The presentation of results from studies in clinical haematology.

Research is based on trying to find answers to specific questions or to test hypotheses. Studies are thus undertaken to generate data which, with appropriate statistical methods, will help to determine the validity of the science under investigation. The aim of this paper is not to provide answers on which statistical methods to use, but will concentrate on suggesting the best ways of presenting the results of appropriately analysed data. And presentation is the key, because however well conducted and analysed a study may be, incorrect or inappropriate presentation of the findings will severely hamper its publication potential. With illustrative examples, the fundamentals required in the presentation of study objectives, population selection, description of characteristics and missing values, survival analyses, unadjusted analyses, multivariate regression models and matched pair analyses, are presented.

The accuracy of haemoglobin A2 measurements in the presence and absence of haemoglobin S.

The quantification of haemoglobin A2 by high-performance liquid chromatography (HPLC) was compared with quantification by capillary electrophoresis for control subjects and patients with sickle cell trait or sickle cell anaemia. Significant differences were found, with estimated values being higher by HPLC for control subjects and higher by capillary electrophoresis for sickle cell trait and sickle cell anaemia patients. There is an ongoing need for improved standardisation and alignment of methods.

Systems medicine dissection of chr1q-amp reveals a novel PBX1-FOXM1 axis for targeted therapy in multiple myeloma.

Understanding the biological and clinical impact of copy number aberrations (CNAs) on the development of precision therapies in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring an adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although several genes across chromosome 1 (chr1q) portend high-risk MM disease, the underpinning molecular etiology remains elusive. Here, with reference to the 3-dimensional (3D) chromatin structure, we integrate multi-omics data sets from patients with MM with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent among these genes, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed superenhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional program. Together, PBX1 and FOXM1 activate a proliferative gene signature that predicts adverse prognosis across multiple types of cancer. Notably, pharmacological disruption of the PBX1-FOXM1 axis with existing agents (thiostrepton) and a novel PBX1 small molecule inhibitor (T417) is selectively toxic against chr1q-amp myeloma and solid tumor cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes, and proposes novel CNA-targeted therapy strategies in MM and other types of cancer.

Diagnostic application of transcripts associated with antibody-mediated rejection in kidney transplant biopsies.

BACKGROUND: The diagnosis of antibody-mediated rejection (AMR) is reached using the Banff Classification for Allograft Pathology, which now includes gene expression analysis. In this study, we investigate the application of 'increased expression of thoroughly validated gene transcripts/classifiers strongly associated with AMR' as diagnostic criteria. METHOD: We used quantitative real-time polymerase chain reaction for 10 genes associated with AMR in a retrospective cohort of 297 transplant biopsies, including biopsies that met the full diagnostic criteria for AMR, even without molecular data (AMR, n = 27), biopsies that showed features of AMR, but that would only meet criteria for AMR with increased transcripts [suspicious for AMR (AMRsusp), n = 49] and biopsies that would never meet criteria for AMR (No-AMR, n = 221). RESULTS: A 10-gene AMR score trained by a receiver-operating characteristic to identify AMR found 16 cases with a high score among the AMRsusp cases (AMRsusp-high) that had significantly worse graft survival than those with a low score (AMRsusp-low; n = 33). In both univariate and multivariate Cox regression analysis, the AMR 10-gene score was significantly associated with an increased hazard ratio (HR) for graft loss (GL) in the AMRsusp group (HR = 1.109, P = 0.004 and HR = 1.138, P = 0.012, respectively), but not in the whole cohort. Net reclassification index and integrated discrimination improvement analyses demonstrated improved risk classification and superior discrimination, respectively, for GL when considering the gene score in addition to histological and serological data, but only in the AMRsusp group, not the whole cohort. CONCLUSIONS: This study provides evidence that a gene score strongly associated with AMR helps identify cases at higher risk of GL in biopsies that are suspicious for AMR but do not meet full criteria.

Fecal Microbiota Transplant Mitigates Adverse Outcomes Seen in Patients Colonized With Multidrug-Resistant Organisms Undergoing Allogeneic Hematopoietic Cell Transplantation.

The gut microbiome can be adversely affected by chemotherapy and antibiotics prior to hematopoietic cell transplantation (HCT). This affects graft success and increases susceptibility to multidrug-resistant organism (MDRO) colonization and infection. We performed an initial retrospective analysis of our use of fecal microbiota transplantation (FMT) from healthy donors as therapy for MDRO-colonized patients with hematological malignancy. FMT was performed on eight MDRO-colonized patients pre-HCT (FMT-MDRO group), and outcomes compared with 11 MDRO colonized HCT patients from the same period. At 12 months, survival was significantly higher in the FMT-MDRO group (70% versus 36% p = 0.044). Post-HCT, fewer FMT-MDRO patients required intensive care (0% versus 46%, P = 0.045) or experienced fever (0.29 versus 0.11 days, P = 0.027). Intestinal MDRO decolonization occurred in 25% of FMT-MDRO patients versus 11% non-FMT MDRO patients. Despite the significant differences and statistically comparable patient/transplant characteristics, as the sample size was small, a matched-pair analysis between both groups to non-MDRO colonized control cohorts (2:1 matching) was performed. At 12 months, the MDRO group who did not have an FMT had significantly lower survival (36.4% versus 61.9% respectively, p=0.012), and higher non relapse mortality (NRM; 60.2% versus 16.7% respectively, p=0.009) than their paired non-MDRO-colonized cohort. Conversely, there was no difference in survival (70% versus 43.4%, p=0.14) or NRM (12.5% versus 31.2% respectively, p=0.24) between the FMT-MDRO group and their paired non-MDRO cohort. Collectively, these data suggest that negative clinical outcomes, including mortality associated with MDRO colonization, may be ameliorated by pre-HCT FMT, even in the absence of intestinal MDRO decolonization. Further work is needed to explore this observed benefit.

Predictors of recovery following allogeneic CD34+-selected cell infusion without conditioning to correct poor graft function.

Poor graft function is a serious complication following allogeneic hematopoietic stem cell transplantation. Infusion of CD34+-selected stem cells without pre-conditioning has been used to correct poor graft function, but predictors of recovery are unclear. We report the outcome of 62 consecutive patients who had primary or secondary poor graft function who underwent a CD34+-selected stem cell infusion from the same donor without further conditioning. Forty-seven of 62 patients showed hematological improvement and became permanently transfusion and growth factor-independent. In multivariate analysis, parameters significantly associated with recovery were shared CMV seronegative status for recipient/donor, the absence of active infection and matched recipient/donor sex. Recovery was similar in patients with mixed and full donor chimerism. Five -year overall survival was 74.4% (95% CI 59-89) in patients demonstrating complete recovery, 16.7% (95% CI 3-46) in patients with partial recovery and 22.2% (CI 95% 5-47) in patients with no response. In patients with count recovery, those with poor graft function in 1-2 lineages had superior 5-year overall survival (93.8%, 95% CI 82-99) than those with tri-lineage failure (53%, 95% CI 34-88). New strategies including cytokine or agonist support, or second transplant need to be investigated in patients who do not recover.

Presence of donor-encoded centromeric KIR B content increases the risk of infectious mortality in recipients of myeloablative, T-cell deplete, HLA-matched HCT to treat AML.

The reported influence of donor Killer-cell Immunoglobulin-like Receptor (KIR) genes on the outcomes of haematopoietic cell transplantation (HCT) are contradictory, in part due to diversity of disease, donor sources, era and conditioning regimens within and between different studies. Here, we describe the results of a retrospective clinical analysis establishing the effect of donor KIR motifs on the outcomes of 119 HLA-matched, unrelated donor HCT for adult acute myeloid leukaemia (AML) using myeloablative conditioning (MAC) in a predominantly T-cell deplete (TCD) cohort. We observed that HCT involving donors with at least one KIR B haplotype were more likely to result in non-relapse mortality (NRM) than HCT involving donors with two KIR A haplotypes (p = 0.019). Upon separation of KIR haplotypes into their centromeric (Cen) and telomeric (Tel) motif structures, we demonstrated that the Cen-B motif was largely responsible for this effect (p = 0.001). When the cause of NRM was investigated further, infection was the dominant cause of death (p = 0.006). No evidence correlating donor KIR B haplotype with relapse risk was observed. The results from this analysis confirm previous findings in the unrelated, TCD, MAC transplant setting and imply a protective role for donor-encoded Cen-A motifs against infection in allogeneic HCT recipients.

Recipients Receiving Better HLA-Matched Hematopoietic Cell Transplantation Grafts, Uncovered by a Novel HLA Typing Method, Have Superior Survival: A Retrospective Study.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next-generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLA-matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

FOXM1 modulates 5-FU resistance in colorectal cancer through regulating TYMS expression.

Resistance to 5-Fluoruracil (5-FU) has been linked to elevated expression of the main target, thymidylate synthase (TYMS), which catalyses the de novo pathway for production of deoxythymidine monophosphate. The potent oncogenic forkhead box transcription factor, FOXM1 is is regulated by E2F1 which also controls TYMS. This study reveals a significant role of FOXM1 in 5-FU resistance. Overexpression and knock-down studies of FOXM1 in colon cancer cells suggest the importance of FOXM1 in TYMS regulation. ChIP and global ChIP-seq data also confirms that FOXM1 can also potentially regulate other 5-FU targets, such as TYMS, thymidine kinase 1 (TK-1) and thymidine phosphorylase (TYMP). In human colorectal cancer tissue specimens, a strong correlation of FOXM1 and TYMS staining was observed. Elevated FOXM1 and TYMS expression was also observed in acquired 5-FU resistant colon cancer cells (HCT116 5-FU Res). A synergistic effect was observed following treatment of CRC cells with an inhibitor of FOXM1, thiostrepton, in combination with 5-FU. The combination treatment decreased colony formation and migration, and induced cell cycle arrest, DNA damage, and apoptosis in CRC cell lines. In summary, this research demonstrated that FOXM1 plays a pivotal role in 5-FU resistance at least partially through the regulation of TYMS.

Impact of route and adequacy of nutritional intake on outcomes of allogeneic haematopoietic cell transplantation for haematologic malignancies.

BACKGROUND: Allogeneic haematopoietic cell transplantation (HCT) is often associated with poor oral intake due to painful mucositis and gastrointestinal sequalae that occur following a preparative regimen of intensive chemotherapy and/or total body radiation. Although attractive to assume that optimal nutrition improves HCT outcomes, there are limited data to support this. It is also unclear whether artificial nutrition support should be provided as enteral tube feeding or parenteral nutrition (PN). METHODS: We analysed day-100 non-relapse mortality (NRM), incidence of acute graft-versus-host disease (GvHD), acute gastrointestinal GvHD, 5-year survival and GvHD-free/relapse-free survival (GRFS) according to both route and adequacy of nutritional intake prior to neutrophil engraftment, together with other known prognostic factors, in a retrospective cohort of 484 patients who underwent allogeneic HCT for haematologic malignancy between 2000 and 2014. RESULTS: Multivariate analyses showed increased NRM with inadequate nutrition (hazard ratio (HR) 4.1; 95% confidence interval (CI) 2.2-7.2) and adequate PN (HR 2.9; 95% CI 1.6-5.4) compared to adequate enteral nutrition (EN) both P < .001. There were increased incidences of gastrointestinal GvHD of any stage and all GvHD ≥ grade 2 in patients who received PN (odds ratio (OR) 2.0; 95% CI 1.2-3.3; P = .006, and OR 1.8; 95% CI 1.1-3.0; P = .018, respectively), compared to adequate EN. Patients who received adequate PN and inadequate nutrition also had reduced probabilities of survival and GRFS at 5 years. CONCLUSION: Adequate EN during the early transplantation course is associated with reduced NRM, improved survival and GRFS at 5 years. Furthermore, adequate EN is associated with lower incidence of overall and gut acute GvHD than PN, perhaps because of its ability to maintain mucosal integrity, modulate the immune response to intensive chemo/radiotherapy and support the gastrointestinal tract environment, including gut microflora.

Harvests from bone marrow donors who weigh less than their recipients are associated with a significantly increased probability of a suboptimal harvest yield.

BACKGROUND: Previous studies have demonstrated the importance of bone marrow (BM) harvest yield in determining transplant outcomes, but little is known regarding donor and procedure variables associated with achievement of an optimal yield. We hypothesized that donor demographics and variables relating to the procedure were likely to impact the yield (total nucleated cells [TNCs]/kg recipient weight) and quality (TNCs/mL) of the harvest. STUDY DESIGN AND METHODS: To test our hypothesis, BM harvests of 110 consecutive unrelated donors were evaluated. The relationship between donor or procedure characteristics and the BM harvest yield was examined. RESULTS: The relationship between donor and recipient weight significantly influenced the harvest yield; only 14% of BM harvests from donors who weighed less than their recipient achieved a TNC count of more than 4 × 10(8) /kg compared to 56% of harvests from donors heavier than their recipient (p = 0.001). Higher-volume harvests were significantly less likely to achieve an optimal yield than lower-volume harvests (32% vs. 78%; p = 0.007), and higher-volume harvests contained significantly fewer TNCs/mL, indicating peripheral blood contamination. BM harvest quality also varied significantly between collection centers adding to recent concerns regarding maintenance of BM harvest expertise within the transplant community. CONCLUSION: Since the relationship between donor and recipient weight has a critical influence yield, we recommend prioritizing this secondary donor characteristic when selecting from multiple well-matched donors. Given the declining number of requests for BM harvests, it is crucial that systems are developed to train operators and ensure expertise in this procedure is retained.

The finer points of writing and refereeing scientific articles.

Writing scientific papers is a skill required by all haematologists. Many also need to be able to referee papers submitted to journals. These skills are not often formally taught and as a result may not be done well. We have reviewed published evidence of errors in these processes. Such errors may be ethical, scientific or linguistic, or may result from a lack of understanding of the processes. The objective of the review is, by highlighting errors, to help writers and referees to avoid them.

A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematological conditions. Acute graft-versus-host disease (aGVHD) is a prevalent immune-mediated complication following HSCT. Current diagnostic biomarkers that correlate with aGVHD severity, progression, and therapy response in graft recipients are insufficient. Here, we investigated whether epigenetic marks measured in peripheral blood of healthy graft donors stratify aGVHD severity in human leukocyte antigen (HLA)-matched sibling recipients prior to T cell-depleted HSCT. METHODS: We measured DNA methylation levels genome-wide at single-nucleotide resolution in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, with Illumina Infinium HumanMethylation450 BeadChips. RESULTS: Using genome-wide DNA methylation profiling, we showed that epigenetic signatures underlying aGVHD severity in recipients correspond to immune pathways relevant to aGVHD etiology. We discovered 31 DNA methylation marks in donors that associated with aGVHD severity status in recipients, and demonstrated strong predictive performance of these markers in internal cross-validation experiments (AUC = 0.98, 95% CI = 0.96-0.99). We replicated the top-ranked CpG classifier using an alternative, clinical DNA methylation assay (P = 0.039). In an independent cohort of 32 HSCT donors, we demonstrated the utility of the epigenetic classifier in the context of a T cell-replete conditioning regimen (P = 0.050). CONCLUSIONS: Our findings suggest that epigenetic typing of HSCT donors in a clinical setting may be used in conjunction with HLA genotyping to inform both donor selection and transplantation strategy, with the ultimate aim of improving patient outcome.

Predonation health-related quality of life scores predict time to recovery in hematopoietic stem cell donors.

The physical reactions to hematopoietic stem cell donation have been extensively studied, but less is known about factors that predict poorer donation experiences. The aim of this prospective study was to examine demographic and health-related quality of life (HRQOL) factors that might be associated with recovery and side effects. We also described the changes in HRQOL during the donation process. In total, 275 peripheral blood stem cell (PBSC) and 37 bone marrow (BM) consecutive donors completed the SF-36 questionnaire predonation and 4 weeks, and 3 months postdonation. Predonation HRQOL markers were the strongest predictors of time to recovery. Poorer predonation physical health was associated with longer recovery (P = .017) and certain side effects in PBSC donors. Poorer predonation mental health was associated with longer recovery in BM donors (P = .03) and pain after PBSC donation (P = .003). Physical HRQOL scores declined significantly from predonation to 4 weeks postdonation. This was shown both for PBSC and BM donors (P < .001 and P = .009, respectively), but the decline was much greater for BM donors. There was a return to predonation HRQOL values 3 months after donation in both groups with values well above the mean of the general population (P < .001).

Female donors and donors who are lighter than their recipient are less likely to meet the CD34+ cell dose requested for peripheral blood stem cell transplantation.

BACKGROUND: It is of clinical relevance to recognize donors who are unlikely to meet the requested stem cell dose for transplantation, as this group may benefit from an alternative mobilization regimen. This study was performed to evaluate the frequency of unrelated donor peripheral blood stem cell (PBSC) collections that meet the target yield and the impact of donor factors on this. STUDY DESIGN AND METHODS: All sequential PBSC collections facilitated by the national registry (n = 323) from January through December 2011 were analyzed. Donor factors analyzed included age, sex, weight, and presence of a central line. RESULTS: In univariate analyses, we found that reaching the target yield was significantly associated with a higher donor weight (85.6 kg vs. 75.3 kg, p < 0.001), male donor sex (55% vs. 19%, p < 0.001), a positive difference in weight between donor and recipient (4.3 kg vs. -8 kg, p < 0.001), and a higher volume of blood processed (13.8 L vs. 11.9 L, p < 0.001). After stepwise binary logistic regression, sex (p < 0.001) and difference between donor and recipient weight (p < 0.005) remained significantly associated with target yield being met after 1 day of collection. CONCLUSIONS: This study shows than women and donors who are lighter than their recipient have a decreased likelihood of meeting the transplant physician's requested dose. New strategies to improve mobilization in such donors are needed. These findings may also impact future donor recruitment strategies.

Night-time immobilization of the distal interphalangeal joint reduces pain and extension deformity in hand osteoarthritis.

OBJECTIVE: DIP joint OA is common but has few cost-effective, evidence-based interventions. Pain and deformity [radial or ulnar deviation of the joint or loss of full extension (extension lag)] frequently lead to functional and cosmetic issues. We investigated whether splinting the DIP joint would improve pain, function and deformity. METHODS: A prospective, radiologist-blinded, non-randomized, internally controlled trial of custom splinting of the DIP joint was carried out. Twenty-six subjects with painful, deforming DIP joint hand OA gave written, informed consent. One intervention joint and one control joint were nominated. A custom gutter splint was worn nightly for 3 months on the intervention joint, with clinical and radiological assessment at baseline, 3 and 6 months. Differences in the change were compared by the Wilcoxon signed rank test. RESULTS: The median average pain at baseline was similar in the intervention (6/10) and control joints (5/10). Average pain (primary outcome measure) and worst pain in the intervention joint were significantly lower at 3 months compared with baseline (P = 0.002, P = 0.02). Differences between intervention and control joint average pain reached significance at 6 months (P = 0.049). Extension lag deformity was significantly improved in intervention joints at 3 months and in splinted joints compared with matched contralateral joints (P = 0.016). CONCLUSION: Short-term night-time DIP joint splinting is a safe, simple treatment modality that reduces DIP joint pain and improves extension of the digit, and does not appear to give rise to non-compliance, increased stiffness or joint restriction. TRIAL REGISTRATION: clinical trials.gov, http://clinicaltrials.gov, NCT01249391.