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Additional file 17. Uncropped electrophoretic gel of Fig. 3. A RT-PCR was used to confirm overexpression of pCYB5Aalt in NALM-6 cell line. Uncropped electrophoretic gel (agarose, 1.6%) of RT-PCR results of Fig. 3 are shown. Cells were transfected with pCYB5Aalt (+) or pEmpty (−). GAPDH was used as control. NTC = non-template-control. Size of PCR bands are shown in bp. Cropped parts of gel that were used for Fig. 3 are indicated by dotted, black rectangles.

Additional file 10. Wildtype CYB5A mRNA expression is lower in pCYB5Aalt NALM-6 than in empty vector controls. Relative mRNA expression measured by qRT-PCR is depicted as ΔCT(GAPDH-CYB5A). GAPDH was used as reference gene. Red dots represent mean expression of pCYB5Aalt NALM-6 (n = 11, mean = − 7.24) and empty vector controls (n = 3, mean = − 6.61). Red lines show standard error of the mean (pCYB5Aalt NALM-6: ±0.185, empty vector controls: ±0.063). The maximum and minimum expression value of CYB5Aalt clones were defined as outliers and excluded from statistical analysis. Mean expression was compared using both-sided t-test (p = 0.01).

Additional file 3. RNAseq pipeline algorithms of cohort 1 and cohort 2. RNAseq workflow is shown for cohort 1 and cohort 2 including most important steps of patient material collection, processing and data analysis.

Additional file 9. CYB5Aalt mRNA expression across molecular subtypes in cohort 3. CYB5Aalt expression (TPM log2) is shown across molecular subgroups in cohort 3. Only samples with detectable CYB5Aalt expression (n = 22) are displayed. Subgroups with CYB5Aalt positive samples include NH/HeH (n = 15), Ph-like (n = 5), LH (n = 1) and unknown (n = 1).

Additional file 12. Gene set enrichment analysis identifying differentially expressed pathways between pCYB5Aalt NALM-6 cells and Empty Vector controls. Gene set enrichment analysis was performed using the log2 foldchange/standard error of log2 foldchange between the transcriptional profiles of pCYB5Aalt and Empty Vector samples. A NES for MsigDB Hallmark pathways (n = 50) is displayed in a descending order, positive scores implying an upregulation. Pathways with a FDR

Additional file 7. Validation of CYB5Aalt in patient samples via RT-PCR and Sanger Sequencing. A RT-PCR with specific primers reaching from alternative Exon 1 to Exon 2 of CYB5A WT confirms expression of CYB5Aalt in cDNA derived from two representative patient RNA samples (AL03 REL, AE02 REL). CXCR4 expression was used as positive control, NTC = non-template control. Size of PCR product is shown in bp. Full length electrophoretic gel is shown in Additional File 16. B Sanger sequencing of CYB5Aalt PCR products in (A), depicted as chromatograms, confirms RNAseq sequence (top) of alternative transcript.

Additional file 11. Sample-to-sample distance between pCYB5Aalt NALM-6 and Empty Vector controls by Principal Component Analysis. Principal component analysis of rlog normalised RNAseq counts from pCYB5Aalt Nalm 6 cells (n = 4) and Empty Vector controls (n = 2) identifies different clusters. Empty vector controls define one cluster (bottom right). Three pCYB5Aalt NALM-6 clones define another (top middle). The fourth pCYB5Aalt clone was treated as an outlier and not included in further analysis.

Additional file 13. Gene set enrichment analysis comparing CYB5Aalt-positive and CYB5A WT samples in the NH/HeH gene expression cluster shows similar downregulated pathways to the overexpression cell line. Gene set enrichment analysis was performed between CYB5Aalt-positive samples (n = 15) and CYB5A WT samples (n = 7) in the NH/HeH cluster (n = 22) of combined cohort 3. NES for MsigDB Hallmark pathways (n = 50) is shown with positive scores implying an upregulation in CYB5Aalt-positive samples. Pathways with a FDR

Additional file 14. Expression of BH3-motif genes in engineered cell lines and combined cohort 3. BH3-motif genes are subdivided according to their reported roles in apoptosis in pro-apoptotic (BAD, BID, BCL2L11), anti-apoptotic (BCL2, BCL2L2, BCL2A1) and isoform-dependent (MCL1, BCL2L1). A BH3-motif gene expression (in TPM) is compared between pCYB5Aalt NALM-6 (n = 3) and empty vector controls (n = 2). Dots represent expression of individual clones. B Gene expression is compared between samples positive for CYB5Aalt (n = 22) and negative samples (n = 198) in combined cohort 3 (n = 220). Dots represent mean expression; vertical lines show standard error of mean. FDR = false discovery rate (Benjamini-Hochberg method).

Additional file 6. Schematic illustration of CYB5A WT- and CYB5Aalt-transcript. The graphic summarises the differences between CYB5A WT (top) and CYB5Aalt (bottom) at a transcript level. The WT ORF (dark grey), starts in exon 1, marked by “ATG” and the arrow, is disrupted in CYB5Aalt due to non-canonical exon usage (dashed boxes), skipping of exon 1 WT and splicing into exon 2 of the WT. This results in an alternative ORF (light grey) starting in WT exon 2, which is exon 3 of CYB5Aalt, lacking the full coding sequence of the heme binding domain, shown in red. The transmembrane region (green) is contained in both transcripts.

Additional file 8. Heatmap of subgroup-specific RNA expression profiles shows subgroup specificity of CYB5Aalt in cohort 2. Unsupervised clustering (Euclidean, Average Linkage) of 500 most variably expressed genes was performed in cohort 2 (n = 140). Samples could be grouped according to known subtypes of BCP-ALL (top). Further, CYB5Aalt occurrence is shown at the top for each patient sample. Columns indicate patient samples, rows indicate gene expression in TPM log2 for each sample. The colour of the heatmap indicates relative expression strength as deviation from the mean of all samples in TPM log2.

Additional file 16. Uncropped electrophoretic gel of Additional file 7. RT-PCR, followed by Sanger Sequencing (Additional Figure 5), was performed to validate CYB5Aalt expression in two representative patient samples (AL03 REL, AE02 REL). A and B show the top and the bottom of the same uncropped electrophoretic gel (agarose, 1.6%) showing the expression of different genes (CYB5A, CYB5Aalt, NTRK1, PEAR1, ZEB2, CXCR4, ZEB2-CXCR4) in two patient samples (AL03 REL, AE02 REL). CXCR4 was used as positive control. NTC = non-template-control. Ladder size is indicated in bp. Cropped parts of gel are indicated by dotted, black rectangles.

Additional file 4. Heatmap of subgroup-specific RNA expression profiles shows subgroup specificity of CYB5Aalt in cohort 1. Unsupervised clustering (Euclidean, Average Linkage) of 500 most variably expressed genes is shown in samples of cohort 1 (n = 80). Subgroup-allocation and occurrence of CYB5Aalt is shown for each patient sample at the top. Columns indicate patient samples, rows represent gene expression in TPM log2 for each sample. Colour of the heatmap cells indicates relative expression strength according to the change from the mean of all samples in TPM log2.

Nature Communications 13(1), 3881 (2022). doi:10.1038/s41467-022-31355-8, Published by Nature Publishing Group UK, [London]

Objectives: Detectable minimal residual disease (MRD) after therapy for acute lymphoblastic leukemia (ALL) is the strongest predictor of hematologic relapse. This study evaluated outcomes of patients with B-cell precursor ALL with MRD of ≥10−4 Methods: Study population was from ALL study groups in Europe managed in national study protocols 2000–2014. MRD was measured by polymerase chain reaction or flow cytometry. Patients were age ≥15 years at initial ALL diagnosis. Patients were excluded if exposed to blinatumomab within 18 months of baseline or prior alloHSCT. Results: Of 272 patients in CR1, baseline MRD was ≥10−1, 10−2 to −1, 10−3 to −2, and 10−4 to −3 in 15 (6%), 71 (26%), 109 (40%), and 77 (28%) patients, respectively. Median duration of complete remission (DoR) was 18.5 months (95% confidence interval [CI], 11.9–27.2), median relapse-free survival (RFS) was 12.4 months (95% CI, 10.0–19.0) and median overall survival (OS) was 32.5 months (95% CI, 23.6–48.0). Lower baseline MRD level (P ≤ .0003) and white blood cell count P ≤ .0053) were strong predictors for better RFS and DoR. Allogeneic hematopoietic stem cell transplantation (alloHSCT) was associated with longer RFS (hazard ratio [HR], 0.59; 95% CI, 0.41–0.84) and DoR (HR, 0.43; 95% CI, 0.29–0.64); the association with OS was not significant (HR, 0.72; 95% CI, 0.50–1.05). Discussion: In conclusion, RFS, DoR, and OS are relatively short in patients with MRD-positive ALL, particularly at higher MRD levels. AlloHSCT may improve survival but has limitations. Alternative approaches are needed to improve outcomes in MRD-positive ALL.

Figure S1. The distribution of lncRNAs and PC gene expression and DNA methylation levels across samples. Figure S2. BCP-ALL subtype-specific differentially expressed lncRNAs. Figure S3. Comparison of molecular pathways from cis and trans based analysis on subtype-specific DE lncRNAs. Figure S4. The subtype-specific lncRNAs co-expressed with oncogenes involved in key signaling pathways in DUX4 and Ph-like subtypes. (PDF 1510 kb)

Additional file 4. Available patient numbers (N) for analysis of distribution profiles of CD8+ and CD4+ T cells and subsets in Fig. 4.

Additional file 6. Available patient numbers (N) for analysis of cytokine and body temperature peak maxima in Fig. 6.

Additional file 3. Available patient numbers (N) for analysis of T-cell redistribution, LFA activation, and CD69 upregulation in Fig. 3.

Additional file 7. Available patient numbers (N) for analysis of cytokine and granzyme B profiles in Fig. 7.

Additional file 1. Available patient numbers (N) for analysis of liver and first-dose parameters in Fig. 1.

Additional file 2. Available patient numbers (N) for analysis of distribution profiles of neutrophils, thrombocytes, T cells and B cells in Fig. 2.

Additional file 5. Available patient numbers (N) for analysis of cytokine profiles in Fig. 5.

OBJECTIVE: Value frameworks in oncology have not been validated for the assessment of treatments in haematological malignancies, but to avoid overlaps and duplications it appears reasonable to build up experience on existing value frameworks, such as the European Society for Medical Oncology-Magnitude of Clinical Benefit Scale (ESMO-MCBS). METHODS: Here we present the results of the first feasibility testing of the ESMO-MCBS v1.1 for haematological malignancies based on the grading of 80 contemporary studies for acute leukaemia, chronic leukaemia, lymphoma, myeloma and myelodysplastic syndromes. The aims were (1) to evaluate the scorability of data, (2) to evaluate the reasonableness of the generated grades for clinical benefit using the current version and (3) to identify shortcomings in the ESMO-MCBS v1.1 that require amendments to improve the efficacy and validity of the scale in grading new treatments in the management of haematological malignancies. RESULTS: In general, the ESMO-MCBS v1.1 was found to be widely applicable to studies in haematological malignancies, generating scores that were judged as reasonable by European Hematology Association (EHA) experts. A small number of studies could either not be graded or were not appropriately graded. The reasons, related to the differences between haematological and solid tumour malignancies, are identified and described. CONCLUSIONS: Based on the findings of this study, ESMO and EHA are committed to develop a version of the ESMO-MCBS that is validated for haematological malignancies. This development process will incorporate all of the usual stringencies for accountability of reasonableness that have characterised the development of the ESMO-MCBS including field testing, statistical modelling, evaluation for reasonableness and openness to appeal and revision. Applying such a scale will support future public policy decision-making regarding the value of new treatments for haematological malignancies and will provide insights that could be helpful in the design of future clinical trials.

Objective: Value frameworks in oncology have not been validated for the assessment of treatments in haematological malignancies, but to avoid overlaps and duplications it appears reasonable to build up experience on existing value frameworks, such as the European Society for Medical Oncology—Magnitude of Clinical Benefit Scale (ESMO-MCBS). Methods: Here we present the results of the first feasibility testing of the ESMO-MCBS v1.1 for haematological malignancies based on the grading of 80 contemporary studies for acute leukaemia, chronic leukaemia, lymphoma, myeloma and myelodysplastic syndromes. The aims were (1) to evaluate the scorability of data, (2) to evaluate the reasonableness of the generated grades for clinical benefit using the current version and (3) to identify shortcomings in the ESMO-MCBS v1.1 that require amendments to improve the efficacy and validity of the scale in grading new treatments in the management of haematological malignancies. Results: In general, the ESMO-MCBS v1.1 was found to be widely applicable to studies in haematological malignancies, generating scores that were judged as reasonable by European Hematology Association (EHA) experts. A small number of studies could either not be graded or were not appropriately graded. The reasons, related to the differences between haematological and solid tumour malignancies, are identified and described. Conclusions: Based on the findings of this study, ESMO and EHA are committed to develop a version of the ESMO-MCBS that is validated for haematological malignancies. This development process will incorporate all of the usual stringencies for accountability of reasonableness that have characterised the development of the ESMO-MCBS including field testing, statistical modelling, evaluation for reasonableness and openness to appeal and revision. Applying such a scale will support future public policy decision-making regarding the value of new treatments for haematological malignancies and will provide insights that could be helpful in the design of future clinical trials.

Allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission is a standard of care for adult patients with Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) and high risk of relapse. However, the stratification systems vary among study groups. Inadequate response at the level of minimal residual disease is the most commonly accepted factor indicating the need for alloHSCT. In this consensus paper on behalf of the European Working Group for AdultAcute Lymphoblastic Leukemia and the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation, we summarize available evidence and reflect current clinical practice in major European study groups regarding both indications for HSCT and particular aspects of the procedure including the choice of donor, source of stem cells and conditioning. Finally, we propose recommendations for daily clinical practice as well as for planning of prospective trials.

Adults with relapsed/refractory acute lymphoblastic leukemia have an unfavourable prognosis, which is influenced by disease and patient characteristics. To further evaluate these characteristics, a retrospective analysis of 1,706 adult patients with Ph-negative relapsed/refractory B-precursor acute lymphoblastic leukemia diagnosed between 1990-2013 was conducted using data reflecting the standard of care from 11 study groups and large centers in Europe and the United States. Outcomes included complete remission, overall survival, and realization of stem cell transplantation after salvage treatment. The overall complete remission rate after first salvage was 40%, ranging from 35%-41% across disease status categories (primary refractory, relapsed with or without prior transplant), and was lower after second (21%) and third or greater (11%) salvage. The overall complete remission rate was higher for patients diagnosed from 2005 onward (45%, 95% CI: 39%-50%). One- and three-year survival rates after first, second, and third or greater salvage were 26% and 11%, 18% and 6%, and 15% and 4%, respectively, and rates were 2%-5% higher among patients diagnosed from 2005. Prognostic factors included younger age, longer duration of first remission, and lower white blood cell counts at primary diagnosis. This large dataset can provide detailed reference outcomes for patients with relapsed/refractory Ph-negative B-precursor acute lymphoblastic leukemia. clinicaltrials.gov identifier: 02003612.

The prognosis for adults with relapsed acute lymphoblastic leukemia is poor. We sought to determine whether inotuzumab ozogamicin, an anti-CD22 antibody conjugated to calicheamicin, results in better outcomes in patients with relapsed or refractory acute lymphoblastic leukemia than does standard therapy.In this phase 3 trial, we randomly assigned adults with relapsed or refractory acute lymphoblastic leukemia to receive either inotuzumab ozogamicin (inotuzumab ozogamicin group) or standard intensive chemotherapy (standard-therapy group). The primary end points were complete remission (including complete remission with incomplete hematologic recovery) and overall survival.Of the 326 patients who underwent randomization, the first 218 (109 in each group) were included in the primary intention-to-treat analysis of complete remission. The rate of complete remission was significantly higher in the inotuzumab ozogamicin group than in the standard-therapy group (80.7% [95% confidence interval {CI}, 72.1 to 87.7] vs. 29.4% [95% CI, 21.0 to 38.8], P0.001). Among the patients who had complete remission, a higher percentage in the inotuzumab ozogamicin group had results below the threshold for minimal residual disease (0.01% marrow blasts) (78.4% vs. 28.1%, P0.001); the duration of remission was longer in the inotuzumab ozogamicin group (median, 4.6 months [95% CI, 3.9 to 5.4] vs. 3.1 months [95% CI, 1.4 to 4.9]; hazard ratio, 0.55 [95% CI, 0.31 to 0.96]; P=0.03). In the survival analysis, which included all 326 patients, progression-free survival was significantly longer in the inotuzumab ozogamicin group (median, 5.0 months [95% CI, 3.7 to 5.6] vs. 1.8 months [95% CI, 1.5 to 2.2]; hazard ratio, 0.45 [97.5% CI, 0.34 to 0.61]; P0.001); the median overall survival was 7.7 months (95% CI, 6.0 to 9.2) versus 6.7 months (95% CI, 4.9 to 8.3), and the hazard ratio was 0.77 (97.5% CI, 0.58 to 1.03) (P=0.04). In the safety population, the most frequent grade 3 or higher nonhematologic adverse events with inotuzumab ozogamicin were liver-related. Veno-occlusive liver disease of any grade occurred in 15 patients (11%) who received inotuzumab ozogamicin and in 1 patient (1%) who received standard therapy.The rate of complete remission was higher with inotuzumab ozogamicin than with standard therapy, and a higher percentage of patients in the inotuzumab ozogamicin group had results below the threshold for minimal residual disease. Both progression-free and overall survival were longer with inotuzumab ozogamicin. Veno-occlusive liver disease was a major adverse event associated with inotuzumab ozogamicin. (Funded by Pfizer; INO-VATE ALL ClinicalTrials.gov number, NCT01564784.).