Semen analysis and reproductive hormones in boys with classical Hodgkin lymphoma treated according to the EuroNet-PHL-C2 protocol.

STUDY QUESTION: What is the impact of the EuroNet-PHL-C2 treatment for boys with classical Hodgkin lymphoma (cHL) on semen parameters? SUMMARY ANSWER: More than half of the patients (52%, n = 16/31) had oligozoospermia or azoospermia at 2 years from cHL diagnosis; particularly boys treated for advanced-stage cHL had low sperm counts and motility. WHAT IS KNOWN ALREADY: Chemotherapy and radiotherapy to the inguinal region or testes can impair spermatogenesis and result in reduced fertility. The EuroNet-PHL-C2 trial aims to minimize radiotherapy in standard childhood cHL treatment, by intensifying chemotherapy. The present study aims to assess the (gonadotoxic) impact of this treatment protocol on semen parameters and reproductive hormones in boys aged ≤18 years. STUDY DESIGN, SIZE, DURATION: This international, prospective, multi-centre cohort study was an add-on study to the randomized phase-3 EuroNet-PHL-C2 trial, where the efficacy of standard cHL treatment with OEPA-COPDAC-28 (OEPA: vincristine, etoposide, prednisone, and doxorubicin; COPDAC-28: cyclophosphamide, vincristine, prednisone, and dacarbazine) was compared to intensified OEPA-DECOPDAC-21 chemotherapy (DECOPDAC-21: COPDAC with additional doxorubicin and etoposide and 25% more cyclophosphamide). Patients were recruited between January 2017 and September 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligibility criteria included male patients, diagnosed with classical HL before or at the age of 18 years, and treated according to the EuroNet-PHL-C2 protocol in any of the 18 participating sites in the Netherlands, Germany, Belgium, Czech Republic, and Austria. Sperm parameters (sperm concentration, progressive motility, sperm volume, and calculated total motile sperm count) were assessed at diagnosis and 2 years after diagnosis in (post)pubertal boys. Laboratory measurements (serum follicle-stimulating hormone (FSH) and inhibin B) were performed in samples drawn at diagnosis, during treatment (2-3 times), and at 2 years post-diagnosis, and (age-adjusted) analyses were conducted separately for pre-pubertal and (post)pubertal boys. Outcomes were compared between the treatment levels (TL1, TL2, and TL3) and consolidation treatment schemes (COPDAC-28 and DECOPDAC-21). MAIN RESULTS AND THE ROLE OF CHANCE: In total, 101 boys were included in the present analysis: 73 were (post)pubertal (median age 15.4 years, (IQR 14.4; 16.6), 10 TL1, 29 TL2, 34 TL3, 62% of TL2/3 patients received COPDAC-28) and 28 boys were pre-pubertal (median age 9.6 years (IQR 6.6; 11.4), 4 TL1, 7 TL2, 17 TL3, 38% of TL2/3 patients received COPDAC-28). The study included six boys who had received pelvic radiotherapy; none were irradiated in the inguinal or testicular area. At diagnosis, 48 (post)pubertal boys delivered semen for cryopreservation; 19 (40%) semen samples were oligospermic and 4 (8%) were azoospermic. Low sperm concentration (<15 mil/ml) appeared to be related to the HL disease itself, with a higher prevalence in boys who presented with B symptoms (76% vs 26%, aOR 2.3 (95% CI 1.0; 3.8), P = 0.001) compared to those without such symptoms. At 2 -years post-diagnosis, 31 boys provided semen samples for analysis, of whom 12 (39%) boys had oligozoospermia and 4 (13%) had azoospermia, while 22 boys (71%) had low total motile sperm counts (TMSC) (<20 mil). Specifically, the eight boys in the TL3 group treated with DECOPDAC-21 consolidation had low sperm counts and low progressive motility after 2 years (i.e. median sperm count 1.4 mil/ml (IQR <0.1; 5.3), n = 7 (88%), low sperm concentration, low median progressive motility 16.5% (IQR 0.0; 51.2), respectively). Age-adjusted serum FSH levels were significantly raised and inhibin B levels (and inhibin B:FSH ratios) were decreased during chemotherapy in (post)pubertal boys, with subsequent normalization in 80% (for FSH) and 60% (for inhibin B) of boys after 2 years. Only 4 out of the 14 (post)pubertal boys (29%) with low sperm concentrations after 2 years had elevated FSH (>7.6 IU/l), while 7 (50%) had low inhibin B levels (<100 ng/l). In pre-pubertal boys, reproductive hormones were low overall and remained relatively stable during chemotherapy. LIMITATIONS, REASONS FOR CAUTION: The present analyses included sperm and laboratory measurements up to 2 years post-diagnosis. Long-term reproductive outcomes and potential recovery of spermatogenesis remain unknown, while recovery was reported up to 5- or even 10-year post-chemotherapy in previous studies.Boys who were pre-pubertal at diagnosis were still too young and/or physically not able to deliver semen after 2 years and we could not assess a potential difference in gonadotoxicity according to pubertal state at the time of treatment. Overall, the statistical power of the analyses on sperm concentration and quality after 2 years was limited. WIDER IMPLICATIONS OF THE FINDINGS: Results of the semen analyses conducted among the 31 boys who had provided a semen sample at 2 years post-treatment were generally poor. However, additional long-term and adequately powered data are crucial to assess the potential recovery and clinical impact on fertility. The participating boys will be invited to deliver a semen sample after 5 years. Until these data become available, benefits of intensified chemotherapy in cHL treatment to reduce radiotherapy and lower risk for development of secondary tumours should be carefully weighed against potentially increased risk of other late effects, such as diminished fertility due to the increased chemotherapy burden. Boys with newly diagnosed cHL should be encouraged to deliver sperm for cryopreservation whenever possible. However, patients and clinicians should also realize that the overall state of disease and inflammatory milieu of cHL can negatively affect sperm quality and thereby reduce chance of successful fertility preservation. Furthermore, the measurement of FSH and inhibin B appears to be of low value in predicting low sperm quality at two years from cHL treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Dutch charity foundation KiKa (project 257) that funds research on all forms of childhood cancer. C.M.-K., D.K., W.H.W., D.H., MC, A.U., and A.B. were involved in the development of the EuroNet-PHL-C2 regimen. The other authors declare no potential conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

The impact of treatment for childhood classical Hodgkin lymphoma according to the EuroNet-PHL-C2 protocol on serum anti-Müllerian Hormone.

STUDY QUESTION: What is the impact of the EuroNet-PHL-C2 treatment protocol for children with classical Hodgkin lymphoma (cHL) on gonadal function in girls, based on assessment of serum anti-Müllerian hormone (AMH)? SUMMARY ANSWER: Serum AMH levels decreased after induction chemotherapy and increased during subsequent treatment and 2 years of follow-up, with lowest levels in patients treated for advanced stage cHL. WHAT IS KNOWN ALREADY: Treatment for cHL, particularly alkylating agents and pelvic irradiation, can be gonadotoxic and result in premature reduction of primordial follicles in females. The current EuroNet-PHL-C2 trial aims to reduce the use of radiotherapy in standard childhood cHL treatment, by intensifying chemotherapy. This study aims to assess the gonadotoxic effect of the EuroNet-PHL-C2 protocol. STUDY DESIGN, SIZE, DURATION: This international, prospective, multicenter cohort study is embedded in the EuroNet-PHL-C2 trial, an European phase-3 treatment study evaluating the efficacy of standard cHL treatment with OEPA-COPDAC-28 (OEPA: vincristine, etoposide, prednisone, and doxorubicin; COPDAC-28: cyclophosphamide, vincristine, prednisone, and dacarbazine) versus intensified OEPA-DECOPDAC-21 (DECOPDAC-21: COPDAC with additional doxorubicin and etoposide and 25% more cyclophosphamide) in a randomized setting. Participants were recruited between January 2017 and September 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Female patients aged ≤18 years, treated according to the EuroNet-PHL-C2 protocol for cHL were recruited across 18 sites in the Netherlands, Belgium, Germany, Austria, and Czech Republic. All parents and patients (aged ≥12 years old) provided written informed consent. Serum AMH levels and menstrual cycle characteristics were evaluated over time (at diagnosis, one to three times during treatment and 2 up to 5 years post-diagnosis) and compared between treatment-levels (TL1, TL2, and TL3) and treatment-arms (OEPA-COPDAC-28 and OEPA-DECOPDAC-21). Serum samples obtained from patients after receiving pelvic radiotherapy were excluded from the main analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 104 females, with median age at diagnosis of 15.6 years (IQR 13.7; 17.0), were included in the analysis. Ninety-nine were (post)pubertal. Eighteen girls were diagnosed with an early stage of cHL (TL1) and 86 with intermediate or advanced stage disease (50 TL2 and 36 TL3, 66% received COPDAC-28 and 34% DECOPDAC-21). Five patients received pelvic radiotherapy. Median AMH level at diagnosis was 1.7 µg/l (IQR 0.9; 2.7). After two courses of OEPA chemotherapy, AMH levels decreased substantially in all patients (98% <0.5 µg/l), followed by a significant increase during the consolidation treatment and follow-up. After 2 years, 68% of patients reached their baseline AMH value, with overall median recovery of 129% (IQR 75.0; 208.9) compared to baseline measurement. Five patients (7%) had AMH <0.5 µg/l. In patients treated for advanced stage disease, AMH levels remained significantly lower compared to early- or intermediate stage disease, with median serum AMH of 1.3 µg/l (IQR 0.8; 2.1) after 2 years. Patients who received DECOPDAC-21 consolidation had lower AMH levels during treatment than patients receiving COPDAC-28, but the difference was no longer statistically significant at 2 years post-diagnosis. Of the 35 postmenarchal girls who did not receive hormonal co-treatment, 19 (54%) experienced treatment-induced amenorrhea, two girls had persisting amenorrhea after 2 years. LIMITATIONS, REASONS FOR CAUTION: The studied population comprises young girls with diagnosis of cHL often concurring with pubertal transition, during which AMH levels naturally rise. There was no control population, while the interpretation of AMH as a biomarker during childhood is complex. The state of cHL disease may affect AMH levels at diagnosis, potentially complicating assessment of AMH recovery as a comparison with baseline AMH. The current analysis included data up to 2-5 years post-diagnosis. WIDER IMPLICATIONS OF THE FINDINGS: The current PANCARE guideline advises to use the cyclophosphamide-equivalent dose score (CED-score, as an estimation of cumulative alkylating agent exposure) with a cut-off of 6000 mg/m2 to identify females aged <25 years at high risk of infertility. All treatment-arms of the EuroNet-PHL-C2 protocol remain below this cut-off, and based on this guideline, girls treated for cHL should therefore be considered low-risk of infertility. However, although we observed an increase in AMH after chemotherapy, it should be noted that not all girls recovered to pre-treatment AMH levels, particularly those treated for advanced stages of cHL. It remains unclear how our measurements relate to age-specific expected AMH levels and patterns. Additional (long-term) data are needed to explore clinical reproductive outcomes of survivors treated according to the EuroNet-PHL-C2 protocol. STUDY FUNDING/COMPETING INTEREST(S): The fertility add-on study was funded by the Dutch charity foundation KiKa (project 257) that funds research on all forms of childhood cancer. C.M-K., D.K., W.H.W., D.H., M.C., A.U., and A.B. were involved in the development of the EuroNet-PHL-C2 regimen. The other authors indicated no potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Prevention of activated brown adipose tissue on 18F-FDG-PET scans of young lymphoma patients: results of an ancillary study within the EuroNet-PHL-C2 trial.

Activated brown fat (aBAT) is known to affect the evaluation of 18F-FDG PET scans, especially in young patients. The aim of this study was to determine factors influencing the occurrence of aBAT, and to investigate the effectiveness of the two preventive measures, warming and beta-blocker (propranolol) administration. Five-hundred-twenty-eight 18F-FDG-PET scans of 241 EuroNet-PHL-C2 trial patients from 41 nuclear medicine departments in Germany and Czech Republic were screened for aBAT. The occurrence of aBAT was analyzed with patient characteristics (age, sex, body mass index, predisposition to aBAT), weather data at the day of 18F-FDG PET scanning as well as the preventive measures taken. Potentially important factors from univariate analyses were included into a logistic regression model. Warming as a preventive measure was used in 243 18F-FDG-PET scans, propranolol was administered in 36, warming and propranolol were combined in 84, and no preventive measures were taken in 165 scans. Whereas age, sex and body mass index had no clear impact, there was an individual predisposition to aBAT. Logistic regression model revealed that the frequency of aBAT mainly depends on the outside temperature (p = 0.005) and can be effectively reduced by warming (p = 0.004), the administration of unselective beta-blocker or the combination of both. Warming is a simple, cheap and non-invasive method to reduce the frequency of aBAT. However, the effect of warming decreases with increasing outside temperatures. Administration of propranolol seems to be equally effective and provides advantages whenever the positive effect of warming is compromised. The combination of both preventive measures could have an additive effect.

Modified Glasgow prognostic score (mGPS) is correlated with sarcopenia and dominates the prognostic role of baseline body composition parameters in advanced gastric and esophagogastric junction cancer patients undergoing first-line treatment from the phase III EXPAND trial.

BACKGROUND: Sarcopenia represents an established adverse prognostic factor in cancer patients. Consequently, different means to counteract sarcopenia have been proposed to improve cancer treatment. Computed tomography (CT)-based measurements, also labor intensive, are well validated for the analysis of sarcopenia. As inflammation plays a key role in the development of sarcopenia, we here studied the role of the modified Glasgow prognostic score (mGPS), consisting of inflammation parameters plasma C-reactive protein (CRP) and albumin, to predicting sarcopenia and adipose tissue-related body composition (BC) parameters at baseline and their changes during treatment and to analyze its prognostic role in conjunction with BC parameters. PATIENTS AND METHODS: CT measurements of BC parameters were carried out at baseline and week 12 in patients with advanced gastric or esophagogastric junction cancer from the phase III EXPAND trial, undergoing first-line platinum-fluoropyrimidine chemotherapy. mGPS was calculated from baseline CRP and albumin plasma levels. Pearson correlation and Cox regression analyses were carried out. RESULTS: mGPS is strongly prognostic for overall survival (OS). Baseline mGPS is significantly correlated with baseline mean muscle attenuation (MA; P < 0.0001). Baseline mGPS did not predict a decline in muscle or adipose tissue parameters during 12 weeks of treatment and a decline in muscle or adipose tissue parameters was not prognostic for OS. MA lost its prognostic role for OS when mGPS or CRP was entered into the Cox models. Eastern Cooperative Oncology Group performance status together with CRP or mGPS remained the sole baseline prognostic factors for OS. CONCLUSIONS: Our findings support a model where tumor-mediated inflammatory response represents a strong prognostic factor, which is causally related to sarcopenia, but with no direct causal path from sarcopenia to survival. Therefore, therapeutic targeting of systemic inflammation should be further explored as a promising strategy to improve both sarcopenia and the efficacy and tolerability of cancer treatment.

Only strongly enhanced residual FDG uptake in early response PET (Deauville 5 or qPET ≥ 2) is prognostic in pediatric Hodgkin lymphoma: Results of the GPOH-HD2002 trial.

PURPOSE: In 2014, we published the qPET method to quantify fluorodeoxyglucose positron emission tomography (FDG-PET) responses. Analysis of the distribution of the quantified signals suggested that a clearly abnormal FDG-PET response corresponds to a visual Deauville score (vDS) of 5 and high qPET values ≥ 2. Evaluation in long-term outcome data is still pending. Therefore, we analyzed progression-free survival (PFS) by early FDG-PET response in a subset of the GPOH-HD2002 trial for pediatric Hodgkin lymphoma (PHL). PATIENTS/METHODS: Pairwise FDG-PET scans for initial staging and early response assessment after two cycles of chemotherapy were available in 93 PHL patients. vDS and qPET measurement were performed and related to PFS. RESULTS: Patients with a qPET value ≥ 2.0 or vDS of 5 had 5-year PFS rates of 44%, respectively 50%. Those with qPET values < 2.0 or vDS 1 to 4 had 5-year PFS rates of 90%, respectively 80%. The positive predictive value of FDG-PET response assessment increased from 18% (9%; 33%) using a qPET threshold of 0.95 (vDS ≤ 3) to 30% (13%; 54%) for a qPET threshold of 1.3 (vDS ≤ 4) and to 56% (23%; 85%) when the qPET threshold was ≥ 2.0 (vDS 5). The negative predictive values remained stable at ≥92% (CI: 82%; 98%). CONCLUSION: Only strongly enhanced residual FDG uptake in early response PET (vDS 5 or qPET ≥ 2, respectively) seems to be markedly prognostic in PHL when treatment according to the GPOH-HD-2002 protocol is given.

Pediatric Nodular Lymphocyte-predominant Hodgkin Lymphoma: Treatment Recommendations of the GPOH-HD Study Group.

Nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL) is a very rare disease in childhood and adolescence. In Germany, about 15 newly diagnosed patients present with this disease annually; this number comprises less than 10% of all pediatric Hodgkin lymphoma cases. Since the EuroNet-PHL-LP1 trial for early stage nLPHL patients stopped recruiting in Germany in October 2014, the GPOH-HD writing committee reviewed the literature and decided to deliver treatment recommendations for childhood and adolescent nLPHL patients. These guidelines shall be applicable to young nLPHL patients in European countries that will no longer be able to participate in nLPHL trials for young patients. Therefore, the EuroNet-PHL-nLPHL-registry will be installed to provide quality assured central review of staging and response assessment for registered patients by the Central Review Board of EuroNet-PHL in Halle/Leipzig, Germany.

Genomic and epigenomic co-evolution in follicular lymphomas.

Follicular lymphoma (FL) with a t(14;18) is a B-cell neoplasm clinically characterized by multiple recurrencies. In order to investigate the clonal evolution of this lymphoma, we studied paired primary and relapse tumor samples from 33 patients with recurrent non-transformed t(14;18)-positive FL. We reconstructed phylogenetic trees of the evolution by taking advantage of the activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) active in the germinal center reaction using sequences of the clonal VHDHJH rearrangements of the immunoglobulin heavy chain (IGH) locus. Mutational analysis of the IGH locus showed evidence for ongoing somatic mutation and for counter-selection of mutations affecting the BCR conformation during tumor evolution. We further followed evolutionary divergence by targeted sequencing of gene loci affected by aberrant SHM as well as of known driver genes of lymphomagenesis, and by array-based genome-wide chromosomal imbalance and DNA methylation analysis. We observed a wide spectrum of evolutionary patterns ranging from almost no evolution to divergent evolution within recurrent non-transformed t(14;18) FL. Remarkably, we observed a correlation of the magnitude of evolutionary divergence across all genetic and epigenetic levels suggesting co-evolution. The distribution of coding mutations in driver genes and the correlation with SHM suggest CREBBP and AID to be potential modifiers of genetic and epigenetic co-evolution in FL.

Detecting Trichinella infections using inverse microscopy and an improved larval counting technique.

Several methods for the detection of Trichinella in meat are legally prescribed in regulation (EC) No 2075/2005, which prescribes the magnetic stirrer method for pooled sample digestion (MSM) as the reference method. However, the MSM's multistage protocol requires several preparatory steps that seem to be accountable for the loss of larvae. Here we present a modified MSM (mMSM) based on: (1) an inversion of the optical path using inverse microscopy; and (2) a modified larval counting basin (mLCB, 'Trichoview'). This enables one to examine samples of up to 40 ml and reduces the examination area from 72 to 10.3 cm2. Preparatory steps that might cause the loss of Trichinella larvae are eliminated from the new protocol. Correspondingly, the overall analytical time is reduced. In a direct and blinded comparison using 60 digest samples containing spiked vital Trichinella larvae (1-90 L1), both methods performed well for both small and large numbers of L1. However, 1278 of 1285 L1 (99.4%) were detected using the mMSM, while MSM recovered only 1225 L1 (95.3%). The improvement stems largely from samples with small numbers of L1: in all samples spiked with fewer than 10 L1, the recovery rate of mMSM was 100% compared to only 93% with MSM. Our data suggest that the use of the mMSM can improve the recovery rate by about 4% and therefore reduce the chances of a false-negative result in a sample containing 5 larvae by a factor of about 4.

The EuroNet paediatric hodgkin network - modern imaging data management for real time central review in multicentre trials.

Since 2007, children and adolescents with Hodgkin lymphomas are treated in the Europe-wide EuroNet-PHL trials. A real time central review process for stratification of the patients enhances quality control and efficient therapy management. This process includes reading of all cross-sectional-images. Since reference evaluation is time critical, a fast, easy to handle and safe data transfer is important. In addition, immediate and constant access to all the data has to be guaranteed in case of queries and for regulatory reasons. To meet the mentioned requirements the EuroNet Paediatric Hodgkin Data Network (funded by the European Union - Project Number: 2007108) was established between 2008 and 2011. A respective tailored data protection plan was formulated. The aim of this article is to describe the networks' mode of operation and the advantages for multi-centre trials that include centralized image review.

Evaluation of the prognostic meaning of C-reactive protein (CRP) in children and adolescents with classical Hodgkin's lymphoma (HL).

BACKGROUND: In adult cancer patients the negative predictive value of elevated CRP levels has been described for several malignancies. Only few studies have analyzed the prognostic role of CRP in children and adolescents with classical HL. In these studies elevated CRP levels correlate with the presence of classical risk factors and adverse outcome. PATIENTS AND METHODS: The prognostic role of CRP for patients with classical HL admitted to the GPOH-HD-2002 study was analyzed retrospectively. RESULTS: CRP levels were documented for 369 of 573 patients. Significant (p<0.05) increased median CRP levels were found in the presence of B-Symptoms (25.7 vs. 5.1 mg/l), extranodal involvement (21.5 vs. 7.5 mg/l), elevated erythrocyte sedimentation rate (ESR, 13.0 vs. 1.0 mg/l) and stage III/IV disease (15.5 vs. 5.3 mg/l). 83.9% of patients with elevated and 45.8% of patients with normal CRP had an ESR >30 mm/h. CONCLUSION: Elevated CRP levels were associated with classical risk factors of HL. CRP and ESR may reflect different biological processes. CRP was prognostic within early stage TG-1 patients treated with reduced treatment, but not within advanced stage TG-2+3.

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.

Chromosomal imbalances and partial uniparental disomies in primary central nervous system lymphoma.

To determine the pattern of genetic alterations in primary central nervous system lymphomas (PCNSL), 19 PCNSL were studied by high-density single-nucleotide polymorphism arrays. Recurrent losses involved 6p21.32, 6q21, 8q12-12.2, 9p21.3, 3p14.2, 4q35.2, 10q23.21 and 12p13.2, whereas gains involved 18q21-23, 19q13.31, 19q13.43 and the entire chromosomes X and 12. Partial uniparental disomies (pUPDs) were identified in 6p and 9p21.3. These genomic alterations affected the HLA locus, the CDKN2A/p16, CDKN2B/p15 and MTAP, as well as the PRDM1, FAS, MALT1, and BCL2 genes. Increased methylation values of the CDKN2A/p16 promoter region were detected in 75% (6/8) PCNSL. Gene expression profiling showed 4/21 (20%) minimal common regions of imbalances to be associated with a differential mRNA expression affecting the FAS, STAT6, CD27, ARHGEF6 and SEPT6 genes. Collectively, this study unraveled novel genomic imbalances and pUPD with a high resolution in PCNSL and identified target genes of potential relevance in the pathogenesis of this lymphoma entity.

A novel view on stem cell development: analysing the shape of cellular genealogies.

OBJECTIVES: The analysis of individual cell fates within a population of stem and progenitor cells is still a major experimental challenge in stem cell biology. However, new monitoring techniques, such as high-resolution time-lapse video microscopy, facilitate tracking and quantitative analysis of single cells and their progeny. Information on cellular development, divisional history and differentiation are naturally comprised into a pedigree-like structure, denoted as cellular genealogy. To extract reliable information concerning effecting variables and control mechanisms underlying cell fate decisions, it is necessary to analyse a large number of cellular genealogies. MATERIALS AND METHODS: Here, we propose a set of statistical measures that are specifically tailored for the analysis of cellular genealogies. These measures address the degree and symmetry of cellular expansion, as well as occurrence and correlation of characteristic events such as cell death. Furthermore, we discuss two different methods for reconstruction of lineage fate decisions and show their impact on the interpretation of asymmetric developments. In order to illustrate these techniques, and to circumvent the present shortage of available experimental data, we obtain cellular genealogies from a single-cell-based mathematical model of haematopoietic stem cell organization. RESULTS AND CONCLUSIONS: Based on statistical analysis of cellular genealogies, we conclude that effects of external variables, such as growth conditions, are imprinted in their topology. Moreover, we demonstrate that it is essential to analyse timing of cell fate-specific changes and of occurrence of cell death events in the divisional context in order to understand the mechanisms of lineage commitment.

Munich Oktoberfest experience: remarkable impact of sex and age in ethanol intoxication.

Approximately 5,000 of 6 million annual visitors of the Oktoberfest in Munich have to undergo medical treatment. Patients with alcohol intoxication without trauma or further complications are all treated in a specialized medical camp. We studied these patients in order to identify risk factors and to assess the relevance of the Glasgow Coma Score (GCS) and of ethanol blood concentrations for patient management. In 2004 totally 405 patients suffering from ethanol intoxication without trauma were treated in the medical camp. A complete set of the following data was obtained from all 405 patients: GCS, ethanol blood concentration, age, sex, blood pressure (mean, systolic and diastolic), body temperature, heart rate, blood sugar, GOT, gamma-GT, and CK. A multivariate logistic regression model was applied to identify risk factors predicting patients at increased risk of hospitalization. Low GCS (< or =8 vs. >8, OR: 4.18, CI: 1.96-8.65) low age (20-29 vs. > or =30 years, OR: 2.35, CI: 1.05-5.65) and male gender (male vs. female, OR: 3.58, CI: 1.36-9.34) independently predicted patients that had to be hospitalized. All other parameters including ethanol blood concentrations were not explanatory. Patients with GCS < or = 8 (n = 66) had a lower median blood pressure (P = 0.0312) and showed a smaller increase in blood pressure during the observation period compared to patients with GCS > 8 (P < 0.001), suggesting that this subgroup may require longer recovery periods. Men aged 20-29 years were at highest risk for hospital admission. Increased risk could not be explained by higher ethanol blood concentrations in this subgroup. Importantly, GCS < 6 does not justify endotracheal intubation in ethanol intoxicated patients, when further complications, such as trauma, can be excluded.

Pathway activation patterns in diffuse large B-cell lymphomas.

Deregulation of cell signaling pathways controlling cell growth and cell survival is a common feature of all cancers. Although a core repertoire of oncogenic mechanisms is widely conserved between various malignancies, the constellation of pathway activities can vary even in patients with the same malignant disease. Modern molecularly targeted cancer drugs intervene in cell signaling compensating for pathway deregulation. Hence characterizing tumors with respect to pathway activation will become crucial for treatment decisions. Here we have used semi-supervised machine learning methodology to generate signatures of eight oncogene-inducible pathways, which are conserved across epithelial and lymphoid tissues. We combined them to patterns of pathway activity called PAPs for pathway activation patterns and searched for them in 220 morphologically, immunohistochemically and genetically well-characterized mature aggressive B-cell lymphomas including 134 cases with clinical data available. Besides Burkitt lymphoma, which was characterized by a unique pattern, the PAPs identified four distinct groups of mature aggressive B-cell lymphomas across independent gene expression studies with distinct biological characteristics, genetic aberrations and prognosis. We confirmed our findings through cross-platform analysis in an independent data set of 303 mature aggressive B-cell lymphomas.

Dose-escalated CHOEP for the treatment of young patients with aggressive non-Hodgkin's lymphoma: I. A randomized dose escalation and feasibility study with bi- and tri-weekly regimens.

BACKGROUND: To determine the maximum tolerated dose of a bi- and tri-weekly combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone plus etoposide (CHOEP) regimen without stem-cell support. PATIENTS AND METHODS: Randomized phase I/II multicenter four-level (cyclophosphamide: 1000-1200-1400-1600 mg/m2; doxorubicin: 55-60-65-70 mg/m2; etoposide: 375-450-525-600 mg/m2) dose escalation study with CHOEP-14 and CHOEP-21 in young patients (18-60 years) with newly diagnosed aggressive non-Hodgkin's lymphoma. Dose-limiting toxicity was defined as thrombocytopenia <80,000/mm3 and leukocytopenia <2500/mm3 on days 16 (CHOEP-14) and 23 (CHOEP-21) or prolonged (>4 days) leukocytopenia (<1000/mm3) or thrombocytopenia (<20,000/mm3). RESULTS: One hundred and thirty-nine patients (high-CHOEP-14: 47, high-CHOEP-21: 92) were randomly allocated to the study. Maximal tolerated dose was level 2 for CHOEP-14 and level 4 for CHOEP-21. With a less favorable profile of patients in CHOEP-14, 4-year event-free survival was 47.9% after high-CHOEP-14 and 66.2% after high-CHOEP-21, 4-year overall survival 62.1% after high-CHOEP-14 and 73.4% after high-CHOEP-21, respectively. CONCLUSION: Significant dose escalations of CHOEP are possible with granulocyte colony-stimulating factor support, with different chemotherapy models favoring the maximally escalated bi- or tri-weekly regimen, respectively. Because a higher total dose can be achieved with six cycles of the tri-weekly compared with the biweekly regimen, CHOEP-21 at dose escalation level 3 was chosen for a nationwide randomized comparison with baseline CHOEP-21 in a subsequent phase III trial.

Role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) for prognosis in endometrial cancer.

BACKGROUND: Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) contribute to the invasiveness of many carcinomas. Here, we studied a possible association between cytosolic uPA and PA-1 concentrations in tumor tissue with prognosis in patients with endometrial cancer. METHODS: Cytosolic concentrations of uPA and PAI-1 were determined in 69 primary endothelial adenocarcinomas using an enzyme-linked immunoassay (ELISA). A possible influence of uPA and PAI-1 was studied by multivariate Cox regression adjusting for the established clinical prognostic factors FIGO-stage, grading, depth of invasion, diabetes mellitus and age. RESULTS: Both uPA (p=0.011) and PAI-1 (p=0.003) were associated with relapse free time using the multivariate proportional hazards model. Association with overall survival was less pronounced with p=0.021 for uPA and p=0.358 for PAI-1. Concentrations of PAI-1 increased with FIGO stage (p=0.003) and with histological grading (p=0.005). Both uPA and PAI-1 concentrations were negatively correlated with estrogen and progesterone receptor levels. CONCLUSION: The combination of high cytosolic concentrations of uPA (>5 ng/mg total protein) and high PAI-1 (>20 ng/mg total protein) may reveal a group of patients with increased risk of progression.

Development and implementation of an analysis tool for array-based comparative genomic hybridization.

OBJECTIVES: Array-comparative genomic hybridization (aCGH) is a high-throughput method to detect and map copy number aberrations in the genome. Multi-step analysis of high-dimensional data requires an integrated suite of bioinformatic tools. In this paper we detail an analysis pipeline for array CGH data. METHODS: We developed an analysis tool for array CGH data which supports single and multi-chip analyses as well as combined analyses with paired mRNA gene expression data. The functions supporting relevant steps of analysis were implemented using the open source software R and combined as package aCGHPipeline. Analysis methods were illustrated using 189 CGH arrays of aggressive B-cell lymphomas. RESULTS: The package covers data input, quality control, normalization, segmentation and classification. For multi-chip analysis aCGHPipeline offers an algorithm for automatic delineation of recurrent regions. This task was performed manually up to now. The package also supports combined analysis with mRNA gene expression data. Outputs consist of HTML documents to facilitate communication with clinical partners. CONCLUSIONS: The R package aCGHPipeline supports basic tasks of single and multi-chip analysis of array CGH data.

Role of the progesterone receptor for paclitaxel resistance in primary breast cancer.

Paclitaxel plays an important role in the treatment of primary breast cancer. However, a substantial proportion of patients treated with paclitaxel does not appear to derive any benefit from this therapy. We performed a prospective study using tumour cells isolated from 50 primary breast carcinomas. Sensitivity of primary tumour cells to paclitaxel was determined in a clinically relevant range of concentrations (0.85-27.2 microg ml(-1) paclitaxel) using an ATP assay. Chemosensitivity data were used to study a possible association with immunohistochemically determined oestrogen and progesterone receptor (ER and PR) status, as well as histopathological parameters. Progesterone receptor (PR) mRNA expression was also determined by quantitative RT-PCR. We observed a clear association of the PR status with chemosensitivity to paclitaxel. Higher levels of immunohistochemically detected PR expression correlated with decreased chemosensitivity (P=0.008). Similarly, high levels of PR mRNA expression were associated with decreased paclitaxel chemosensitivity (P=0.007). Cells from carcinomas with T-stages 3 and 4 were less sensitive compared to stages 1 and 2 (P=0.013). Multiple regression analysis identified PR receptor status and T-stage as independent predictors of paclitaxel chemosensitivity, whereas the ER, N-stage, grading and age were not influential. In conclusion, in vitro sensitivity to paclitaxel was higher for PR-negative compared with PR-positive breast carcinoma cells. Thus, PR status should be considered as a possible factor of influence when designing new trials and chemotherapy protocols.

Evaluation of insulin-like growth factor II, cyclooxygenase-2, ets-1 and thyroid-specific thyroglobulin mRNA expression in benign and malignant thyroid tumours.

OBJECTIVE: We evaluated three markers (insulin-like growth factor II (IGF-II), cyclooxygenase-2 (COX-2) and ets-1) of thyroid growth stimulation and cell transformation together with a thyroid-specific marker (thyroglobulin (Tg)) for their potential to differentiate benign and malignant follicular thyroid neoplasia (FN). DESIGN AND METHODS: mRNA expression levels were determined by real-time PCR in 100 snap-frozen thyroid samples: 36 benign thyroid nodules with different histology and function (19 cold (CTN) and 17 toxic thyroid nodules (TTN)), 36 corresponding normal thyroid tissues of the same patients, eight Graves' disease (GD) thyroids, 10 follicular thyroid carcinomas (FTC) and 10 papillary thyroid carcinomas (PTC). RESULTS: Mean IGF-II and COX-2 levels were not significantly altered between benign and malignant thyroid nodules (IGF-II) or nodular (FTC, TTN, CTN) and normal thyroid tissues (COX-2). In contrast, eight- to tenfold upregulation of ets-1 was observed in PTC and three- to fourfold upregulation of ets-1 was observed in FTC (and GD) compared with benign thyroid nodules and normal thyroid tissues. In addition, thyroglobulin mRNA expression was markedly downregulated (50- to 100-fold) in FTC, PTC and GD samples compared with benign nodular and normal thyroid tissues. Hence an ets-1/Tg ratio >20 distinguished differentiated thyroid cancer from benign nodular or normal thyroid tissue. We then studied ets1- and Tg mRNA expression levels in fine needle aspiration cytology (FNAC) samples. However, in a consecutive series of 40 FNAC samples only equivocal results were obtained on 38 benign and two malignant (FTC) thyroid tumour samples. CONCLUSIONS: Upregulation of ets-1 and downregulation of Tg mRNA expression occur in differentiated thyroid cancer and may facilitate pre-operative identification of thyroid malignancy depending on further evaluation of these potentially promising markers in a larger series of benign and malignant thyroid tumours and their FNAC samples.