginal zone B-cell lymphomas from the three geographical regions have common clinicopathological features, however, moderate/marked tissue eosinophilia is a feature found almost exclusively in Asian cases. Borrelia infection and API2–MALT1 fusion are not significant in this tumor. Methylation of DAPK and p16INK4a genes is a frequent event in this lymphoma at its initial presentation, but may not be associated with tumor progression.

Nodal peripheral T-cell lymphomas and, in particular, their lymphoepithelioid (Lennert's) variant are often derived from CD8(+) cytotoxic T-cells.

Nodal peripheral T-cell lymphomas are not well understood, and most of them are classified in the “not otherwise specified group” (PTCL-NOS). Data on their normal cellular derivation are ambiguous. Most peripheral T-cell lymphomas are composed of tumor cells and a (sometimes dominant) reactive background, which also includes resting and activated T-lymphocytes. We defined the phenotype of the tumor cells in 101 PTCL-NOS based on their cytological atypia and using immunohistochemical double stains on paraffin sections with CD4/Ki67 and CD8/Ki67. The results were correlated to clinical presentation and outcome. Lineage could be defined in 98 cases (97%). Tumor cells were CD4+ in 43 cases and CD8+ in 38. These presented at a younger age but a higher clinical stage compared with the CD4+ lymphomas. In 15 cases, the atypical cells were CD4−CD8−; two cases were CD4+CD8+. Of 17 lymphoepithelioid (Lennert’s) lymphomas, 15 expressed CD8, one each was CD4+ and CD4−CD8−.

Primary cutaneous marginal zone lymphomas with plasmacytic differentiation show frequent IgG4 expression.

The expression of IgG4 in malignant B-cell lymphomas has only partially been studied. Recent reports described single cases of marginal zone lymphomas arising in the ocular adnexae that express IgG4. Moreover, a subset of dura-associated marginal zone lymphomas appear to express IgG4 as well. We investigated IgG4 expression in a more systematic manner in a large cohort of marginal zone lymphoma specimens derived from the archive of our institute. Overall, we examined 169 marginal zone lymphomas of various primary sites that displayed a distinct plasmacytic differentiation and light chain restriction, allowing for a detailed investigation of the immunoglobulin heavy chain expression in these tumors by immunohistochemistry. Unexpectedly, primary cutaneous marginal zone lymphomas showed frequent IgG4 expression. Although only 1 out of 120 noncutaneous marginal zone lymphomas, located in the ocular adnexae, expressed IgG4, 19 of 49 (39%) primary cutaneous marginal zone lymphomas showed this feature, constituting the highest expression rate of IgG4 reported to date in any B-cell lymphoma. None of the IgG4-positive cutaneous marginal zone lymphomas with available clinical data showed evidence of a preexisting systemic IgG4-related disease, suggesting a localized immunologic IgG4-driven pathogenetic process at early stages of the disease. IgG4-positive and IgG4-negative primary cutaneous marginal zone lymphomas did not significantly differ in architectural features of the infiltrate or the composition of the reactive T-cell infiltrate as determined by analysis of T-cell content, CD4/CD8 ratio, and content of FOXP3- and PD1-positive T cells. Although the pathogenetic role of IgG4 expression in a significant subset of primary cutaneous marginal zone lymphomas with plasmacytic differentiation remains unclear at present, the demonstration of IgG4 expression in a marginal zone lymphoma involving the skin might be a helpful clue in the routine diagnostic setting, as these tumors will almost invariably be of primary cutaneous origin with an extremely low risk of spread to noncutaneous sites and an excellent prognosis.

WTX inactivation is a frequent, but late event in Wilms tumors without apparent clinical impact

Wilms tumor (WT) is one of the most common solid tumors in childhood. Mutations in WT1 and CTNNB1 are well established as causal alterations in about 10–15% of cases. Recently, WTX (WT gene on the X‐chromosome), a gene implicated in WNT signaling, has been identified as a third WT gene. We determined the mutation status of WTX, CTNNB1, and WT1 in a large set of 429 tumors. Genomic WTX alterations were identified in 17% of WTs, equally distributed between males and females. Analysis of 104 WT samples for WTX point mutations revealed a rate of only 2%. An additional 11.5% of tumor samples lacked expression of WTX mRNA. These WTX alterations can occur in parallel to WT1 or CTNNB1 mutations. However, we could not find a significant correlation between WTX deletion status or expression level and clinical parameters suggesting that WTX mutations apparently have little direct impact on tumor behavior and presentation. Incomplete deletions of WTX in several cases suggested heterogeneity in tumors. In a small number of cases, we could analyze separate tumor fragments or microdissected regions with different histology of tumors with heterozygous point mutations. Despite complete allele losses at other sites in the genome, we detected varying degrees of WTX mutation. This suggests that WTX alteration is not an essential and early mutation needed to drive tumorigenesis, but rather a later event that may affect only a fraction of cells with unclear clinical relevance.

Activation of STAT5 triggers proliferation and contributes to anti-apoptotic signalling mediated by the oncogenic Xmrk kinase

Extensive studies of primary tumors and tumor derived cell lines revealed that inappropriate activation of specific STATs (particularly of STAT3 and STAT5) occurs with high frequency in a wide variety of human cancers. We reported recently that the melanoma inducing EGFR-related receptor Xmrk specifically induces constitutive activation of STAT5 in fish melanoma cells. However, little is known about the role of STAT5 in solid tumours in general and its function in melanoma in particular. Recent examinations suggest that activated STAT signalling participates in oncogenesis by stimulating cell proliferation and preventing apoptosis. As an initial approach to understanding the consequences of Xmrk induced STAT5 signalling we used the well characterized pro B-cell line Ba/F3 as a sensitive system to analyse mitogenic as well as anti-apoptotic signalling. We identified STAT5 activation as being involved in both growth and survival signalling triggered by the Xmrk kinase possibly due to STAT5 induced expression of pim-1 and bcl-x. We also found a new mechanism of activation of STAT5 by receptor tyrosine kinases, whereby direct interaction of the receptor kinase domain with the STAT protein in a phosphotyrosine independent way led to activation of STAT5 in terms of DNA binding and target gene expression.

Disturbed expression of the T-cell receptor/CD3 complex and associated signaling molecules in CD30+ T-cell lymphoproliferations

Background CD30+ T-cell lymphoproliferations comprise a spectrum of clinically heterogeneous entities, including systemic anaplastic large cell lymphomas (ALK− and ALK+) and primary cutaneous CD30+ T-cell lymphoproliferative disorders. While all these entities are characterized by proliferation of highly atypical, anaplastic CD30+ T cells, the expression of T-cell specific antigens in the tumor cells is not consistently detectable. Design and Methods We evaluated biopsies from 19 patients with primary cutaneous CD30+ lymphoproliferative disorders, 38 with ALK− and 33 with ALK+ systemic anaplastic large cell lymphoma. The biopsies were examined for the expression of T-cell receptorαβ/CD3 complex (CD3γ, δ, ɛ, ζ), transcription factors regulating T-cell receptor expression (ATF1, ATF2, TCF-1, TCF-1α/LEF-1, Ets1), and molecules of T-cell receptor-associated signaling cascades (Lck, ZAP-70, LAT, bcl-10, Carma1, NFATc1, c-Jun, c-Fos, Syk) using immunohistochemistry. Results In comparison to the pattern in 20 peripheral T-cell lymphomas, not otherwise specified, we detected a highly disturbed expression of the T-cell receptor/CD3 complex, TCF-1, TCF-1α/LEF-1, Lck, ZAP-70, LAT, NFATc1, c-Jun, c-Fos and Syk in most of the systemic anaplastic large cell lymphomas. In addition, primary cutaneous CD30+ lymphoproliferative disorders showed such a similar expression pattern to that of systemic anaplastic large cell lymphomas, that none of the markers we investigated can reliably distinguish between these CD30+ T-cell lymphoproliferations. Conclusions Severely altered expression of the T-cell receptor/CD3 complex, T-cell receptor-associated transcription factors and signal transduction molecules is a common characteristic of systemic and cutaneous CD30+ lymphoproliferations, although the clinical behavior of these entities is very different. Since peripheral T-cell lymphomas, not otherwise specified retain the full expression program required for functioning T-cell receptor signaling, the differential expression of a subset of these markers might be of diagnostic utility in distinguishing peripheral T-cell lymphomas, not otherwise specified from the entire group of CD30+ lymphoproliferations.

Mutation analysis of the tyrosine phosphatase PTPN2 in Hodgkin's lymphoma and T-cell non-Hodgkin's lymphoma.

We recently reported deletion of the protein tyrosine phosphatase gene PTPN2 in T-cell acute lymphoblastic leukemia. Functional analyses confirmed that PTPN2 acts as classical tumor suppressor repressing the proliferation of T cells, in part through inhibition of JAK/STAT signaling. We investigated the expression of PTPN2 in leukemia as well as lymphoma cell lines. We identified bi-allelic inactivation of PTPN2 in the Hodgkin’s lymphoma cell line SUP-HD1 which was associated with activation of the JAK/STAT pathway. Subsequent sequence analysis of Hodgkin’s lymphoma and T-cell non-Hodgkin’s lymphoma identified bi-allelic inactivation of PTPN2 in 2 out of 39 cases of peripheral T-cell lymphoma not otherwise specified, but not in Hodgkin’s lymphoma. These results, together with our own data on T-cell acute lymphoblastic leukemia, demonstrate that PTPN2 is a tumor suppressor gene in T-cell malignancies.