Effect of light-induced changes in leaf anatomy on intercellular and cellular components of mesophyll resistance for CO2 in Fagus sylvatica.

Mesophyll resistance for CO2 diffusion (rm) is one of the main limitations for photosynthesis and plant growth. Breeding new varieties with lower rm requires knowledge of its distinct components. We tested new method for estimating the relative drawdowns of CO2 concentration (c) across hypostomatous leaves of Fagus sylvatica. This technique yields values of the ratio of the internal CO2 concentrations at the adaxial and abaxial leaf side, cd/cb, the drawdown in the intercellular air space (IAS), and intracellular drawdown between IAS and chloroplast stroma, cc/cbd. The method is based on carbon isotope composition of leaf dry matter and epicuticular wax isolated from upper and lower leaf sides. We investigated leaves from tree-canopy profile to analyse the effects of light and leaf anatomy on the drawdowns and partitioning of rm into its inter- (rIAS) and intracellular (rliq) components. Validity of the new method was tested by independent measurements of rm using conventional isotopic and gas exchange techniques. 73% of investigated leaves had adaxial epicuticular wax enriched in 13C compared to abaxial wax (by 0.50‰ on average), yielding 0.98 and 0.70 for average of cd/cb and cc/cbd, respectively. The rIAS to rliq proportion were 5.5:94.5% in sun-exposed and 14.8:85.2% in shaded leaves. cc dropped to less than half of the atmospheric value in the sunlit and to about two-thirds of it in shaded leaves. This method shows that rIAS is minor but not negligible part of rm and reflects leaf anatomy traits, i.e. leaf mass per area and thickness.

Impact of risk factors on outcomes in early-stage Hodgkin's lymphoma: an analysis of international staging definitions.

BACKGROUND: In early-stage Hodgkin's lymphoma (HL), treatment according to the early favorable or unfavorable subgroup is guided by staging definitions, which differ between various study groups worldwide. We analyzed risk factors used in different international staging systems and their impact on the outcome of early-stage HL patients. PATIENTS AND METHODS: In 1173 early-stage HL patients treated homogenously within the German Hodgkin Study Group (GHSG) trials HD10 and HD11, the impact of three staging systems developed and used by the GHSG, the European Organization for Research and Treatment of Cancer (EORTC), and the National Comprehensive Cancer Network (NCCN) in discriminating risk groups for progression-free survival (PFS) and overall survival (OS) was assessed and the relevance of their single risk factors was investigated. RESULTS: All the three staging systems defined an unfavorable risk group out of early-stage patients of comparable size (56%, 55%, and 57%), having a significantly poorer PFS and OS as compared with the corresponding favorable group; 5-year differences between early favorable and early unfavorable in terms of PFS were 9.4% (HR 2.61, 95% CI 1.74-3.91), 6.7% (HR 2.10, 95% CI 1.41-3.13), and 8.6% (HR 2.14, 95% CI 1.45-3.16) with the GHSG, EORTC, and NCCN definition, respectively. Sensitivity was high for all systems (84%, 79%, and 83%); however, there was a low specificity with high rates of false-positive results (1-specificity 54%, 53%, and 55%, respectively). Models of high sensitivity included risk factors associated with large tumor burden and high tumor activity. Most risk factors for tumor-specific end points were also predictive of OS. CONCLUSIONS: Differentiating between a favorable and an unfavorable risk group has significant impact on PFS and OS in early-stage HL patients in the modern treatment era. Risk-adapted treatment strategies using new risk factors with higher specificity are needed.

An individual patient-data comparison of combined modality therapy and ABVD alone for patients with limited-stage Hodgkin lymphoma.

BACKGROUND: Treatment options for patients with nonbulky stage IA-IIA Hodgkin lymphoma include combined modality therapy (CMT) using doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) plus involved-field radiation therapy (IFRT), and chemotherapy with ABVD alone. There are no mature randomized data comparing ABVD with CMT using modern radiation techniques. PATIENTS AND METHODS: Using German Hodgkin Study Group HD10/HD11 and NCIC Clinical Trials Group HD.6 databases, we identified 588 patients who met mutually inclusive eligibility criteria from the preferred arms of HD10 or 11 (n = 406) and HD.6 (n = 182). We evaluated time to progression (TTP), progression-free (PFS) and overall survival, including in three predefined exploratory subset analyses. RESULTS: With median follow-up of 91 (HD10/11) and 134 (HD.6) months, respective 8-year outcomes were for TTP, 93% versus 87% [hazard ratio (HR) 0.44, 95% confidence interval (CI) 0.24-0.78]; for PFS, 89% versus 86% (HR 0.71, 95% CI 0.42-1.18) and for overall survival, 95% versus 95% (HR 1.09, 95% CI 0.49-2.40). In the exploratory subset analysis including HD10 eligible patients who achieved complete response (CR) or unconfirmed complete response (CRu) after two cycles of ABVD, 8-year PFS was 87% (HD10) versus 95% (HD.6) (HR 2.8; 95% CI 0.64-12.5) and overall survival 96% versus 100%. In contrast, among those without CR/CRu after two cycles of ABVD, 8-year PFS was 88% versus 74% (HR 0.35; 95% CI 0.16-0.79) and overall survival 95% versus 91%, respectively (HR 0.42; 95% CI 0.12-1.44). CONCLUSIONS: In patients with nonbulky stage IA-IIA Hodgkin lymphoma, CMT provides better disease control than ABVD alone, especially among those not achieving complete response after two cycles of ABVD. Within the follow-up duration evaluated, overall survivals were similar. Longer follow-up is required to understand the implications of radiation and chemotherapy-related late effects. CLINICAL TRIALS: The trials included in this analysis were registered at ClinicalTrials.gov: HD10 - NCT00265018, HD11 - NCT00264953, HD.6 - NCT00002561.

Comparing long-term toxicity and efficacy of combined modality treatment including extended- or involved-field radiotherapy in early-stage Hodgkin's lymphoma.

BACKGROUND: To evaluate long-term toxicity and efficacy of a combined modality strategy including extended-field radiotherapy (EF-RT) or involved-field radiotherapy (IF-RT), the German Hodgkin Study Group carried out a follow-up analysis in patients with early unfavorable Hodgkin's lymphoma (HL). PATIENTS AND METHODS: One thousand two hundred and four patients were randomized to four cycles of chemotherapy followed by either 30 Gy EF- or 30 Gy IF-RT (HD8 trial); 532 patients in each treatment arm were eligible. RESULTS: At 10 years, no arm differences were revealed with respect to freedom from treatment failure (FFTF) (79.8% versus 79.7%), progression-free survival (79.8% versus 80.0%), and overall survival (86.4% versus 87.3%). Non-inferiority of IF-RT was demonstrated for the primary end point FFTF (95% confidence interval for hazard ratio 0.72-1.25). Elderly patients had a poorer outcome when treated with EF-RT. So far, 15.0% of patients in arm A and 12.2% in arm B died, mostly due to secondary malignancies (5.3% versus 3.4%) or HL (3.2% versus 3.4%). After EF-RT, there were more secondary malignancies overall (58 versus 45), especially acute myeloid leukemias (11 versus 4). CONCLUSION: Radiotherapy intensity reduction to IF-RT does not result in poorer long-term outcome but is associated with less acute toxicity and might be associated with less secondary malignancies.

Fertility and gonadal function in female survivors after treatment of early unfavorable Hodgkin lymphoma (HL) within the German Hodgkin Study Group HD14 trial.

BACKGROUND: In the HD14 trial, 2×BEACOPPescalated+2×ABVD (2+2) has improved the primary outcome. Compared with 4×ABVD, this benefit might be compromised by more infertility in women. Therefore, we analyzed gonadal function and fertility. PATIENTS AND METHODS: Women≤45 years in ongoing remission at least 1 year after therapy were included. Hormone parameters, menopausal symptoms, measures to preserve fertility, menstrual cycle, pregnancies, and offspring were evaluated. RESULTS: Three hundred and thirty one of 579 women addressed participated (57.2%) and 263 per-protocol treated patients qualified (A=ABVD: 137, B=2+2: 126, mean time after therapy 42 and 43 months, respectively). Regular menstrual cycle after treatment (A: 87%, B: 83%) and time to recovery (≤12 months) were not different. Follicle-stimulating hormone and anti-Muellerian hormone were significantly better in arm A. However, pregnancies after therapy favored arm B (A: 15%, B: 26%, P=0.043) and motherhood rates were equivalent to the German normal population. Multivariate analysis revealed prophylactic use of gonadotropin-releasing hormone (GnRH) analogues as highly significant prognostic factor for preservation of fertility (odds ratio=12.87, P=0.001). Severe menopausal symptoms were frequent in women≥30 years (A: 21%, B: 25%). CONCLUSIONS: Hormonal levels after 2+2 indicate a reduced ovarian reserve. However, 2+2 in combination with GnRH analogues does not compromise fertility within the evaluated observation time.

Impact of first- and second-line treatment for Hodgkin's lymphoma on the incidence of AML/MDS and NHL--experience of the German Hodgkin's Lymphoma Study Group analyzed by a parametric model of carcinogenesis.

BACKGROUND: Using a parametric carcinogenesis model, we disentangle the superimposing effects of primary and relapse therapies of Hodgkin's disease on secondary neoplasias. PATIENTS AND METHODS: We analyze eight randomized trials of the German Hodgkin's lymphoma study group [5357 individuals, 67 secondary acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and 97 secondary non-Hodgkin's lymphoma (NHL)]. Primary therapies were divided into four groups: radiotherapy alone, moderately dosed COPP/ABVD-like chemotherapies for intermediate and advanced stages and BEACOPP escalated. RESULTS: For secondary AML/MDS, the hazards after primary therapies are proportional (maximum at 3.4 years), while the hazard after relapse therapy is more peaked (maximum at 1.8 years). Intermediate and advanced stage chemotherapy resulted in a cumulative risk of 1.5%, while the risk after BEACOPP escalated is higher (4.4%, P = 0.004) and comparable with that after relapse therapy (4.5%). For secondary NHL, there are no differences in cumulative risk between the primary therapies (2.9%), while the risk after relapse therapy is increased (6.6%, P = 0.002). CONCLUSIONS: BEACOPP escalated moderately increases the risk of secondary AML/MDS but not NHL. No differences were found between other chemotherapies of advanced stages and intermediate stages. Secondary AML/MDS occurs faster after relapse treatment than after primary treatment.

Nitrogen depletion enhances endodermal suberization without restricting transporter-mediated root NO3- influx.

Roots vary their permeability to aid radial transport of solutes towards xylem vessels in response to nutritional cues. Nitrogen (N) depletion was previously shown to induce early suberization of endodermal cell walls and reduce hydraulic conductivity of barley roots suggesting reduced apoplastic transport of ions (Armand et al., 2019). Suberization may also limit transcellular ion movement by blocking access to transporters (Barberon et al., 2016). The aim of this study was to confirm that N depletion induced suberization in the roots of barley and demonstrate that this was a specific effect in response to NO3- depletion. Furthermore, in roots with early and enhanced suberization, we assessed their ability for transporter-mediated NO3- influx. N depletion induced lateral root elongation and early and enhanced endodermal suberization of the seminal root of each genotype. Both root to shoot NO3- translocation and net N uptake was half that of plants supplied with steady-state NO3-. Genes with predicted functions in suberin synthesis (HvHORST) and NO3- transport (HvNRT2.2) were induced under N-deplete conditions. N-deplete roots had a higher capacity for high-affinity NO3- influx in early suberized roots than under optimal NO3-. In conclusion, NO3- depletion induced early and enhanced suberization in the roots of barley, however, suberization did not restrict transcellular NO3- transport.

Somatic mutation of the CD95 gene in human B cells as a side-effect of the germinal center reaction.

Somatic hypermutation specifically modifies rearranged immunoglobulin (Ig) genes in germinal center (GC) B cells. However, the bcl-6 gene can also acquire somatic mutations during the GC reaction, indicating that certain non-Ig genes can be targeted by the somatic hypermutation machinery. The CD95 gene, implicated in negative selection of B lymphocytes in GCs, is specifically expressed by GC B cells and was recently identified as a tumor suppressor gene being frequently mutated in (post) GC B cell lymphomas. In this study, the 5' region (5'R) and/or the last exon coding for the death domain (DD) of the CD95 gene were investigated in naive, GC, and memory B cells from seven healthy donors. About 15% of GC and memory, but not naive, B cells carried mutations within the 5'R (mutation frequency 2.5 x 10(-4) per basepair). Mutations within the DD were very rare but could be efficiently selected by inducing CD95-mediated apoptosis: in 22 apoptosis-resistant cells, 12 DD mutations were found. These results indicate that human B cells can acquire somatic mutations of the CD95 gene during the GC reaction, which potentially confers apoptosis resistance and may counteract negative selection through the CD95 pathway.

Relation between Epstein-- Barr viral and cell membrane immunofluorescence in Burkitt tumor cells. II. Comparison of cells and sera from patients with Burkitt's lymphoma and infectious mononucleosis.

Blastoid cell cultures derived from leukocytes of patients in the acute stage of infectious mononucleosis (IM) and harboring Epstein-Barr (EB) virus in at least 1% of the cells were found to possess antigens in their membranes which presently are indistinguishable from those detected in Burkitt's lymphoma (BL) cells by the techniques employed. It was noted that, in the course of IM, antibodies are formed which react in indirect immunofluorescence tests with membrane antigens of live cells from Burkitt tumor lines as well as from IM leukocyte cultures, including an autochthonous line in the case of one patient. Sets of sera from IM patients were tested which included a serum collected weeks to years before onset of illness. In the majority of these the pre-IM serum failed to react in membrane immunofluorescence (MIF) tests with any of several Burkitt tumor cell lines employed, but in some cases the presera reacted with cells of some but not others of the lines. Possible explanations for these discrepant results have been discussed. The antibodies involved in the MIF test are evidently distinct from those responsible for the EBV and heterophile reactions. Maximal MIF activity is attained long after the other two antibodies have reached maximal titers and antibodies to EBV and membrane antigens seem to persist for years whereas the heterophile reaction turns negative within a few months.

Clonal deleterious mutations in the IkappaBalpha gene in the malignant cells in Hodgkin's lymphoma.

Members of the nuclear factor (NF)-kappaB family of transcription factors play a crucial role in cellular activation, immune responses, and oncogenesis. In most cells, they are kept inactive in the cytosol by complex formation with members of the inhibitor of NF-kappaB (IkappaB) family, whose degradation activates NF-kappaB in response to diverse stimuli. In Hodgkin's lymphoma (HL), high constitutive nuclear activity of NF-kappaB is characteristic of the malignant Hodgkin and Reed-Sternberg (H/RS) cells, which occur at low number in a background of nonneoplastic inflammatory cells. In single H/RS cells micromanipulated from histological sections of HL, we detect clonal deleterious somatic mutations in the IkappaBalpha gene in two of three Epstein-Barr virus (EBV)-negative cases but not in two EBV-positive cases (in which a viral oncogene may account for NF-kappaB activation). There was no evidence for IkappaBalpha mutations in two non-HL entities or in normal germinal center B cells. This study establishes deleterious IkappaBalpha mutations as the first recurrent genetic defect found in H/RS cells, indicating a role of IkappaBalpha defects in the pathogenesis of HL and implying that IkappaBalpha is a tumor suppressor gene.

No protection of the ovarian follicle pool with the use of GnRH-analogues or oral contraceptives in young women treated with escalated BEACOPP for advanced-stage Hodgkin lymphoma. Final results of a phase II trial from the German Hodgkin Study Group.

BACKGROUND: The reduction of treatment-related toxic effects is the main goal in the current trials of the German Hodgkin Study Group (GHSG). In this regard, the protection of the ovarian reserve in young women is very important. Therefore, the GHSG investigated the use of gonadotropin-releasing hormone-analogues (GnRH-a) and oral contraceptives (OC) in young women with advanced-stage Hodgkin lymphoma (HL). PATIENTS AND METHODS: Women (18-40 years) were randomly assigned either to receive daily OC or monthly GnRH-a during escalated combination therapy with bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, and prednisone (BEACOPPesc). Hormonal levels were determined at baseline, during therapy, and at follow-up. RESULTS: The study was closed prematurely after an interim analysis of 12 patients in arm A (OC) and 11 in arm B (GnRH-a), 9 and 10 are assessable for the primary end point. Women's median age was 25 years in both arms. The anti-Mullerian hormone level after at least 12 months was reduced in all patients. For the entire study cohort, the respective ovarian follicle preservation rate was 0% (95% confidence interval 0% to 12%). CONCLUSION: We observed no protection of the ovarian reserve with hormonal co-treatment during BEACOPPesc. This result supports efforts of ongoing trials to reduce chemotherapy intensity and toxicity. Alternative strategies for the protection of fertility must be offered to young female HL patients before the start of BEACOPPesc therapy.

Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells.

Cultured cells derived from male patients with Burkitt's lymphoma and harboring herpes-type virus particles were lethally irradiated. These irradiated cells induced normal peripheral leukocytes of female infants to grow within 2 to 4 weeks after mixed cultivation. Cells of a line free of this agent failed to stimulate growth. If either type of cell was cultured separately, it did not survive under the experimental conditions. Herpes-type viral antigen and C-group chromosomal marker previously described in cultured Burkitt cells were found in all of the female cell cultures that were obtained.

Cure of xenografted human tumors by bispecific monoclonal antibodies and human T cells.

Tumor immunotherapy should increase both the number of T cells that kill the tumor and the likelihood that those cells are activated at the tumor site. Bispecific monoclonal antibodies (Bi-mAbs) were designed that bound to a Hodgkin's tumor-associated antigen (CD30) on the tumor and to either CD3 or CD28 on the T cell. Immunodeficient mice were cured of established human tumors when mice were treated with both the CD3-CD30 and the CD28-CD30 Bi-mAbs and then given human peripheral blood lymphocytes that had been incubated with the CD3-CD30 Bi-mAb and cells that expressed CD30. The enrichment of human T cells within the tumor and the fact that established tumors can be cured may indicate in situ activation of both the T cell receptor and the costimulatory pathway.

Assessment of male fertility in patients with Hodgkin's lymphoma treated in the German Hodgkin Study Group (GHSG) clinical trials.

BACKGROUND: Infertility is one of the most significant side-effects in long-term survivors of successfully treated Hodgkin's lymphoma (HL). PATIENTS AND METHODS: The fertility status was assessed in male HL patients enrolled into trials of the German Hodgkin Study Group from 1988 to 2003. RESULTS: In pre-treatment analysis (n = 202), 20% of patients had normozoospermia, 11% azoospermia and 69% had other dyspermia. In post-treatment analysis (n = 112), 64% of patients had azoospermia, 30% other dyspermia and 6% normozoospermia (P < 0.001). Azoospermia was observed in 90% of patients treated with chemotherapy alone, 67% of those treated with combined modality and 11% of those treated with radiotherapy alone (P < 0.001). Azoospermia was more frequent after 4x cyclophosphamide, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, vinblastine, dacarbazine (COPP/ABVD) (91%), 8x bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone (BEACOPP) baseline (93%) and 8x BEACOPP escalated (87%) compared with 2x COPP/ABVD (56%; P = 0.003). There was a statistically significant difference in post-treatment follicle-stimulating hormone (FSH) levels between patients with azoospermia and those with preserved spermatogenesis (P = 0.001). CONCLUSIONS: Depending on the treatment received, male HL patients are at high risk of infertility after treatment. FSH might be used as surrogate parameter for male fertility in future studies.

Unique antigen of cultured Hodgkin's cells. A putative sialyltransferase.

Hodgkin's disease-derived giant cell lines (HD-cells) express high levels of ectosialyltransferase activity presumed to be a galactose-specific lectin recognizing the desialylated 3-fucosyl-N-acetyllactosamine structure (X-hapten). Both the anti-X-hapten monoclonal antibody VIM-D5 and a polyclonal antiserum to another galactose-lectin, the hepatic asialoglycoprotein receptor (HBP), recognize a 55,000-mol wt HD-cell protein (Paietta, E., R. J. Stockert, A. G. Morell, V. Diehl, and P. H. Weirnik. 1986. Proc. Natl. Acad. Sci. USA. 83:3451-3455.) That the expression of the 55,000-mol wt protein is restricted to HD-cells among X-hapten positive cells lines is confirmed in this study. The 55,000-mol wt protein is shown to be present on the cell surface and intracellularly, where an additional immunocrossreactive 150,000-mol wt protein is recognized. Extraction of the 55,000 mol wt protein from HD-cell lysates by affinity chromatography results in the loss of sialyltransferase activity. While evidence for a single protein possessing both the antigenic and the enzymatic activity is not direct, these results suggest that the ectosialyltransferase unique to HD-cells is a 55,000-mol wt membrane glycoprotein possessing the X-hapten oligosaccharide.

L-428 nodular sclerosing Hodgkin's cell secretes a unique transforming growth factor-beta active at physiologic pH.

Nodular sclerosing Hodgkin's disease is characterized by dense collagen fibrosis. Although transforming growth factor-beta (TGF-beta) is an important bifunctional growth factor for fibroblasts and is stored and released by many cells, it requires acidification to pH 2.0-3.0 before it becomes a biologically active growth factor. We show here that the L-428 Hodgkin's cell releases a high molecular weight TGF that competes for the TGF-beta cell membrane receptor but not the TGF-alpha receptor. This growth factor is most active at physiologic pH and is 97% inactivated by acidification. Hodgkin's TGF is also inactivated by proteases and can be preserved by protease inhibitors. The Hodgkin's TGF can be separated from an autocrine growth factor using either column chromatography or electroelution from gels and is shown to have a molecular weight of approximately 350,000. Incubation of the Hodgkin's TGF in SDS releases a 25,000-D protein with reduced biological activity but which cross-reacts with anti-TGF-beta IgG. We propose that L-428 nodular sclerosing Hodgkin's disease fibrosis is mediated by a potent high molecular weight TGF-beta which, unlike TGF-beta characterized to date, is secreted in a form most active at physiologic pH.

Cytolysis of leukemic B-cells by T-cells activated via two bispecific antibodies.

Bispecific monoclonal antibodies can be used in the activation of effector cells to lyse autologous tumor cells. We analyzed the activation of human T-cells in vitro with bispecific monoclonal antibodies, which were generated by hybridoma-hybridoma fusion. Preactivated allogeneic and autologous T-cells could be triggered to lyse tumoral B-cells in the presence of CD3 x CD19 bispecific antibodies. In addition, the combined use of two CD3 x CD19 plus CD28 x CD22 bispecific antibodies induced optimal interleukin 2 secretion by Jurkat T-cell acute lymphocytic leukemia cells in the presence of target B-cells. The same antibody combination was able to generate cytolytic effector cells without prior activation, when resting T-cells were cocultured with freshly isolated autologous leukemic B-cells in the presence of the bispecific antibodies. The results suggest that signals required to activate cytolytic T-cell precursors can be provided by the two bispecific antibodies. Although activation of resting T-cells can be achieved by CD3 x CD19 bispecific antibodies in association with monospecific bivalent CD28 antibodies, the second bispecific antibody, CD28 x CD22, further increases the specificity of the target cell dependent activation of T-cells. When used for immunotherapy of B-cell malignancies, the CD3 x CD19 and CD28 x CD22 bispecific antibody combination may avoid the need for ex vivo activated effector cells, because the antibodies may induce T-cell activation directly at the tumor site.

Hodgkin's cell lectin: an ectosialyltransferase and lymphocyte agglutinant related to the hepatic asialoglycoprotein receptor.

The galactophilic lectin expressed on the surface of cultured Hodgkin's cells, recently described by this laboratory, has binding characteristics similar to those of the hepatic asialoglycoprotein receptor (HBP), and has been recognized as a Mr 55,000 (p55) membrane glycoprotein by a polyclonal antiserum to rat HBP. This study confirms the close structural relationship between the two lectins showing immunological cross-reactivity of monoclonal and polyclonal antibodies recognizing distinct epitopes on rat or human HBP. In support of the suggested dual nature of p55 as lectin and ectosialyltransferase, enzyme activity is inhibited by the monoclonal anti-HBP antibody, anti-HA 116. Cultured Hodgkin's cells, as purified HBP, agglutinate T-lymphocytes expressing hyposialylated membrane glycosyl determinants. This cell-cell interaction mediated by p55 results in the incorporation of sialic acid into lymphocyte surface asialo-glycans. The function of the Hodgkin's lectin as lymphocyte agglutinant in vitro suggests its role as an immunomodulator contributing to the immunodeficiencies associated with Hodgkin's disease.

Somatic mutations of the CD95 gene in Hodgkin and Reed-Sternberg cells.

Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) are thought to be derived from preapoptotic germinal center B cells. However, little is known about the transforming events rescuing the precursor of the H/RS cells from apoptosis. Given the importance of CD95 (Apo-1/Fas)-mediated apoptosis for negative selection within the germinal center, single micromanipulated H/RS cells from 10 cases of cHD were analyzed for somatic mutations within the CD95 gene. Three clonal mutations within the 5' regions were amplified from single H/RS cells in one case. From H/RS cells of another case, two mutations within the last exon coding for the death domain were detected. About half of these H/RS cells carried a monoallelic stop-codon; the remaining tumor cells harbored a monoallelic replacement mutation. Both mutations likely impair CD95 function. Because all these H/RS cells also bear clonal mutations inactivating the IkappaB alpha gene, the IkappaB alpha mutations occurred earlier than those of the CD95 gene in the sequence of transforming events leading to cHD. In conclusion, somatic mutations of the CD95 gene occur in a fraction of cHD cases and may favor the escape of the precursor of the H/RS clone from apoptosis.