Tool evaluation for the detection of variably sized indels from next generation whole genome and targeted sequencing data.

Insertions and deletions (indels) in human genomes are associated with a wide range of phenotypes, including various clinical disorders. High-throughput, next generation sequencing (NGS) technologies enable the detection of short genetic variants, such as single nucleotide variants (SNVs) and indels. However, the variant calling accuracy for indels remains considerably lower than for SNVs. Here we present a comparative study of the performance of variant calling tools for indel calling, evaluated with a wide repertoire of NGS datasets. While there is no single optimal tool to suit all circumstances, our results demonstrate that the choice of variant calling tool greatly impacts the precision and recall of indel calling. Furthermore, to reliably detect indels, it is essential to choose NGS technologies that offer a long read length and high coverage coupled with specific variant calling tools.

Efficacy of conventional-dose cytarabine, idarubicin and thioguanine versus intermediate-dose cytarabine and idarubicin in the induction treatment of acute myeloid leukemia: Long-term results of the prospective randomized nationwide AML-2003 study by the Finnish Leukemia Group.

OBJECTIVES: AML-2003 study sought to compare the long-term efficacy and safety of IAT and IdAraC-Ida in induction chemotherapy of acute myeloid leukemia (AML) and introduce the results of an integrated genetic and clinical risk classification guided treatment strategy. METHODS: Patients were randomized to receive either IAT or IdAraC-Ida as the first induction treatment. Intensified postremission strategies were employed based on measurable residual disease (MRD) and risk classification. Structured questionnaire forms were used to gather data prospectively. RESULTS: A total of 356 AML patients with a median age of 53 years participated in the study. Long-term overall survival (OS) and relapse-free survival (RFS) were both 49% at 10 years. The median follow-up was 114 months. No significant difference in remission rate, OS or RFS was observed between the two induction treatments. Risk classification according to the protocol, MRD after the first and the last consolidation treatment affected the OS and RFS significantly (p < .001). CONCLUSIONS: Intensified cytarabine dose in the first induction treatment was not better than IAT in patients with AML. Intensification of postremission treatment in patients with clinical risk factors or MRD seems reasonable, but randomized controlled studies are warranted in the future.

Measurable residual disease assessment by qPCR in peripheral blood is an informative tool for disease surveillance in childhood acute myeloid leukaemia.

Serial assessments of measurable (or minimal) residual disease (MRD) by qPCR may identify nascent relapse in children with acute myeloid leukaemia (AML) and enable pre-emptive therapy. We investigated the kinetics and prognostic impact of recurrent fusion transcripts (RUNX1-RUNX1T1, CBFB-MYH11, KMT2A-MLLT3 or KMT2A-ELL) in 774 post-induction samples from bone marrow (BM, 347) and peripheral blood (PB, 427) from 75 children with AML. BM MRD persistence during consolidation did not increase the risk of relapse, and MRD at therapy completion did not correlate to outcome (HR = 0·64/MRD log reduction (CI: 0·32-1·26), P = 0·19). In contrast, 8/8 patients with detectable MRD in PB after first consolidation relapsed. Persistence (n = 4) and shifting from negative to positive (n = 10) in PB during follow-up predicted relapse in 14/14 patients. All 253 PB samples collected during follow-up from 36 patients in continuous complete remission were MRD negative. In core-binding factor AML, persistent low-level MRD positivity in BM during follow-up was frequent but an increment to above 5 × 10-4 heralded subsequent haematological relapse in 12/12 patients. We demonstrate that MRD monitoring in PB after induction therapy is highly informative and propose an MRD increment above 5 × 10-4 in PB and BM as a definition of molecular relapse since it always leads to haematological relapse.

Discontinuation of tyrosine kinase inhibitor therapy in chronic myeloid leukaemia (EURO-SKI): a prespecified interim analysis of a prospective, multicentre, non-randomised, trial.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) have improved the survival of patients with chronic myeloid leukaemia. Many patients have deep molecular responses, a prerequisite for TKI therapy discontinuation. We aimed to define precise conditions for stopping treatment. METHODS: In this prospective, non-randomised trial, we enrolled patients with chronic myeloid leukaemia at 61 European centres in 11 countries. Eligible patients had chronic-phase chronic myeloid leukaemia, had received any TKI for at least 3 years (without treatment failure according to European LeukemiaNet [ELN] recommendations), and had a confirmed deep molecular response for at least 1 year. The primary endpoint was molecular relapse-free survival, defined by loss of major molecular response (MMR; >0·1% BCR-ABL1 on the International Scale) and assessed in all patients with at least one molecular result. Secondary endpoints were a prognostic analysis of factors affecting maintenance of MMR at 6 months in learning and validation samples and the cost impact of stopping TKI therapy. We considered loss of haematological response, progress to accelerated-phase chronic myeloid leukaemia, or blast crisis as serious adverse events. This study presents the results of the prespecified interim analysis, which was done after the 6-month molecular relapse-free survival status was known for 200 patients. The study is ongoing and is registered with ClinicalTrials.gov, number NCT01596114. FINDINGS: Between May 30, 2012, and Dec 3, 2014, we assessed 868 patients with chronic myeloid leukaemia for eligibility, of whom 758 were enrolled. Median follow-up of the 755 patients evaluable for molecular response was 27 months (IQR 21-34). Molecular relapse-free survival for these patients was 61% (95% CI 57-64) at 6 months and 50% (46-54) at 24 months. Of these 755 patients, 371 (49%) lost MMR after TKI discontinuation, four (1%) died while in MMR for reasons unrelated to chronic myeloid leukaemia (myocardial infarction, lung cancer, renal cancer, and heart failure), and 13 (2%) restarted TKI therapy while in MMR. A further six (1%) patients died in chronic-phase chronic myeloid leukaemia after loss of MMR and re-initiation of TKI therapy for reasons unrelated to chronic myeloid leukaemia, and two (<1%) patients lost MMR despite restarting TKI therapy. In the prognostic analysis in 405 patients who received imatinib as first-line treatment (learning sample), longer treatment duration (odds ratio [OR] per year 1·14 [95% CI 1·05-1·23]; p=0·0010) and longer deep molecular response durations (1·13 [1·04-1·23]; p=0·0032) were associated with increasing probability of MMR maintenance at 6 months. The OR for deep molecular response duration was replicated in the validation sample consisting of 171 patients treated with any TKI as first-line treatment, although the association was not significant (1·13 [0·98-1·29]; p=0·08). TKI discontinuation was associated with substantial cost savings (an estimated €22 million). No serious adverse events were reported. INTERPRETATION: Patients with chronic myeloid leukaemia who have achieved deep molecular responses have good molecular relapse-free survival. Such patients should be considered for TKI discontinuation, particularly those who have been in deep molecular response for a long time. Stopping treatment could spare patients from treatment-induced side-effects and reduce health expenditure. FUNDING: ELN Foundation and France National Cancer Institute.

Combination of pegylated IFN-α2b with imatinib increases molecular response rates in patients with low- or intermediate-risk chronic myeloid leukemia.

Biologic and clinical observations suggest that combining imatinib with IFN-α may improve treatment outcome in chronic myeloid leukemia (CML). We randomized newly diagnosed chronic-phase CML patients with a low or intermediate Sokal risk score and in imatinib-induced complete hematologic remission either to receive a combination of pegylated IFN-α2b (Peg-IFN-α2b) 50 µg weekly and imatinib 400 mg daily (n = 56) or to receive imatinib 400 mg daily monotherapy (n = 56). The primary endpoint was the major molecular response (MMR) rate at 12 months after randomization. In both arms, 4 patients (7%) discontinued imatinib treatment (1 because of blastic transformation in imatinib arm). In addition, in the combination arm, 34 patients (61%) discontinued Peg-IFN-α2b, most because of toxicity. The MMR rate at 12 months was significantly higher in the imatinib plus Peg-IFN-α2b arm (82%) compared with the imatinib monotherapy arm (54%; intention-to-treat, P = .002). The MMR rate increased with the duration of Peg-IFN-α2b treatment (< 12-week MMR rate 67%, > 12-week MMR rate 91%). Thus, the addition of even relatively short periods of Peg-IFN-α2b to imatinib markedly increased the MMR rate at 12 months of therapy. Lower doses of Peg-IFN-α2b may enhance tolerability while retaining efficacy and could be considered in future protocols with curative intent.

Mono/oligoclonal T and NK cells are common in chronic myeloid leukemia patients at diagnosis and expand during dasatinib therapy.

In a proportion of patients with chronic myeloid leukemia (CML) being treated with dasatinib, we recently observed large granular lymphocyte (LGL) expansions carrying clonal T-cell receptor (TCR) gamma/delta gene rearrangements. To assess the prevalence and role of clonal lymphocytes in CML, we collected samples from patients (n = 34) at the time of diagnosis and during imatinib and dasatinib therapies and analyzed lymphocyte clonality with a sensitive polymerase chain reaction-based method of TCR gamma and delta genes. Surprisingly, at CML diagnosis, 15 of 18 patients (83%) had a sizeable clonal, BCR-ABL1 negative lymphocyte population, which was uncommon in healthy persons (1 of 12; 8%). The same clone persisted at low levels in most imatinib-treated patients. In contrast, in a distinct population of dasatinib-treated patients, the diagnostic phase clone markedly expanded, resulting in absolute lymphocytosis in blood. Most patients with LGL expansions (90%) had TCR delta rearrangements, which were uncommon in patients without an LGL expansion (10%). The TCR delta clones were confined to gammadelta(+) T- or natural killer-cell compartments and the TCR gamma clones to CD4(+)/CD8(+) alphabeta(+) fractions. The functional importance of clonal lymphocytes as a part of leukemia immune surveillance and the putative anergy-reversing role of dasatinib require further evaluation.

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Gene fusions and oncogenic mutations in MLH1 deficient and BRAFV600E wild-type colorectal cancers.

Gene fusions can act as oncogenic drivers and offer targets for cancer therapy. Since fusions are rare in colorectal cancer (CRC), their universal screening seems impractical. Our aim was to investigate gene fusions in 62 CRC cases with deficient MLH1 (dMLH1) and BRAFV600E wild-type (wt) status from a consecutive real-life series of 2079 CRCs. First, gene fusions were analysed using a novel FusionPlex Lung v2 RNA-based next-generation sequencing (NGS) panel, and these results were compared to a novel Idylla GeneFusion assay and pan-TRK immunohistochemistry (IHC). NGS detected seven (7/62, 11%) NTRK1 fusions (TPM3::NTRK1, PLEKHA6::NTRK1 and LMNA::NTRK1, each in two cases, and IRF2BP2::NTRK1 in one case). In addition, two ALK, four RET and seven BRAF fusions were identified. Idylla detected seven NTRK1 expression imbalances, in line with the NGS results (overall agreement 100%). Furthermore, Idylla detected the two NGS-identified ALK rearrangements as one specific ALK fusion and one ALK expression imbalance, whilst only two of the four RET fusions were discovered. However, Idylla detected several expression imbalances of ALK (n = 7) and RET (n = 1) that were found to be fusion negative with the NGS. Pan-TRK IHC showed clearly detectable, fusion partner-dependent staining patterns in the seven NTRK1 fusion cases. Overall agreement for pan-TRK antibody clone EPR17341 was 98% and for A7H6R 100% when compared to the NGS. Of the 62 CRCs, 43 were MLH1 promoter hypermethylated (MLH1ph) and 39 were RASwt. All fusion cases were both MLH1ph and RASwt. Our results show that kinase fusions (20/30, 67%) and most importantly targetable NTRK1 fusions (7/30, 23%) are frequent in CRCs with dMLH1/BRAFV600Ewt/MLH1ph/RASwt. NGS was the most comprehensive method in finding the fusions, of which a subset can be screened by Idylla or IHC, provided that the result is confirmed by NGS.

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study.

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.

Comparison of imatinib 400 mg and 800 mg daily in the front-line treatment of high-risk, Philadelphia-positive chronic myeloid leukemia: a European LeukemiaNet Study.

Imatinib mesylate (IM), 400 mg daily, is the standard treatment of Philadelphia-positive (Ph(+)) chronic myeloid leukemia (CML). Preclinical data and results of single-arm studies raised the suggestion that better results could be achieved with a higher dose. To investigate whether the systematic use of a higher dose of IM could lead to better results, 216 patients with Ph(+) CML at high risk (HR) according to the Sokal index were randomly assigned to receive IM 800 mg or 400 mg daily, as front-line therapy, for at least 1 year. The CCgR rate at 1 year was 64% and 58% for the high-dose arm and for the standard-dose arm, respectively (P = .435). No differences were detectable in the CgR at 3 and 6 months, in the molecular response rate at any time, as well as in the rate of other events. Twenty-four (94%) of 25 patients who could tolerate the full 800-mg dose achieved a CCgR, and only 4 (23%) of 17 patients who could tolerate less than 350 mg achieved a CCgR. This study does not support the extensive use of high-dose IM (800 mg daily) front-line in all CML HR patients. This trial was registered at www.clinicaltrials.gov as #NCT00514488.

Depth of response assessed by quantitative ASO-PCR predicts the outcome after stem cell transplantation in multiple myeloma.

Achievement of complete response (CR) is a new goal of therapy for multiple myeloma (MM). By sensitive methods, the depth of response can be measured even among the patients in CR. We used a sensitive real-time quantitative polymerase chain reaction by allele-specific primers (qASO-PCR) to assess the level of minimal residual disease (MRD) in bone marrow of 37 patients with myeloma who had achieved CR/near-to-CR after autologous or allogeneic stem cell transplantation (SCT). Allele-specific primers could be successfully designed for 86% of patients. Three to six months after autotransplantation, the PCR target was not detectable in 53% of patients (16/30 patients), and the respective figure after allotransplantation was 71% (5/7 patients); the median sensitivity of PCR assay was <0.002%. The proportion of patients without detectable PCR target was 22% of all autotransplanted patients. A threshold level of 0.01% in the qASO-PCR assay 3-6 months after SCT was found to be a useful cut-off limit to divide the patients into two prognostic groups: MRD low/negative vs. MRD high. Low/negative MRD after SCT was a significant predictive factor for the prolongation of progression free (70 vs. 19 months; P = 0.003) and suggestively also for overall survival. We conclude that not only CR but also its depth is important for the long-term outcome in MM.

Association of injury severity, MRI-results and ApoE genotype with 1-year outcome in mainly mild TBI: a preliminary study.

PRIMARY OBJECTIVE: To study the ability of MRI findings, apolipoprotein E (ApoE) genotype, the Glasgow Coma Score (GCS) and duration of post-traumatic amnesia (PTA) to predict the 1-year outcome in traumatic brain injury (TBI). RESEARCH DESIGN: A prospective study in unselected emergency room patients with an acute TBI, followed for 1 year. METHODS AND PROCEDURES: Thirty-three consecutive patients were studied. The TBI severity was assessed with GCS and duration of PTA. Brain MRI scans were obtained within approximately 1 week post-injury. The ApoE genotypes were determined by standard methods. The outcome was evaluated with the Head Injury Symptom Checklist and the Glasgow Outcome Scale (extended) 1 year after the injury. The prognostic value of the explanatory variables was evaluated with multiple linear regression analysis. MAIN OUTCOMES AND RESULTS: The presence of traumatic axonal injury lesions or contusions in MRI and duration of PTA were independent predictors of poor outcome. The ApoE genotype and GCS were not associated with outcome. CONCLUSIONS: In multivariate models, the duration of PTA and acute MRI are the best predictors of 1-year outcome in TBI, whereas the prognostic values of GCS and ApoE are modest. The dominating role of GCS in assessing TBI severity should be questioned.

Arpp19 Promotes Myc and Cip2a Expression and Associates with Patient Relapse in Acute Myeloid Leukemia.

Disease relapse from standard chemotherapy in acute myeloid leukemia (AML) is poorly understood. The importance of protein phosphatase 2A (PP2A) as an AML tumor suppressor is emerging. Therefore, here, we examined the potential role of endogenous PP2A inhibitor proteins as biomarkers predicting AML relapse in a standard patient population by using three independent patient materials: cohort1 (n = 80), cohort2 (n = 48) and The Cancer Genome Atlas Acute Myeloid Leukemia (TCGA LAML) dataset (n = 160). Out of the examined PP2A inhibitors (CIP2A, SET, PME1, ARPP19 and TIPRL), expression of ARPP19 mRNA was found to be independent of the current AML risk classification. Functionally, ARPP19 promoted AML cell viability and expression of oncoproteins MYC, CDK1, and CIP2A. Clinically, ARPP19 mRNA expression was significantly lower at diagnosis (p = 0.035) in patients whose disease did not relapse after standard chemotherapy. ARPP19 was an independent predictor for relapse both in univariable (p = 0.007) and in multivariable analyses (p = 0.0001) and gave additive information to EVI1 expression and risk group status (additive effect, p = 0.005). Low ARPP19 expression was also associated with better patient outcome in the TCGA LAML cohort (p = 0.019). In addition, in matched patient samples from diagnosis, remission and relapse phases, ARPP19 expression was associated with disease activity (p = 0.034), indicating its potential usefulness as a minimal residual disease (MRD) marker. Together, these data demonstrate the oncogenic function of ARPP19 in AML and its risk group independent role in predicting AML patient relapse tendency.

Comparison of bone marrow high mitotic index metaphase fluorescence in situ hybridization to peripheral blood and bone marrow real time quantitative polymerase chain reaction on the International Scale for detecting residual disease in chronic myeloid leukemia.

BACKGROUND: Recently, an International Scale was proposed for standardizing BCR-ABL transcript measurements and reporting in the assessment of minimal residual disease by real-time quantitative polymerase chain reaction (RQ-PCR). Here we present the setting up of the International Scale conversion factors for a national laboratory by performing both a cross-analysis of a set of standard samples from a reference laboratory and an analysis of bone marrow and peripheral blood samples at diagnosis (from 32 and 27 patients, respectively). DESIGN AND METHODS: A total of 222 bone marrow and 173 peripheral blood mononuclear cell samples from 96 patients with chronic myeloid leukemia were analyzed with RQ-PCR according to Europe Against Cancer protocols. Additionally, 291 bone marrow samples were analyzed with high mitotic index metaphase fluorescence in situ hybridization (metaphase FISH). RESULTS: Major molecular response according to the International Scale in BCR-ABL/GUS transcript levels corresponded to a ratio of 0.035% in peripheral blood and 0.034% in bone marrow, yielding the same conversion factor of 2.86 for both types of sample. Based on metaphase FISH, values of 10%/-1.0 log, 1%/-2.0 log and 0.1%/-3.0 log on the International Scale, corresponded to 13%, 2%, and 0.3% of Philadelphia chromosome positive cells in bone marrow, respectively. CONCLUSIONS: In conclusion, conversion factors can be determined either by cross-analyzing a number of samples with a laboratory that has already established the International Scale or utilizing sufficient numbers of reference samples from chronic myeloid leukemia patients at diagnosis, or using the upcoming international standards.

Wilms tumour gene 1 overexpression in bone marrow as a marker for minimal residual disease in acute myeloid leukaemia.

OBJECTIVES: Wilms tumour gene 1 (WT1) is overexpressed in leucocytes of most acute myeloid leukaemia (AML) patients. However, the clinical relevance of WT1 gene expression as minimal residual disease (MRD) marker in AML has been questioned. METHODS: We determined the expression of WT1 gene in bone marrow (BM) mononuclear cells of 100 AML patients at diagnosis and compared it with other MRD markers during follow up in 16 patients using quantitative reverse transcription-polymerase chain reaction. RESULTS: The median WT1 gene expression was 9.7% of K562 cell line WT1 expression (lower quartile 1.5%, upper quartile 29.9%, n = 100) at diagnosis and, 0.053% (lower quartile 0.022%, upper quartile 0.125%, n = 87) in molecular or immunophenotypic remission. Median WT1 expression in control BM was 0.029% (lower quartile 0.013%, upper quartile 0.061%, n = 22). The upper 99% percentile of remission samples was 0.3%, which was regarded as the cut-off of increased WT1 gene expression in AML and was exceeded in 87% of all AML patients at diagnosis. WT1 and the other MRD markers showed only minor differences in profiles during follow-up. WT1 expression at diagnosis with median value 9.7% as the cut-off level or as a continuous variable had no prognostic significance for 2-yr survival. CONCLUSIONS: The sensitivity of WT1 as a MRD marker was low due to the relatively high background WT1 gene expression in BM cells at remission and in subjects without haematological malignancies. Therefore, WT1 gene expression analysis would be beneficial only in those patients who do not have a more specific and sensitive MRD marker.

Stem cell transplantation in poor-risk chronic lymphocytic leukemia: assessment of post-transplant minimal residual disease using four- and six-color flow cytometry and allele-specific RQ-PCR.

A total of 178 bone marrow samples were taken for minimal residual disease (MRD) analysis after 34 stem cell transplantations for poor-risk chronic lymphocytic leukemia, and 86 of them were analyzed in parallel by flow cytometry and allele-specific oligonucleotide-PCR (ASO-PCR). ASO primer was successfully designed for all patients whose frozen diagnosis samples were available. Flow cytometry and ASO-PCR were concordant, i.e. both either positive or both negative, in 78% of the analyses. Flow cytometry did not detect MRD in any of the samples that were PCR-negative cases. In contrast, ASO-PCR detected MRD in samples that were negative for MRD by flow cytometry in 22% of the analyses. In one patient, the immunophenotype but not the IgV(H) gene sequence had changed during a course of the disease, and MRD could not be followed by flow cytometry. In the remaining cases, the discrepancy was due to a higher sensitivity of ASO-PCR. Autologous stem cell transplantation resulted in clinical complete response in 87% (20/23) of the patients. By flow cytometry, 35% (8/23) of autotransplanted patients became MRD-negative, but only 12.5% (2/16) PCR-negative (sensitivity of ASO-PCR <0.001 and <0.01, respectively). All allotransplanted patients achieved or maintained hematological CR, and five out of nine patients (56%) became PCR-negative (sensitivity of PCR between <0.001 and <0.003), two of them having non-myeloablative conditioning. None of the patients who became PCR-negative after allogeneic transplantation have relapsed.

Outcome of traumatic brain injury after three decades--relationship to ApoE genotype.

Significant traumatic brain injury (TBI) is nearly always associated with cognitive deficits, but in a highly variable manner. Apolipoprotein E (ApoE) plays a pivotal role in CNS response to injury. To examine the association of ApoE genotype with long-term outcome in TBI patients, we determined the ApoE genotype from 61 TBI patients who had been injured over three decades earlier. All patients had been studied neuropsychologically after their injuries. The long-term outcome was evaluated with repeated neuropsychological testing and by applying various measures of everyday functioning and quality of life. After three decades, TBI patients with the ApoE epsilon4 allele showed significantly poorer general cognitive level than those without this allele. This decline was wholly accounted for by a subgroup of these patients who had developed incident or clinical dementia, while the majority of the ApoE epsilon4 positive patients showed no decline at all. The other outcome measures describing vocational, physical, or subjective symptom outcome did not show significant relationships to the ApoE genotype. A portion of the TBI patients with the ApoE epsilon4 allele seem to be at risk of long-term cognitive decline.

Chronic myeloid leukemia patients in prolonged remission following interferon-α monotherapy have distinct cytokine and oligoclonal lymphocyte profile.

Before the era of tyrosine kinase inhibitors (TKIs), interferon-alpha (IFN-α) was the treatment of choice in chronic myeloid leukemia (CML). Curiously, some IFN-α treated patients were able to discontinue therapy without disease progression. The aim of this project was to study the immunomodulatory effects of IFN-α in CML patients in prolonged remission and isolate biological markers predicting response. Due to rarity of patients on IFN-α monotherapy, a relatively small cohort of patients still on treatment (IFN-ON, n = 10, median therapy duration 11.8 years) or had discontinued IFN-α therapy but remained in remission for >2 years (IFN-OFF, n = 9) were studied. The lymphocyte immunophenotype was analyzed with a comprehensive flow cytometry panel and plasma cytokine levels were measured with multiplex bead-based assay. In addition, the clonality status of different lymphocyte subpopulations was analyzed by TCR γ/δ rearrangement assay. Median NK-cell absolute number and proportion from lymphocytes in blood was higher in IFN-OFF patients as compared to IFN-ON patients or controls (0.42, 0.19, 0.21×10(9)/L; 26%, 12%, 11%, respectively, p<0.001). The proportion of CD8+ T-cells was significantly increased in both patient groups and a larger proportion of T-cells expressed CD45RO. Most (95%) patients had significant numbers of oligoclonal lymphocytes characterized by T-cell receptor γ/δ rearrangements. Strikingly, in the majority of patients (79%) a distinct clonal Vγ9 gene rearrangement was observed residing in γδ(+) T-cell population. Similar unique clonality pattern was not observed in TKI treated CML patients. Plasma eotaxin and MCP-1 cytokines were significantly increased in IFN-OFF patients. Despite the limited number of patients, our data indicates that IFN-α treated CML patients in remission have increased numbers of NK-cells and clonal γδ(+) T-cells and a unique plasma cytokine profile. These factors may relate to anti-leukemic effects of IFN-α in this specific group of patients and account for prolonged therapy responses even after drug discontinuation.