The human embryonic genome is karyotypically complex, with chromosomally abnormal cells preferentially located away from the developing fetus.

STUDY QUESTION: Are chromosome abnormalities detected at Day 3 post-fertilization predominantly retained in structures of the blastocyst other than the inner cell mass (ICM), where chromosomally normal cells are preferentially retained? SUMMARY ANSWER: In human embryos, aneuploid cells are sequestered away from the ICM, partly to the trophectoderm (TE) but more significantly to the blastocoel fluid within the blastocoel cavity (Bc) and to peripheral cells (PCs) surrounding the blastocyst during Day 3 to Day 5 progression. WHAT IS KNOWN ALREADY: A commonly held dogma in all diploid eukaryotes is that two gametes, each with 'n' chromosomes (23 in humans), fuse to form a '2n' zygote (46 in humans); a state that remains in perpetuity for all somatic cell divisions. Human embryos, however, display high levels of chromosomal aneuploidy in early stages that reportedly declines from Day 3 (cleavage stage) to Day 5 (blastocyst) post-fertilization. While this observation may be partly because of aneuploid embryonic arrest before blastulation, it could also be due to embryo 'normalization' to a euploid state during blastulation. If and how this normalization occurs requires further investigation. STUDY DESIGN, SIZE, DURATION: A total of 964 cleavage-stage (Day 3) embryos underwent single-cell biopsy and diagnosis for chromosome constitution. All were maintained in culture, assessing blastulation rate, both for those assessed euploid and aneuploid. Pregnancy rate was assessed for those determined euploid, blastulated and subsequently transferred. For those determined aneuploid and blastulated (174 embryos), ICM (all 174 embryos), TE (all 174), Bc (47 embryos) and PC (38 embryos) were analyzed for chromosome constitution. Specifically, concordance with the original Day 3 diagnosis and determination if any 'normalized' to euploid karyotypes within all four structures was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients (144 couples) were undergoing routine preimplantation genetic testing for aneuploidy in three IVF clinical settings. Cleavage-stage biopsy preceded chromosome analysis by next-generation sequencing. All patients provided informed consent. Additional molecular testing was carried out on blastocyst embryos and was analyzed for up to four embryonic structures (ICM, TE, Bc and PC). MAIN RESULTS AND THE ROLE OF CHANCE: Of 463/964 embryos (48%) diagnosed as euploid at Day 3, 70% blastulated (leading to a 59% pregnancy rate) and 30% degenerated. Conversely, of the 501 (52%) diagnosed as aneuploid, 65% degenerated and 35% (174) blastulated, a highly significant difference (P < 0.0001). Of the 174 that blastulated, the ratio of '(semi)concordant-aneuploid' versus 'normalized-euploid' versus 'other-aneuploid' embryos was, respectively, 39%/57%/3% in the ICM; 49%/48%/3% in the TE; 78%/21%/0% in the PC; and 83%/10%/5% in the Bc. The TE karyotype therefore has a positive predictive value of 86.7% in determining that of the ICM, albeit with marginally higher aneuploid rates of abnormalities (P = .071). Levels of abnormality in Bc/PC were significantly higher (P < 0.0001) versus the ploidy of the ICM and TE and nearly all chromosome abnormalities were (at least partially) concordant with Day 3 diagnoses. LIMITATIONS, REASONS FOR CAUTION: The results only pertain to human IVF embryos so extrapolation to the in vivo situation and to other species is not certain. We acknowledge (rather than lineage-specific survival, as we suggest here) the possibility of other mechanisms, such as lineage-specific movement of cells, during blastulation. Ethical considerations, however, make investigating this mechanism difficult on human embryos. WIDER IMPLICATIONS OF THE FINDINGS: Mosaic human cleavage-stage embryos can differentiate into a euploid ICM where euploid cell populations predominate. Sequestering of aneuploid cells/nuclei to structures no longer involved in fetal development has important implications for preimplantation and prenatal genetic testing. These results also challenge previous fundamental understandings of mitotic fidelity in early human development and indicate a complex and fluid nature of the human embryonic genome. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Organon Pharmaceuticals and Merck Serono by grants to W.G.K. W.G.K. is also an employee of AdvaGenix, who could, potentially, indirectly benefit financially from publication of this manuscript. R.C.M. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133747. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.K.G. provides paid consultancy services for Care Fertility. TRIAL REGISTRATION NUMBER: : N/A.

The association of new-onset diabetes with subsequent diagnosis of pancreatic cancer-novel use of a large administrative database.

BACKGROUND: Screening options for pancreatic ductal adenocarcinoma (PDAC) are limited. New-onset type 2 diabetes (NoD) is associated with subsequent diagnosis of PDAC in observational studies and may afford an opportunity for PDAC screening. We evaluated this association using a large administrative database. METHODS: Patients were identified using claims data from the OptumLabs® Data Warehouse. Adult patients with NoD diagnosis were matched 1:3 with patients without NoD using age, sex and chronic obstructive pulmonary disease (COPD) status. The event of PDAC diagnosis was compared between cohorts using the Kaplan-Meier method. Factors associated with PDAC diagnosis were evaluated with Cox's proportional hazards modeling. RESULTS: We identified 640 421 patients with NoD and included 1 921 263 controls. At 3 years, significantly more PDAC events were identified in the NoD group vs control group (579 vs 505; P < 0.001). When controlling for patient factors, NoD was significantly associated with elevated risk of PDAC (HR 3.474, 95% CI 3.082-3.920, P < 0.001). Other factors significantly associated with PDAC diagnosis were increasing age, increasing age among Black patients, and COPD diagnosis (P ≤ 0.05). CONCLUSIONS: NoD was independently associated with subsequent diagnosis of PDAC within 3 years. Future studies should evaluate the feasibility and benefit of PDAC screening in patients with NoD.

Genetic variants in DNA repair genes as potential predictive markers for oxaliplatin chemotherapy in colorectal cancer.

Oxaliplatin-based chemotherapy exerts its effects through generating DNA damage. Hence, genetic variants in DNA repair pathways could modulate treatment response. We used a prospective cohort of 623 colorectal cancer patients with stage II-IV disease treated with adjuvant/palliative chemotherapy to comprehensively investigate 1727 genetic variants in the DNA repair pathways as potential predictive markers for oxaliplatin treatment. Single nucleotide polymorphisms (SNP) associations with overall survival and recurrence-free survival were assessed using a Cox regression model. Pathway analysis was performed using the gamma method. Patients carrying variant alleles of rs3783819 (MNAT1) and rs1043953 (XPC) experienced a longer overall survival after treatment with oxaliplatin than patients who did not carry the variant allele, while the opposite association was found in patients who were not treated with oxaliplatin (false discovery rate-adjusted P-values for heterogeneity 0.0047 and 0.0237, respectively). The nucleotide excision repair (NER) pathway was found to be most likely associated with overall survival in patients who received oxaliplatin (P-value=0.002). Our data show that genetic variants in the NER pathway are potentially predictive of treatment response to oxaliplatin.

Tumour-site-dependent expression profile of angiogenic factors in tumour-associated stroma of primary colorectal cancer and metastases.

BACKGROUND: Tumour-associated stroma has a critical role in tumour proliferation. Our aim was to determine a specific protein expression profile of stromal angiogenic cytokines and matrix metalloproteinases (MMPs) to identify potential biomarkers or new therapy targets. METHODS: Frozen tissue of primary colorectal cancer (n=25), liver (n=25) and lung metastases (n=23) was laser-microdissected to obtain tumour epithelial cells and adjacent tumour-associated stroma. Protein expression of nine angiogenic cytokines and eight MMPs was analysed using a multiplex-based protein assay. RESULTS: We found a differential expression of several MMPs and angiogenic cytokines in tumour cells compared with adjacent tumour stroma. Cluster analysis displayed a tumour-site-dependent stromal expression of MMPs and angiogenic cytokines. Univariate analysis identified stromal MMP-2 and MMP-3 in primary colorectal cancer, stromal MMP-1, -2, -3 and Angiopoietin-2 in lung metastases and stromal MMP-12 and VEGF in liver metastases as prognostic markers (P>0.05, respectively). Furthermore, stroma-derived Angiopoietin-2 proved to be an independent prognostic marker in colorectal lung metastases. CONCLUSION: Expression of MMPs and angiogenic cytokines in tumour cells and adjacent tumour stroma is dependent on the tumour site. Stroma-derived MMPs and angiogenic cytokines may be useful prognostic biomarkers. These data can be helpful to identify new agents for a targeted therapy in patients with colorectal cancer.

The impact of allogeneic stem cell transplantation on the natural course of poor-risk chronic lymphocytic leukemia as defined by the EBMT consensus criteria: a retrospective donor versus no donor comparison.

BACKGROUND: In a single-center retrospective donor versus no-donor comparison, we investigated if allogeneic stem cell transplantation (alloSCT) can improve the dismal course of poor-risk chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: All patients with CLL who were referred for evaluation of alloSCT within a 7-year time frame and had a donor search indication according to the EBMT criteria or because of Richter's transformation were included. Patients for whom a matched donor could be found within 3 months (matches) were compared with patients without such a donor (controls). Primary end point was overall survival measured from the 3-month landmark after search initiation. RESULTS: Of 105 patients with donor search, 97 (matches 83; controls 14) were assessable at the 3-month landmark. Matches and controls were comparable for age, gender, time from diagnosis, number of previous regimens, and remission status. Disregarding if alloSCT was actually carried out or not, survival from the 3-month landmark was significantly better in matches versus controls [hazard ratio 0.38, 95% confidence interval (CI) 0.17-0.85; P = 0.014]. The survival benefit of matches remained significant on multivariate analysis. CONCLUSION: This study provides first comparative evidence that alloSCT may have the potential to improve the natural course of poor-risk CLL as defined by the EBMT criteria.

Low tumor interleukin-1ß expression predicts a limited effect of adjuvant platinum-based chemotherapy for patients with completely resected lung adenocarcinoma: An identification and validation study.

INTRODUCTION AND OBJECTIVES: Adjuvant platinum-based chemotherapy for completely resected non-small cell lung cancer is associated with modest improvement in survival; nevertheless, no validated biomarker exists for predicting the benefit or harm of adjuvant platinum-based chemotherapy. MATERIALS AND METHODS: We simultaneously measured 27 cytokines in operative tumor specimens from a discovery cohort (n = 97) by multiplex immunoassay; half of the patients received adjuvant platinum-based chemotherapy, and the other half were observed. We tested possible prognostic and predictive factors in multivariate Cox models for overall survival (OS) and relapse-free survival (RFS), and a tree-based method was applied to detect predictive factors with respect to RFS. The results were validated in an independent validation cohort (n = 93). RESULTS: Fifty-two of 97 (54 %) patients in the discovery cohort and 50 of 93 (54 %) in the validation cohort received adjuvant chemotherapy; forty-four (85 %) patients in the discovery cohort and 37 (74 %) in the validation cohort received four cycles as planned. In patients with low IL-1ß-expressing tumors, RFS and OS were worse after adjuvant chemotherapy than after observation. The limited effect of adjuvant chemotherapy for patients with low IL-1ß-expressing tumors was confirmed in the validation cohort. Additionally, RFS and OS were prolonged by adjuvant chemotherapy only in patients with high IL-1ß-expressing tumors in the validation cohort. CONCLUSIONS: This study identified and validated low tumor IL-1ß expression as a potential biomarker of a limited response to adjuvant platinum-based chemotherapy after complete resection of pulmonary adenocarcinoma. This finding has the potential to inform adjuvant treatment decisions.

Tailoring sparse multivariable regression techniques for prognostic single-nucleotide polymorphism signatures.

When seeking prognostic information for patients, modern technologies provide a huge amount of genomic measurements as a starting point. For single-nucleotide polymorphisms (SNPs), there may be more than one million covariates that need to be simultaneously considered with respect to a clinical endpoint. Although the underlying biological problem cannot be solved on the basis of clinical cohorts of only modest size, some important SNPs might still be identified. Sparse multivariable regression techniques have recently become available for automatically identifying prognostic molecular signatures that comprise relatively few covariates and provide reasonable prediction performance. For illustrating how such approaches can be adapted to the specific features of SNP data, we propose different variants of a component-wise likelihood-based boosting approach. The latter links SNP measurements to a time-to-event endpoint by a regression model that is built up in a large number of steps. The variants allow for strategic choices in dealing with SNPs that differ in variance because of their variation in minor allele frequencies. In addition, we propose a heuristic that allows computationally efficient handling of millions of covariates, thus opening the door for incorporating SNP × treatment interactions. We illustrate this using data from patients with acute myeloid leukemia. We judge the resulting models according to prediction error curves and using resampling data sets. We obtain increased stability by moving interpretation from the SNP to the gene level. By considering these different aspects, we outline a more general strategy for linking SNP measurements to a time-to-event endpoint by means of sparse multivariable regression models.

Randomized phase-III study of low-dose cytarabine and etoposide + /- all-trans retinoic acid in older unfit patients with NPM1-mutated acute myeloid leukemia.

The aim of this randomized clinical trial was to evaluate the impact of all-trans retinoic acid (ATRA) in combination with non-intensive chemotherapy in older unfit patients (> 60 years) with newly diagnosed NPM1-mutated acute myeloid leukemia. Patients were randomized (1:1) to low-dose chemotherapy with or without open-label ATRA 45 mg/m2, days 8-28; the dose of ATRA was reduced to 45 mg/m2, days 8-10 and 15 mg/m2, days 11-28 after 75 patients due to toxicity. Up to 6 cycles of cytarabine 20 mg/day s.c., bid, days 1-7 and etoposide 100 mg/day, p.o. or i.v., days 1-3 with (ATRA) or without ATRA (CONTROL) were intended. The primary endpoint was overall survival (OS). Between May 2011 and September 2016, 144 patients (median age, 77 years; range, 64-92 years) were randomized (72, CONTROL; 72, ATRA). Baseline characteristics were balanced between the two study arms. The median number of treatment cycles was 2 in ATRA and 2.5 in CONTROL. OS was significantly shorter in the ATRA compared to the CONTROL arm (p = 0.023; median OS: 5 months versus 9.2 months, 2-years OS rate: 7% versus 10%, respectively). Rates of CR/CRi were not different between treatment arms; infections were more common in ATRA beyond treatment cycle one. The addition of ATRA to low-dose cytarabine plus etoposide in an older, unfit patient population was not beneficial, but rather led to an inferior outcome.The clinical trial is registered at clinicaltrialsregister.eu (EudraCT Number: 2010-023409-37, first posted 14/12/2010).

Microsatellite instability and Beta2-Microglobulin mutations as prognostic markers in colon cancer: results of the FOGT-4 trial.

BACKGROUND: High-level microsatellite instability (MSI-H) has been reported as a prognostic marker in colon cancer. We here analysed the prognostic significance of MSI and mutations of the Beta2-Microglobulin (B2M) gene, which occur in about 30% of MSI-H colon cancer, in the cohort of the prospective FOGT-4 (Forschungsruppe Onkologie Gastrointestinale Tumoren, FOGT) trial. METHODS: Microsatellite instability status was determined using standard protocols (NCI/ICG-HNPCC panel and CAT25) in 223 colon cancer lesions. Beta2-Microglobulin mutation status was evaluated by exon-wise sequencing in all MSI-H lesions. RESULTS: Patients with MSI-H (n=34) colon cancer presented with a significantly lower risk of relapse after 12 months of follow-up compared with MSS (n=189) colon cancer patients (5 year time to relapse: MSI-H 0.82 vs MSS 0.66, P=0.03). No significant difference in overall survival was detected. Beta2-Microglobulin mutations were identified in 10 (29.4%) out of 34 MSI-H colon cancers and were associated with a complete absence of disease relapse or tumour-related death events (P=0.09). CONCLUSION: The risk of late disease relapse was significantly lower in patients with MSI-H compared with MSS colon cancer. Moreover, B2M mutations may contribute to the favourable outcome of MSI-H colon cancer patients and should therefore be evaluated as a potential prognostic marker in future clinical trials.

Rationale and design of the CRAFT (Continuous ReAssessment with Flexible ExTension in Rare Malignancies) multicenter phase II trial.

BACKGROUND: Approvals of cancer therapeutics are primarily disease entity specific. Current molecular diagnostic approaches frequently identify actionable alterations in rare cancers or rare subtypes of common cancers for which the corresponding treatments are not approved and unavailable within clinical trials due to entity-related eligibility criteria. Access may be negotiated with health insurances. However, approval rates vary, and critical information required for a scientific evaluation of treatment-associated risks and benefits is not systematically collected. Thus clinical trials with optimized patient selection and comprehensive molecular characterization are essential for translating experimental treatments into standard care. PATIENTS AND METHODS: Continuous ReAssessment with Flexible ExTension in Rare Malignancies (CRAFT) is an open-label phase II trial for adults with pretreated, locally advanced, or metastatic solid tumors. Based on the evaluation by a molecular tumor board, patients are assigned to combinations of six molecularly targeted agents and a programmed death-ligand 1 (PD-L1) antagonist within seven study arms focusing on (i) BRAF V600 mutations; (ii) ERBB2 amplification and/or overexpression, activating ERBB2 mutations; (iii) ALK rearrangements, activating ALK mutations; (iv and v) activating PIK3CA and AKT mutations, other aberrations predicting increased PI3K-AKT pathway activity; (vi) aberrations predicting increased RAF-MEK-ERK pathway activity; (vii) high tumor mutational burden and other alterations predicting sensitivity to PD-L1 inhibition. The primary endpoint is the disease control rate (DCR) at week 16; secondary and exploratory endpoints include the progression-free survival ratio, overall survival, and patient-reported outcomes. Using Simon's optimal two-stage design, 14 patients are accrued for each study arm. If three or fewer patients achieve disease control, the study arm is stopped. Otherwise, 11 additional patients are accrued. If the DCR exceeds 7 of 25 patients, the null hypothesis is rejected for the respective study arm. CONCLUSIONS: CRAFT was activated in October 2021 and will recruit at 10 centers in Germany. TRIAL REGISTRATION NUMBERS: EudraCT: 2019-003192-18; ClinicalTrials.gov: NCT04551521.

FGFR4 Arg388 genotype is associated with pathological complete response to neoadjuvant chemotherapy for primary breast cancer.

BACKGROUND: A single-nucleotide polymorphism (SNP) in the FGFR4 gene is associated with poor prognosis in solid tumors. A recent study presented the first evidence that FGFR4 Arg388 could predict resistance to adjuvant chemotherapy in breast cancer. The present study evaluates the potential of this SNP to predict response to neoadjuvant chemotherapy (NCT) for primary breast cancer (PBC). METHODS: As part of a randomized phase II trial, 257 patients received either doxorubicin-cyclophosphamide (AC) or doxorubicin-pemetrexed (AP) followed by docetaxel (Doc; Taxotere) as NCT for T2-4/N0-2/M0 PBC. FGFR4 genotype analyzed on germline DNA was correlated with clinicopathologic variables, clinical response, and pathological complete response (pCR) using univariate and multivariate analyses. RESULTS: Only axillary lymph node status was associated with FGFR4 Arg388 [odds ratio (OR) 1.82, P = 0.03]. Joint analysis of both treatment arms revealed a correlation of FGFR4 Arg388 with clinical response (OR 2.14, P = 0.03) but not with pCR. In the AC-Doc arm, however, FGFR4 Arg388 was a strong predictor of pCR in the multivariate analysis (OR 3.79, P = 0.03). A significant interaction between FGFR4 genotype and treatment (P = 0.01) was found, indicating a therapy-specific effect. CONCLUSION: We provide the evidence that FGFR4 388Arg is an independent predictor of pCR following AC-Doc as NCT in PBC.

On the prognostic value of survival models with application to gene expression signatures.

As part of the validation of any statistical model, it is a good statistical practice to quantify the prediction accuracy and the amount of prognostic information represented by the model; this includes gene expression signatures derived from high-dimensional microarray data. Several approaches exist for right-censored survival data measuring the gain in prognostic information compared with established clinical parameters or biomarkers in terms of explained variation or explained randomness. They are either model-based or use estimates of prediction accuracy.As these measures differ in their underlying mechanisms, they vary in their interpretation, assumptions and properties, in particular in how they deal with the presence of censoring. It remains unclear, under what conditions and to what extent they are comparable. We present a comparison of several common measures and illustrate their behaviour in high-dimensional situations in simulation examples as well as in applications to real gene expression microarray data sets. An overview of available software implementations in R is given.

Active and inactive genes localize preferentially in the periphery of chromosome territories.

The intranuclear position of a set of genes was analyzed with respect to the territories occupied by the whole chromosomes in which these genes are localized. Genes and their respective chromosome territories were simultaneously visualized in three-dimensionally preserved nuclei applying dual color fluorescence in situ hybridization. Three coding (DMD, MYH7, and HBB) and two noncoding sequences (D1Z2 and an anonymous sequence) were analyzed in four different cell types, including cells where DMD and MYH7 are actively transcribed. Spatial analysis by confocal laser scanning microscopy revealed that the genes are preferentially located in the periphery of chromosome territories. This positioning was independent from the activity of the genes. In contrast, the non-expressed anonymous fragment was found randomly distributed or localized preferentially in the interior of the corresponding chromosome territory. Furthermore, the distribution of the analyzed genes within the territorial peripheries was found to be highly characteristic for each gene, and, again, independent from its expression. The impact of these findings with regard to the three-dimensional arrangement of the linear DNA string within chromosome territories, as well as with respect to a putative nuclear subcompartment confining gene expression, are discussed.

Randomized phase-II trial evaluating induction therapy with idarubicin and etoposide plus sequential or concurrent azacitidine and maintenance therapy with azacitidine.

The aim of this randomized phase-II study was to evaluate the effect of substituting cytarabine by azacitidine in intensive induction therapy of patients with acute myeloid leukemia (AML). Patients were randomized to four induction schedules for two cycles: STANDARD (idarubicin, cytarabine, etoposide); and azacitidine given prior (PRIOR), concurrently (CONCURRENT), or after (AFTER) therapy with idarubicin and etoposide. Consolidation therapy consisted of allogeneic hematopoietic-cell transplantation or three courses of high-dose cytarabine followed by 2-year maintenance therapy with azacitidine in the azacitidine-arms. AML with CBFB-MYH11, RUNX1-RUNX1T1, mutated NPM1, and FLT3-ITD were excluded and accrued to genotype-specific trials. The primary end point was response to induction therapy. The statistical design was based on an optimal two-stage design applied for each arm separately. During the first stage, 104 patients (median age 62.6, range 18-82 years) were randomized; the study arms PRIOR and CONCURRENT were terminated early due to inefficacy. After randomization of 268 patients, all azacitidine-containing arms showed inferior response rates compared to STANDARD. Event-free and overall survival were significantly inferior in the azacitidine-containing arms compared to the standard arm (p < 0.001 and p = 0.03, respectively). The data from this trial do not support the substitution of cytarabine by azacitidine in intensive induction therapy.

Hypermethylation and transcriptional downregulation of the CITED4 gene at 1p34.2 in oligodendroglial tumours with allelic losses on 1p and 19q.

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and have been associated with sensitivity to radio- and chemotherapy as well as favourable prognosis. By using microarray-based expression profiling, we found that oligodendroglial tumours with 1p and 19q losses showed significantly lower expression of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 as compared to tumours without 1p and 19q losses. Mutational analysis showed no CITED4 mutations in gliomas. However, 1p and 19q losses as well as low expression of CITED4 transcripts were significantly associated with hypermethylation of the CITED4-associated CpG island. In line with the latter finding, treatment of CITED4 hypermethylated glioma cell lines with 5-aza-2'-deoxycytidine and trichostatine A resulted in a marked increase of the CITED4 transcript levels. Furthermore, CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival of patients with oligodendroglial tumours. Taken together, our results indicate that CITED4 is epigenetically silenced in the vast majority of oligodendroglial tumours with 1p and 19q deletions and suggest CITED4 hypermethylation as a novel prognostic marker in oligodendroglioma patients.

High density of FOXP3-positive T cells infiltrating colorectal cancers with microsatellite instability.

High-level microsatellite instability (MSI-H) in colorectal cancer accounts for about 12% of colorectal cancers and is typically associated with a dense infiltration with cytotoxic CD8-positive lymphocytes. The role of regulatory T cells that may interfere with the host's antitumoural immune response in MSI-H colorectal cancers has not been analysed yet. Using an antibody directed against the regulatory T-cell marker transcription factor forkhead box P3 (FOXP3), regulatory T cells were examined in 70 colorectal cancers with known MSI status (MSI-H, n=37; microsatellite stable, n=33). In MSI-H colorectal cancers, we found a significantly higher intraepithelial infiltration with FOXP3-positive cells (median: 8.5 cells per 0.25 mm(2) vs 3.1 cells per 0.25 mm(2) in microsatellite stable, P<0.001), and a significantly elevated ratio of intraepithelial to stromal infiltration (0.05 vs 0.01 in microsatellite stable, P<0.001). CD8-positive cell counts were related positively to the number of FOXP3-positive cells (Spearman's rho=0.56 and 0.55, respectively). Our results show that the elevated number of CD8-positive lymphocytes found in MSI-H colorectal cancers is paralleled by an enhanced infiltration with CD8-negative FOXP3-positive cells. These data suggest that FOXP3-positive cells may play a role in the regulation of the immune response directed against MSI-H colorectal cancers at the primary tumour site.

Lack of CD56 expression on myeloma cells is not a marker for poor prognosis in patients treated by high-dose chemotherapy and is associated with translocation t(11;14).

Lack of CD56 expression was reported to be associated with a poor prognosis in multiple myeloma (MM) patients treated with conventional chemotherapy. Aim of our retrospective study was to analyse whether CD56 expression on MM cells reveals as a prognostic factor in patients treated with high-dose chemotherapy. MM cells of 99 patients prior to treatment with high-dose chemotherapy were analysed for CD56 expression by flow cytometry. Multivariable analysis of event-free survival in these patients showed no statistically significant difference between the CD56(-) (n=28) and the CD56(+) (n=71) group. The lack of CD56 expression on MM cells of these patients correlated significantly with the presence of translocation (11;14) (t(11;14)) (estimated correlation coefficient=0.655 95%, confidence interval (0.481; 0.779)). In summary, our results indicate that lack of CD56 expression on MM cells is not a prognostic marker in patients treated with high-dose chemotherapy, but is associated with t(11;14).

Proposals for sample size calculation programs.

OBJECTIVES: Numerous sample size calculation programs are available nowadays. They include both commercial products as well as public domain and open source applications. We propose modifications for these programs in order to even better support statistical consultation during the planning stage of a two-armed clinical trial. METHODS: Directional two-sided tests are commonly used for two-armed clinical trials. This may lead to a non-negligible Type III error risk in a severely underpowered study. In the case of a reasonably sized study the question for the so-called auxiliary alternative may evolve. RESULTS: We propose that sample size calculation programs should be able to compute i) Type III errors and the so-called q-values, ii) minimum sample sizes required to keep the q-values below pre-specified levels, and iii) detectable effect sizes of the so-called auxiliary alternatives. CONCLUSIONS: Proposals i and ii are intended to help prevent irresponsibly underpowered clinical trials, whereas the proposal iii is meant as additional assistance for the planning of reasonably sized clinical trials.

Transplant-associated thrombotic microangiopathy is an endothelial complication associated with refractoriness of acute GvHD.

There is increasing evidence that endothelial dysfunction is involved in refractoriness of acute GvHD (aGvHD). Here we investigated the hypothesis that another endothelial complication, transplant-associated thrombotic microangiopathy (TMA), contributes to the pathogenesis of aGvHD refractoriness. TMA was retrospectively assessed in 771 patients after allogeneic stem cell transplantation (alloSCT). Incidences of TMA and refractory aGvHD were correlated with biomarkers of endothelial damage obtained before alloSCT for patients receiving or not receiving statin-based endothelial prophylaxis (SEP). Diagnostic criteria for TMA and refractory aGvHD were met by 41 (5.3%) and 76 (10%) patients, respectively. TMA was overrepresented in patients with refractory aGvHD (45.0 vs 2.3% in all other patients, P<0.001). TMA independently increased mortality. Elevated pretransplant suppressor of tumorigenicity-2 and nitrates along with high-risk variants of the thrombomodulin gene were associated with increased risk of TMA. In contrast, SEP abolished the unfavorable outcome predicted by pretransplant biomarkers on TMA risk. Patients on SEP had a significantly lower risk of TMA (P=0.001) and refractory aGvHD (P=0.055) in a multivariate multistate model. Our data provide evidence that TMA contributes to the pathogenesis of aGvHD refractoriness. Patients with an increased TMA risk can be identified pretransplant and may benefit from pharmacological endothelium protection.

Pomalidomide in myeloproliferative neoplasm-associated myelofibrosis.

Myeloproliferative neoplasm (MPN)-associated myelofibrosis is a MPN characterized by bone marrow fibrosis, cytopenias, splenomegaly and constitutional symptoms. Pomalidomide, an immune-modifying drug, is reported to improve anaemia and thrombocytopenia in some patients with MPN-associated myelofibrosis. We designed a phase 2 study of pomalidomide in patients with MPN-associated myelofibrosis and anaemia and/or thrombocytopenia and/or neutropenia. Subjects received pomalidomide 2.0 mg/day in cohort 1 (n=38) or 0.5 mg/day in cohort 2 (n=58). Prednisolone was added if there was no response after 3 months in cohort 1 and based on up-front randomization in cohort 2 if there was no response at 3 or 6 months. Response rates were 39% (95% confidence interval (CI), 26-55%) in cohort 1 and 24% (95% CI, 15-37%) in cohort 2. In a multivariable logistic regression model pomalidomide at 2.0 mg/day (odds ratio (OR), 2.62; 95% CI, 1.00-6.87; P=0.05) and mutated TET2 (OR, 5.07; 95% CI, 1.16-22.17; P=0.03) were significantly associated with responses. Median duration of responses was 13.0 months (range 0.9-52.7). There was no significant difference in response rates or duration in subjects receiving or not receiving prednisolone. Clinical trial MPNSG 01-09 is registered at ClinicalTrials.gov (NCT00949364) and clinicaltrialsregister.eu (EudraCT Number: 2009-010738-23).