Hodgkin And Reed-Sternberg Cells Research Articles

Detection of ABCC1 expression in classical Hodgkin lymphoma is associated with increased risk of treatment failure using standard chemotherapy protocols

BackgroundThe mechanisms responsible for chemoresistance in patients with refractory classical Hodgkin lymphoma (CHL) are unknown. ATP-binding cassette (ABC) transporters confer multidrug resistance in various cancers and ABCC1 overexpression has been shown to contribute to drug resistance in the CHL cell line, KMH2.FindingsWe analyzed for expression of five ABC transporters ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2 using immunohistochemistry in 103 pre-treatment tumor specimens obtained from patients with CHL. All patients received first-line standard chemotherapy with doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine (ABVD) or equivalent regimens. ABCC1 was expressed in Hodgkin and Reed-Sternberg (HRS) cells in 16 of 82 cases (19.5%) and ABCG2 was expressed by HRS cells in 25 of 77 cases (32.5%). All tumors were negative for ABCB1, ABCC2 and ABCC3. ABCC1 expression was associated with refractory disease (p = 0.01) and was marginally associated with poorer failure-free survival (p = 0.06). Multivariate analysis after adjusting for hemoglobin and albumin levels and age showed that patients with CHL with HRS cells positive for ABCC1 had a higher risk of not responding to treatment (HR = 2.84, 95%, CI: 1.12-7.19 p = 0.028).ConclusionsExpression of ABCC1 by HRS cells in CHL patients predicts a higher risk of treatment failure and is marginally associated with poorer failure-free survival using standard frontline chemotherapy regimens.

High frequency of t(14;18) in Hodgkin's lymphoma associated with follicular lymphoma

t(14;18) is a common cytogenetic abnormality in B-cell lymphoma, especially in follicular lymphoma (FL), but is rarely seen in Hodgkin's lymphoma (HL). However, due to the small number of cases, the incidence of t(14;18) in HL associated with FL remains unclear. In this study, we applied chromogenic in situ hybridization and fluorescence in situ hybridization for t(14;18) on paraffin-embedded tissue from four HL associated with FL cases and 11 HL cases without a history of FL for comparison. t(14;18) was present in all of the three successfully-tested HL associated with FL cases and one case without a history of FL. The frequency of t(14;18) was significantly high in HL associated with FL (p = 0.013). All Hodgkin's and Reed-Sternberg (HRS) cells having t(14;18) showed immunoreactivity for BCL2 and were negatively stained for nuclear factor-κB (NF-κB), even in Epstein-Barr virus (EBV)-infected cases. However, HRS cells without t(14;18) showed BCL2 and NF-κB immunoreactivity in 33% and 57% of cases, respectively. There was an inverse correlation between t(14;18) and NF-κB. In conclusion, we assume the incidence of t(14;18) in HL associated with FL is higher than previously believed and BCL2 expression derived from t(14;18) may play a role in the pathogenesis of HL associated with FL.

ETS1 encoding a transcription factor involved in B-cell differentiation is recurrently deleted and down-regulated in classical Hodgkin's lymphoma

The neoplastic B-cell derived Hodgkin and Reed-Sternberg cells (HRS) in classical Hodgkin's lymphoma (cHL) undergo a substantial loss of B-cell identity. It has been suggested that epigenetic mechanisms such as DNA methylation are involved in this cHL-associated reprogramming. [1][1] In a recent

Hodgkin and Reed-Sternberg cells modulate endothelial cell function in classical Hodgkin lymphoma (61.9)

Abstract Growth and survival of neoplastic Hodgkin and Reed-Sternberg cells (H-RS) cells in classical Hodgkin lymphoma (cHL) is dependent on cell-cell contact and soluble mediator signals derived from the inflammatory infiltrate within the lesion. However, the mechanisms underlying recruitment of the inflammatory infiltrate, particularly T cells, into lesional lymph nodes remain unclarified. In this study, we test the hypothesis that H-RS cells secrete soluble mediators to induce endothelial cell (EC) activation to modulate T cell recruitment in cHL. We examined in-vitro interactions, under static and flow conditions, between freshly isolated T cell subsets and EC treated with conditioned medium (CM) harvested from the H-RS cell line, KM-H2. Both CM-stimulated EC and TNFα-stimulated EC support CD4+ naive and memory T-cell interactions under flow conditions. Both CD4+ naive and memory T cell demonstrated enhanced transmigration across CM-stimulated EC monolayers in response to CXCL12 as compared to TNFα-stimulated or unstimulated EC. Our data show that the upregulation of inducible adhesion molecules on CM-stimulated EC is dependent on the NFκB pathway; but the dominant mediator involved is not TNFα. However, blockade of cyclooxygenase activity in KM-H2 cells partially attenuated the efficacy of the CM in activating EC. These findings suggest that H-RS cells secrete soluble mediators that can modulate endothelial cell function and influence recruitment of T cells into the cHL lesion.

Multiple primary enoral soft tissue manifestations of a Hodgkin lymphoma—case report and literature review

Hodgkin lymphoma (HL) are lymphoproliferative neoplasms, histologically comprising of mononuclear and multinucleated Hodgkin and Reed Sternberg cells (HRS). About 4 % of all lymphatic malignancies of the head and neck are HL. The typical disease presents itself as a nodal lesion. Extranodal, enoral soft tissue involvement by HL is very rare. A 73-year-old man with a suspect, ulcerating lesion in the left retromolar region of the mandible was assigned to our hospital. Prior anti-inflammatory therapy has been without success. Subsequently, three biopsies were taken which could only show inflammation. Finally, two biopsies from the left retromolar region and the left inner cheek showed HRS cells with positive expressions of CD15 and CD30 corresponding to a Hodgkin lymphoma. No lymphatic node or bone involvement could be detected. The patient was designated to receive radio-chemotherapy, but died 3 weeks after diagnosis of multiple organ failure. In a literature review, together with this report, nine cases were found concerning primary HL of the oral mucosa. Accordingly, this is the first case of primary multiple extranodal HL in the oral mucosa in absence of lymphatic node involvement. Neither clinical features nor radiological appearances of HL presenting as primary enoral lesions are pathognomonic. Especially when only small biopsy specimens are available, histological diagnosis remains challenging, may lead to a delay in therapy and may result in a significant worse prognosis.

Genetic lesions of the TRAF3 and MAP3K14 genes in classical Hodgkin lymphoma

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) show constitutive activation of nuclear factor (NF)-κB. Several genetic lesions contribute to this deregulated NF-κB activity. Here, we analysed two further NF-κB regulators for genetic lesions, the inhibitory factor TRAF3 and the key signalling component of the alternative NF-κB pathway, MAP3K14 (NIK). Single nucleotide polymorphism (SNP) array analysis of cHL cell lines revealed a uniparental disomy of the long arm of chromosome 14 associated with a biallelic deletion of TRAF3 located on this chromosome in cell line U-HO1. Cloning of the deletion breakpoint showed a 123371 bp deletion. No inactivating mutations of TRAF3 were found in six other cHL cell lines or in microdissected HRS cells from seven cHL. However, in primary cHL samples interphase cytogenetic analyses revealed signal patterns indicating monoallelic deletion of TRAF3 in 3/20 other cases. SNP array analysis revealed a gain of copy number for MAP3K14 in three cHL cell lines. Gains of MAP3K14 were detected in 5/16 cases of primary cHL. In conclusion, in rare instances, HRS cells harbour inactivating mutations of the TRAF3 gene and recurrently show gains of MAP3K14, indicating that more components of NF-κB signalling show genetic lesions in HRS cells than previously known.

Expression of β‐tubulin isotypes in classical Hodgkin's lymphoma

Microtubules consist of heterodimers of α- and β-tubulin. Aberrant expression of specific β-tubulin isotype is associated with resistance to chemotherapy in malignant tumors. In this study, we examined the expressions of β-tubulin isotypes in classical Hodgkin's lymphoma (cHL) by immunohistochemistry. Among the β-tubulin isotypes, class II β-tubulin (31/34, 91%) was most frequently overexpressed in the cytoplasm of almost all Hodgkin's and Reed-Sternberg (HRS) cells, followed by class I β-tubulin (18/34, 53%) and class III β-tubulin (12/34, 35%). Class IV β-tubulin was not expressed in any cHL case. Class I β-tubulin was expressed in the background lymphoid cells as well as the HRS cells. Thus, our results indicate that class II β-tubulin may be very useful for immunohistochemical diagnosis of cHL, and provide valuable information for the potential application of β-tubulin isotype-specific targeting.

Notch is an essential upstream regulator of NF-κB and is relevant for survival of Hodgkin and Reed–Sternberg cells

A major pathogenetic mechanism in classical Hodgkin lymphoma (cHL) is constitutive activation of canonical nuclear factor-κB (NF-κB) p50/p65 signaling, controlling lymphoma cell proliferation and survival. Recently, we demonstrated that aberrant Notch1 activity is a negative regulator of the B cell program in B cell-derived Hodgkin and Reed-Sternberg (HRS) cells. Despite abundant evidence for a complex context-dependent cross talk between Notch and NF-κB signaling in hematopoietic cells, it is unknown whether these pathways interact in HRS cells. Here, we show that Notch-signaling inhibition in HRS cells by the γ-secretase inhibitor (GSI) XII results in decreased alternative p52/RelB NF-κB signaling, interfering with processing of the NF-κB2 gene product p100 into its active form p52. As a result, expression of Notch and NF-κB target genes is reduced, and survival of HRS cells is impaired. Stimulation of alternative NF-κB signaling in the Hodgkin cell line L540cy by activation of the CD30 receptor rescued GSI-mediated loss of cell viability and apoptosis induction. Our data reveal that Notch is an essential upstream regulator of alternative NF-κB signaling and indicate cross talk between both the pathways in HRS cells. Therefore, we suggest that targeting the Notch pathway is a promising therapeutic option in cHL.

Abstract A10: L1236, a Hodgkin and Reed-Sternberg cell line, stimulates monocytes in vitro to differentiate into M2-like macrophages

Abstract The microenvironment of classical Hodgkin lymphoma (cHL) is characterized by a small number of malignant Hodgkin and Reed-Sternberg (HRS) cells embedded in a background rich with infiltrating immune cells. HRS cells and the surrounding cellular infiltrates are believed to produce soluble and membrane-bound factors that are essential for the survival and proliferation of HRS cells. Steidl et al (2010) have reported that an increase in the number of tumor associated macrophages in cHL correlates with poor prognosis. However, little is known about the molecular crosstalk between macrophages and HRS cells in cHL. This study aims to examine in-vitro the influence of HRS cells on monocyte activation and differentiation. Freshly isolated human monocytes were co-cultured with L1236 cells, a HRS cell-line, under standard culture conditions. At the end of 8 days in culture, we examined the influence of HRS cells on monocyte survival and differentiation. In addition, we determined the activation and differentiation status of the co-cultured monocyte by assessing the cells for CD206 and IL-10 expression. The role of IL-4Rα and Macrophage colony-stimulating factor (M-CSF) receptor on HRS cell-induced monocyte differentiation was examined using antibody blocking assay. We also examined consented cHL patient samples for the presence of CD206/CD68 double positive macrophages by immunofluorescent staining. Our data showed that L1236 cells are able to enhance the survival of monocytes in vitro. In the presence of L1236 cells, monocytes differentiated into alternatively activated, M2-like macrophages which expressed CD206 and IL-10. The observed enhanced survival, and differentiation to M2-like macrophages, was mediated by soluble factors secreted by the L1236 cells. The survival of co-cultured monocytes is enhanced by signalling through the M-CSF receptor. In contrast, the differentiation to M2-like macrophages is in part, regulated by signals generated via IL-4Rα. Finally, the presence of CD68/CD206 double positive macrophages was detected in three out of four cHL patient samples. We conclude that HRS cells secrete soluble factors that can in vitro directly modulate monocyte survival and differentiation to a CD206+ M2 macrophage phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A10.

Multicenter phase II study of rituximab-ABVD in classic Hodgkin lymphoma (cHL).

8039 Background: Unlike Hodgkin and Reed Sternberg (HRS) cells, cHL cancer stem cells (CSC) may have a CD20+, memory B cell phenotype and circulate in high frequencies (Jones RJ et al, Blood 2009;113:5920), potentially explaining rituximab’s activity in cHL. Methods: An inter-SPORE study of rituximab-ABVD, to explore the activity of rituximab in cHL using both correlative assays of cHL CSC and clinical outcome, was conducted and accrual completed in 6/2009. Eligibility criteria included stage II, III or IV untreated cHL; age ≥ 18 y; HIV and hepatitis B surface antigen negative; creatinine ≤ 2 mg/dL; bilirubin ≤ 5 mg/dL. Planned treatment was 6 cycles of ABVD with addition of rituximab 375 mg/m2 1 week prior to first ABVD; on days 1, 8, 15, 22 of cycle 1 ABVD; and on day 1 of cycles 2, 4, 6. Dose delays, growth factors and radiation were at physician discretion. Results: Clinical outcomes of all 49 evaluable patients (pts) are presented. Median age was 33 (range 18-66); 26 (53%) were male; stage was IIA in 15 pts, IIB in 5, III-IV in 29 (59%); and 17 (35%) had bulky mediastinal disease. HRS cells were CD20+ in 4 of 47 cases assessed (8.5%). International Prognostic Score (IPS) was 0-1 in 20 pts (41%); 2 in 14 (29%); ≥ 3 in 15 (31%). In urgent cases omission of first rituximab was permitted, and this occurred in 17 pts (35%). Addition of rituximab was generally well tolerated. One death from sepsis occurred during cycle 2. Mean delivered relative dose intensity with 6 cycles was high: 92% for the ABVD combination (96% for AVD, 80% for bleomycin). After 6 cycles, 39 pts (81%) had CR, 6 (12.5%) PR, 1 SD (2%), 2 (4%) progressive disease by 2007 IWG criteria. Only 4 pts received radiation. On centralized review of cycle 2 PET (41 pts), 36 (88%) were PET negative (tumor uptake < liver) and 5 (12%) were PET positive by modified London criteria. Pts with positive interim PET had inferior EFS (P < 0.004). With median 28 month follow-up (range 1.5-48 months), actuarial EFS after treatment initiation is 92% at 2 y and 83% at 3 y (90% at 3 y if IPS ≥ 2). Actuarial overall survival is 98%. Analysis of biomarkers in relation to disease status is ongoing to define cHL CSC biology. Conclusions: Rituximab-ABVD yields encouraging clinical results in cHL, despite a minority of tumors expressing CD20.

Notch and NF-&amp;#954;B Signaling Pathways in the Biology of Classical Hodgkin Lymphoma

Classical Hodgkin lymphoma (cHL) is now recognized as a B-cell-derived lymphoma which is characterized by only about 1% malignant pathognomonic Hodgkin and Reed-Sternberg (HRS) cells and an abundant infiltrate of reactive bystander cells. HRS cells are unique with respect to their lost B-cell-specific gene expression pattern and recurrent genetic lesions. Aberrant activity of Notch signaling, a highly conserved developmental pathway, acts as a negative regulator of the B cell program in HRS cells and thereby contributes to their reprogramming. Another striking feature and the major pathogenetic mechanism in HRS cells is constitutive NF-κB activation. A number of aberrations that contribute to canonical NF-κB activity in HRS cells have been described such as genetic lesions, deregulated receptor signaling and Epstein-Barr virus (EBV) infection. The importance of Notch and NF-κB signaling for cHL pathogenesis, their potential cross-talk and implications for future therapeutic applications are being discussed.

The molecular pathogenesis of Hodgkin lymphoma

Hodgkin lymphoma (HL) is an unusual malignancy in that the tumour cells, the Hodgkin and Reed-Sternberg (HRS) cells, are a minor component of the tumour mass, the bulk of which is a mixed cellular infiltrate. There is compelling evidence that HRS cells are clonal B cells that have lost their B cell phenotype. Mature B cells lacking B cell receptors would normally die by apoptosis, and therefore HRS cells must have developed mechanisms to facilitate survival. The escape from apoptosis and transcriptional reprogramming of HRS cells are interlinked and appear central to disease pathogenesis. Epstein-Barr virus (EBV) is present in the HRS cells of a proportion of cases and expresses genes with a plausible oncogenic function. It is likely that EBV plays a role in reprogramming and survival through dysregulation of several signalling networks and transcription factors, including nuclear factor (NF)-κB. Activation of NF-κB is a feature of all HRS cells and gene mutations affecting this pathway appear common in EBV-negative HL. The HRS cell furthers its own survival by attracting a supportive microenvironment of immune and stromal cells, and suppressing local immune responsiveness. Although many questions remain unanswered, the last two decades have witnessed a considerable increase in our knowledge of this complex disease.

Refined Prognostic Role of CD68-Positive Tumor Macrophages in the Context of the Cellular Micromilieu of Classical Hodgkin Lymphoma

Objective: Classical Hodgkin lymphoma (HL) consists of neoplastic Hodgkin and Reed-Sternberg cells (HRSC) and a nonneoplastic micromilieu that greatly outnumbers the HRSC. Studies on HRSC-related prognostic biomarkers have been unsuccessful, but the microenvironmental composition is of prognostic importance. Recently, the number of CD68-positive macrophages was correlated with adverse survival in HL, and there was a call to validate these results. Methods: We utilized immunohistochemistry to analyze the prognostic importance of the CD68-positive macrophage number compared to other cellular environmental components in an unselected series of 105 HLs in tissue microarrays. Results: Applying a cutoff score of >0.82% tumor macrophages, cases with increased numbers showed worse overall survival (mean 185 months, median 192) compared to cases with lower numbers (mean 285 months, median not reached). Eleven of 62 patients with ≤0.82% tumor macrophages died, compared to 19 of 43 with >0.82% (p < 0.001). The number of macrophages correlated with a low FOXP3-/high granzyme B-/high PD-1-positive micromilieu and patient age, but did not have independent prognostic significance. A combination background score taking into consideration all negative prognostic microenvironmental components (CD68-, PD-1- and granzyme B-positive cells) was of independent prognostic significance (p = 0.002). Conclusion: Increased numbers of CD68-positive tumor macrophages indicate an adverse overall outcome in HL.

Aberrant Cytoplasmic Expression of Cyclin B1 Protein and its Correlation with EBV-LMP1, P53 and P16(INK4A) in Classical Hodgkin Lymphoma in China

The relationships between the expression of cyclin B1, EBV-LMP1, P53 and P16(INK4A) in Chinese classical Hodgkin lymphoma are unknown and need exploring. Samples of classical Hodgkin lymphoma from 60 Chinese patients were analyzed for the expression of cyclin B1, EBV-LMP1, P53 and P16(INK4A) proteins by immunohistochemistry. Cyclin B1 protein was overexpressed in 90.0% (54/60) of this group of classical Hodgkin lymphoma, staining mainly and strongly in cytoplasm but also sparsely and weakly in nucleus of the Hodgkin and Reed-Sternberg (HRS) cells. EBV-LMP1, P53 and P16(INK4A) were overexpressed in 85.0%, 96.7% and 71.7% of Hodgkin lymphoma, respectively. EBV-LMP1, P53 and P16(INK4A) were was noted in the nucleus of HRS cells. Microscopically, cyclin B1 and P53 staining distinguished the HRS cells from the complex background of lymphocytes. Cyclin B1 was positively correlated with EBV-LMP1(P < 0.001) and P53(P < 0.001), but was inversely related to P16(INK4A) (P < 0.05). It is suggested that overexpression of cyclin B1 could play an important role in the evolution of classical Hodgkin lymphoma, and cyclin B1 may be considered as a potential adjunct marker to identify HRS cells in diagnosis and be served as Hodgkin lymphoma-associated antigen in the near future.

Mirna EXPRESSION SIGNATURES as PROGNOSTIC Markers IN ADVANCED Classical HODGKIN LYMPHOMA

AbstractAbstract 4157In spite of the progress in the therapy of advanced stage classic Hodgkin lymphoma (cHL), still a significant proportion of patients fail to respond or relapse after complete remission. MicroRNAs (miRNA) are essential regulators of cell differentiation, emerging as robust predictor and prognostic markers. Specific miRNA signatures from the Hodgkin and Reed Sternberg cells (HRS) cells and their microenvironment have been proposed, but the potential prognostic role of them remains unclear. Here, we investigated whether miRNA signatures can allow to a better understanding of cHL pathogenesis and being applied in patient outcome prognostication.We have used mirRNA gene expression data from 32 samples of advanced cHL patients and 5 cHL derived cell lines (L540, L1236, L428, HDLM2, and KHMZ) to identify specific miRNA profiles from the tumoral cells and their non-tumoral microenvironment. A miRNA signature was identified in the cHL cell lines, probably reflecting functional properties of the HRS, whereas the others informed from properties of the reactive infiltrate. Selected miRNAs were further validated by performing laser capture microdissection using frozen samples in order to look for specific miRNAS preferentially expressed for either HRS cells and their surrounding microenvironment.In addition, logistic regression and Cox analysis allowed us to identify a set of 102 miRNAs differentially expressed (p<0.05) among patients with favourable and unfavourable outcome that included members of the miR-17-92 cluster such us mir-17, mir-92a and mir-92b* and other miRNAs known to be involved in cancerogenesis. A restricted group of 32 miRNAS with better prognostic capacity was further selected to set up a quantitative RT-PCR assay and explore the potential diagnostic application of their expression in an independent series of 96 formalin-fixed paraffin-embedded (FFPE) cHL samples.Bioinformatics target prediction (miRBase and TargetScan) and the web based computational tool DIANA-mirPath, were used in the identification of molecular pathways and target genes potentially regulated by the expression of these mirRNAs. Combined analysis of miRNAs, paired with bioinformatic target prediction, revealed a series of genes and pathways targeted by a small number of miRNAs, including essential pathways for lymphoma survival like MAPK signaling pathway, cell cycle, Jak-STAT signaling pathway and others.In conclusion, specific miRNA signatures for the HRS cells and their microenvironment can be identified in cHL samples, and improve our understanding in cHL pathogenesis. Selected miRNAs can be used in paraffin-embedded tissue for prognosis of advanced cHL cases, and can be incorporated in an RT-PCR assay with potential clinical application in advanced cHL. Disclosures:No relevant conflicts of interest to declare.

High-Dose Cyclophosphamide (Cy), Rituximab, and a Cancer Vaccine for Relapsed Classical Hodgkin's Lymphoma (cHL)

AbstractAbstract 3954Background: Curative but less toxic regimens are needed for relapsed cHL, as autologous stem cell transplantation (auto SCT) carries ∼ 5% mortality risk and nearly 100% risk of infertility. Work at our institution showed that 1) high-dose Cy, equivalent to transplantation doses, can be given safely without SCT and the potential of reinfusing tumor cells, does not preclude any form of salvage SCT, and can spare fertility; and 2) unlike Hodgkin and Reed Sternberg (HRS) cells, cHL cancer stem cells may have a CD20+, memory B cell phenotype and circulate in surprisingly high frequencies (Jones RJ et al, Blood 2009;113:5920), potentially explaining rituximab's activity in this disease. Derived from the K562 cell line, the HL vaccine is a novel, allogeneic (allo), GM-CSF producing vaccine that expresses antigens commonly present in cHL including survivin. In addition, the vaccine expresses the EBV antigens LMP2 and EBNA1, commonly present in EBV+ cHL. Patients and Methods: A phase I/II study of high-dose therapy and immunotherapy for relapsed cHL was developed with curative intent. Eligibility included chemosensitive relapse (chemosensitivity testing was not required for low-volume relapse), no prior SCT, no primary refractory disease, and adequate end-organ function. Chemotherapy consisted of rituximab 375 mg/m2 IV on d 1 and 4, high-dose Cy (50 mg/kg/d IV on d 8–11) with mesna, filgrastim, and rituximab 375 mg/m2 IV weekly for 4 weeks upon platelet recovery. HL vaccine (1 × 10exp8 cells/dose) was administered 1 d after Cy completion and 4, 8, 12, 16, and 24 weeks later. Consolidative radiation was permitted. All treatment was intended to be outpatient. Results: Clinical outcomes of the first 25 pts accrued are described. Median age was 40 (range 18–67) y; 10 were female. Stage was IIB-IV in 14 pts (56%) upon diagnosis, and 9 pts (36%) relapsed with extranodal disease. HRS cells were CD20+ in 4/25 cases (16%), EBV+ in 6/24 evaluable cases (25%). Median time between first-line therapy completion and relapse was 12 (range 2–94) mo; 22 pts had CR or PR and 2 had stable disease to salvage therapy consisting chiefly of ICE or ESHAP (median 2 cycles, range 1–4), and 1 had untested relapse. With median 31 (range 7–53) mo follow-up for all pts, actuarial 1 y and 2 y overall survival is 100%, while actuarial event-free survival is 79% at 1 y and 66% at 2 y following high-dose Cy (Figure). The regimen was well-tolerated, and there was no treatment-related mortality. Eleven admissions occurred, 10 for infections (8 neutropenic, 2 non-neutropenic) which all resolved without sequelae, with 2 other neutropenic fevers managed without admission. One pt developed idiopathic protracted pancytopenia (gradually improving), cardiomyopathy, and chronic kidney disease. Vaccine toxicities were largely limited to mild local skin reactions. Median time to neutrophil recovery after the last dose of Cy was 17 (range 10–26) d; median time to sustained platelets >= 20,000/μL, without transfusion in the preceeding week, was 21 (range 0–46) d, with 5 pts not requiring platelet transfusion. Grade 3 or 4, late-onset neutropenia, presumably rituximab-related, was documented in 11 pts (44%; grade 4 in 5 pts) at a median of 3 (range 1–18) mo following study initiation. In the 7 relapsed pts, median time to relapse was 7 (range 5–16) mo after high-dose Cy; 1 died of disease after auto and allo SCT, 1 died in the course of allo SCT, 1 is alive with disease after allo SCT, 3 are event-free 7 mo - 3 y after allo SCT, and 1 has allo SCT planned. Early stopping rules for safety and efficacy have not been met. One pt had a successful pregnancy 33 mo after high-dose Cy. Conclusion: A novel outpatient regimen incorporating high-dose Cy and rituximab produces encouraging outcomes on preliminary analysis in relapsed cHL pts. Early efficacy appears comparable to that of auto SCT, with potentially less toxicity. Analyses of immunologic endpoints and HL biomarkers are in progress. [Display omitted] Disclosures:Kasamon:Genentech: Research Funding. Off Label Use: Rituximab, cyclophosphamide: off-label use. Cancer vaccine: investigational. Levitsky:HL vaccine: Patents & Royalties. Borrello:HL vaccine: Patents & Royalties. Swinnen:Genentech: Consultancy, Research Funding.

Mechanisms of aberrant GATA3 expression in classical Hodgkin lymphoma and its consequences for the cytokine profile of Hodgkin and Reed/Sternberg cells

AbstractThe transcription factor network in Hodgkin lymphoma (HL) represents a unique composition of proteins found in no other hematopoietic cell. Among these factors, an aberrant expression of the T-cell transcription factor GATA3 is observed in B cell–derived Hodgkin and Reed/Sternberg (HRS) tumor cells. Herein, we elucidate the regulation and function of this factor in HL. We demonstrate binding of NFκB and Notch-1, 2 factors with deregulated activity in HL to GATA3 promoter elements. Interference with NFκB and Notch-1 activity led to decreased GATA3 expression, indicating a dependency of deregulated GATA3 expression on these transcription factors. Down-regulation of GATA3 in HL cell lines demonstrated its role in the regulation of IL-5, IL-13, STAT4, and other genes. A correlation between GATA3 and IL-13 expression was confirmed for HRS cells in HL tissues. Thus, GATA3 shapes the cytokine expression and signaling that is typical of HL. Conclusively, aberrant GATA3 expression in HRS cells is stimulated by the deregulated constitutive activity of NFκB and Notch-1, indicating a complex network of deregulated transcription factors in these cells. GATA3 activity significantly contributes to the typical cytokine secretion of and signaling in HRS cells, which presumably plays an essential role in HL pathogenesis.

Flow cytometric analysis of CD123 is useful for immunophenotyping classical Hodgkin lymphoma

Although the diagnosis of classical Hodgkin lymphoma (CHL) is traditionally made morphologically in tissue sections, it is now possible to diagnose CHL by flow cytometry (FC). In an effort to identify additional antigens on Hodgkin and Reed-Sternberg (HRS) cells that might be useful for immunophenotyping this lymphoma by FC, we examined the expression of CD123 (α chain of the IL-3R) on HRS cells and compared this with the expression of CD123 in other lymph node samples (372 tissue specimens, including 98 reactive and 274 neoplastic cases), using a nine-color FC reagent combination including anti-CD123. CD123 was found to be expressed on the majority of HRS cell populations (59% of 59 CHL lymph nodes examined), rarely on B-cell non-Hodgkin lymphomas (NHLs) (3 of 3 hairy cell leukemia cases, 3 of 29 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) cases, 1 of 55 large B-cell lymphoma cases, but not on 95 other B-cell NHL examined), and in none of the 16 T-cell NHL cases examined. In reactive lymph nodes, CD123 expression was largely limited to plasmacytoid dendritic cells and rare histiocyte populations that can readily be distinguished from HRS populations. As the expression of this antigen is shown to be relatively limited in B- and T-NHL and in reactive lymph node populations, assessment of CD123 expression is useful for supporting the FC diagnosis of CHL.

Rare occurrence of biallelic CYLD gene mutations in classical Hodgkin lymphoma

Survival of the malignant Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is dependent on constitutive activation of the nuclear factor kappaB (NF-kappaB) transcription factor. The deubiquitinating enzyme CYLD is a negative regulator of NF-kappaB and known to function as a tumor suppressor. To determine whether CYLD mutations play a role in cHL pathogenesis, we sequenced the gene in cHL cell lines and microdissected HRS cells obtained from lymph-node biopsies. A biallelic inactivation by mutations was found in the cHL cell-line KM-H2. However, the other seven cHL cell lines analyzed and HRS cells of 10 primary cHL cases did not show any mutations. By interphase cytogenetics, a (sub)clonal biallelic CYLD deletion was observed by interphase cytogenetics in 1 of 29 primary cHL, whereas signal patterns indicating decreased CYLD copy numbers were observed in a total of 10 of 29 primary cases. Our results suggest that biallelic CYLD mutations are rarely involved in cHL pathogenesis. Nevertheless, it is remarkable that KM-H2 cells, besides the CYLD mutations, also carry inactivating mutations in the genes of two other NF-kappaB inhibitors, that is, NFKBIA and TNFAIP3, exemplifying that multiple lesions in regulators of this signaling pathway can likely cooperatively contribute to the strong NF-kappaB activity of these cells.

Does Rituximab Have a Place in Treating Classic Hodgkin Lymphoma?

Monoclonal antibodies targeting surface proteins on the malignant Hodgkin and Reed-Sternberg (HRS) cells are currently under intensive investigation with promising early results. Of particular interest is the CD20 antigen, because it is expressed not only on a small fraction of HRS cells but also on the benign reactive B cells in the microenvironment. The rationale of using rituximab in classic Hodgkin lymphoma (cHL) comprises several points: 1) HRS cells infrequently express CD20; 2) elimination of CD20-positive reactive B cells supporting HRS cells would deprive the malignant cells of survival signals; 3) elimination of reactive B cells may also potentially increase host immune response against HRS cells; 4) HRS stem cells express CD20. Although this rationale has not generally been confirmed in patients, promising results in managing cHL have occurred in early-phase clinical trials of rituximab, including trials of rituximab as a single agent in refractory or recurrent cHL, rituximab plus gemcitabine in refractory or recurrent cHL, and rituximab plus ABVD in newly diagnosed cHL. Based on the results of these trials, several prospective clinical trials using rituximab in the management of advanced-stage cHL and early-stage cHL are ongoing. These trials further clarify the role of rituximab in cHL. Enrollment of patients with this "classic" disease in clinical trials is encouraged.