F18-FDG PET/CT imaging early predicts pathologic complete response to induction chemoimmunotherapy of locally advanced head and neck cancer: preliminary single-center analysis of the checkrad-cd8 trial.

AIM: In the CheckRad-CD8 trial patients with locally advanced head and neck squamous cell cancer are treated with a single cycle of induction chemo-immunotherapy (ICIT). Patients with pathological complete response (pCR) in the re-biopsy enter radioimmunotherapy. Our goal was to study the value of F-18-FDG PET/CT in the prediction of pCR after induction therapy. METHODS: Patients treated within the CheckRad-CD8 trial that additionally received FDG- PET/CT imaging at the following two time points were included: 3-14 days before (pre-ICIT) and 21-28 days after (post-ICIT) receiving ICIT. Tracer uptake in primary tumors (PT) and suspicious cervical lymph nodes (LN +) was measured using different quantitative parameters on EANM Research Ltd (EARL) accredited PET reconstructions. In addition, mean FDG uptake levels in lymphatic and hematopoietic organs were examined. Percent decrease (Δ) in FDG uptake was calculated for all parameters. Biopsy of the PT post-ICIT acquired after FDG-PET/CT served as reference. The cohort was divided in patients with pCR and residual tumor (ReTu). RESULTS: Thirty-one patients were included. In ROC analysis, ΔSUVmax PT performed best (AUC = 0.89) in predicting pCR (n = 17), with a decline of at least 60% (sensitivity, 0.77; specificity, 0.93). Residual SUVmax PT post-ICIT performed best in predicting ReTu (n = 14), at a cutpoint of 6.0 (AUC = 0.91; sensitivity, 0.86; specificity, 0.88). Combining two quantitative parameters (ΔSUVmax ≥ 50% and SUVmax PT post-ICIT ≤ 6.0) conferred a sensitivity of 0.81 and a specificity of 0.93 for determining pCR. Background activity in lymphatic organs or uptake in suspected cervical lymph node metastases lacked significant predictive value. CONCLUSION: FDG-PET/CT can identify patients with pCR after ICIT via residual FDG uptake levels in primary tumors and the related changes compared to baseline. FDG-uptake in LN + had no predictive value. TRIAL REGISTRY: ClinicalTrials.gov identifier: NCT03426657.

A MDR1 (ABCB1) gene single nucleotide polymorphism predicts outcome of temozolomide treatment in glioblastoma patients.

BACKGROUND: Some patients with glioblastoma multiform do not respond to temozolomide even though they have aberrant promoter methylation of the DNA repair enzyme O(6)-methylguanine methyltransferase (MGMT). This suggests that additional factors hamper temozolomide cytotoxicity. We aimed to confirm first that temozolomide is a target for the multidrug resistance transporter MDR1/ABCB1 and second to investigate whether genetic variants of the MDR1 gene are associated with the survival of glioblastoma patients treated with temozolomide. MATERIALS AND METHODS: Temozolomide-mediated cytotoxicity was determined by the colorimetric methyl-thiazol-tetrazolium assay in MDR-expressing and MDR-nonexpressing cell lines. Genotypes of three single nucleotide polymorphisms (SNPs) of the MDR1 gene (C1236T, G2677T, and C3435T), MDR1 mRNA expression levels, and the MGMT promoter methylation status were analyzed in 112 glioblastoma patients who had been treated either by surgery plus radiotherapy alone or by additional temozolomide chemotherapy. RESULTS: In vitro analysis revealed that temozolomide-mediated cytotoxicity is dependent on MDR1 expression. Multivariate analysis of MDR1 genotypes showed that the C/C variant of the exon12 C1236T SNP is predictive for survival of patients treated with temozolomide. This effect was independent of the MGMT methylation status. Patients with the C/C genotype had a 2-year overall survival of 37% compared with 8% and 10% for patients with C/T and T/T genotypes, respectively (P=0.02). No influence was seen in the group of patients with radiotherapy only. CONCLUSION: The genotype of the MDR1 exon12 C1236T SNP is a novel independent predictive factor for outcome of temozolomide treatment in glioblastoma patients.

Targeting mycophenolate mofetil for graft-versus-host disease prophylaxis after allogeneic blood stem cell transplantation.

As low trough levels of mycophenolic acid (MPA) have been measured in recipients of allo-SCTs, we performed a pilot study targeting mycophenolate mofetil (MMF) doses according to the MPA area under the concentration (AUC) levels. Twenty-nine patients were transplanted from matched sibling (n=7) and unrelated donors (n=22). Tacrolimus was given orally from day -1 to achieve trough blood levels of 5-10 ng/ml. MMF was started on day 0 at 1500 mg intravenously b.i.d. AUC measurements of MPA by HPLC were scheduled on days 3, 7 and 11 after transplantation. The MMF dose was modified to achieve an MPA AUC of 35-60 microg/ml/h. With the respective adjustments 66 and 75% surpassed the lower AUC target on days 7 and 11, respectively. The cumulative incidence of grade III-IV acute GVHD was 28% (8/29). Eight out of 24 evaluable patients (33%) suffer from limited (n=3) or extensive (n=5) chronic GVHD. Overall, the results of this study suggest that targeting of MPA exposure is feasible early after transplantation. A simplified MMF targeting strategy based on MPA C(max) or C(2h) levels seems to be warranted in future trials involving more patients at later time points in the outpatient setting.

Expression of the CTLA-4 ligand CD86 on plasmacytoid dendritic cells (pDC) predicts risk of disease recurrence after treatment discontinuation in CML.

This corrects the article DOI: 10.1038/leu.2017.9.

Inhibition of platelet-derived growth factor receptorbeta by imatinib mesylate suppresses proliferation and alters differentiation of human mesenchymal stem cells in vitro.

OBJECTIVES: Recent data show that Imatinib mesylate (IM) also affects haematopoietic stem cells (HSC), T lymphocytes and dendritic cells that do not harbour constitutively active tyrosine kinases. MATERIALS AND METHODS: We evaluated possible effects of IM on human bone marrow-derived mesenchymal stem cells (MSC) in vitro. RESULTS: Screening the activity of 42 receptor tyrosine kinases revealed an exclusive inhibition of platelet-derived growth factor receptorbeta (PDGFRbeta). Analysis of downstream targets of PDGFRbeta demonstrated IM-mediated reduction of Akt and Erk1/2 phosphorylation. Culture of MSC with IM led to the reversible development of perinuclear multi-vesicular bodies. The proliferation and clonogenicity of MSC were significantly reduced compared to control cultures. IM favoured adipogenic differentiation of MSC whereas osteogenesis was suppressed. The functional deficits described led to a 50% reduction in the support of clonogenic haematopoietic stem cells, cultured for 1 month on a monolayer of MSC with IM. CONCLUSION: In summary, inhibition of PDGFRbeta and downstream Akt and Erk signalling by IM has a significant impact on proliferation and differentiation of human MSC in vitro.

F-ara-A pharmacokinetics during reduced-intensity conditioning therapy with fludarabine and busulfan.

Fludarabine is commonly used in combination with busulfan as part of conditioning regimens before allogeneic stem cell transplantation. So far, no data are available on busulfan-fludarabine drug interactions in transplant recipients. The pharmacokinetic (PK) properties of F-ara-A (9-beta-D-arabinosyl-2-fluoradenine) before and after application of busulfan were prospectively investigated in 16 patients with hematological malignancies. The conditioning regimen consisted of intravenous fludarabine 30 mg/m(2) over 30 min from day -6 to day -3, and oral busulfan given at 1 mg/kg every 6 h from day -5 to day -2. PK parameters of F-ara-A, derived from plasma and urine on day -6, -5, -4 and -3, were determined using high-performance liquid chromatography (HPLC). AUC, C(max), t(1/2), Cl(total) and V(SS) were 21.9 microMh, 3.5 microM, 13.0 h, 4.3 l/h/m(2), 60.0 l/m(2) on day -6 and 22.4 microMh, 3.5 microM, 14.0 h, 4.7 l/h/m(2), 69.0 l/m(2) on day -5 to (-2), respectively. Cl(renal) and the urine-recovery were 4.8 l/h, 43.7% of the fludarabine dose on day -6 and 3.9 l/h, 44.2% of the fludarabine dose on day -5 to (-2), respectively. There were no changes in PK parameters of fludarabine given before and after intake of busulfan. This implies that a clinically relevant busulfan-fludarabine drug interaction is unlikely.

Reduced intensity conditioning allows for up-front allogeneic hematopoietic stem cell transplantation after cytoreductive induction therapy in newly-diagnosed high-risk acute myeloid leukemia.

There is substantial need to improve the outcome of patients with high-risk acute myeloid leukemia (AML). The clinical trial reported here investigated a new approach of up-front allogeneic hematopoietic stem cell transplantation (HSCT), provided a median of 40 days (range 22-74) after diagnosis, in twenty-six consecutive patients with newly-diagnosed high-risk AML characterized by poor-risk cytogenetics (n = 19) or inadequate blast clearance by induction chemotherapy (IC, n = 7). The median age was 49 years (range 17-68). During IC-induced aplasia after the 1st (n = 11) or 2nd (n = 15) cycle, patients received allogeneic peripheral blood stem cells (PBSC) from related (n = 11) or unrelated (n = 15) donors following a fludarabine-based reduced-intensity regimen. Seventeen patients were not in remission before HSCT with a median marrow blast count of 34% (range 6-70). All patients achieved rapid engraftment and went into remission with complete myeloid and lymphatic chimerism. Grades II to IV acute GvHD occurred in 14 (56%) and extensive chronic GvHD was documented in 8 (35%) patients. The probability of disease-free survival was 61% with only three patients relapsing 5, 6 and 7 months after transplantation, respectively. Up-front allogeneic HSCT as part of primary induction therapy seems to be an effective strategy in high-risk AML patients and warrants further investigation.

Expression of the CTLA-4 ligand CD86 on plasmacytoid dendritic cells (pDC) predicts risk of disease recurrence after treatment discontinuation in CML.

It is unknown, why only a minority of chronic myeloid leukemia (CML) patients sustains treatment free remission (TFR) after discontinuation of tyrosine kinase inhibitor (TKI) therapy in deep molecular remission (MR). Here we studied, whether expression of the T-cell inhibitory receptor (CTLA-4)-ligand CD86 (B7.2) on plasmacytoid dendritic cells (pDC) affects relapse risk after TKI cessation. CML patients in MR displayed significantly higher CD86+pDC frequencies than normal donors (P<0.0024), whereas TFR patients had consistently low CD86+pDC (n=12). This suggested that low CD86+pDC might be predictive of TFR. Indeed, in a prospective analysis of 122 patients discontinuing their TKI within the EURO-SKI trial, the one-year relapse-free survival (RFS) was 30.1% (95% CI 15.6-47.9) for patients with >95 CD86+pDC per 105 lymphocytes, but 70.0% (95% CI 59.3-78.3) for patients with <95 CD86+pDC (hazard ratio (HR) 3.4, 95%-CI: 1.9-6.0; P<0.0001). Moreover, only patients with <95 CD86+pDC derived a significant benefit from longer (>8 years) TKI exposure before discontinuation (HR 0.3, 95% CI 0.1-0.8; P=0.0263). High CD86+pDC counts significantly correlated with leukemia-specific CD8+ T-cell exhaustion (Spearman correlation: 0.74, 95%-CI: 0.21-0.92; P=0.0098). Our data demonstrate that CML patients with high CD86+pDC counts have a higher risk of relapse after TKI discontinuation.

Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells.

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.

Graft clonogenicity and intensity of pre-treatment: factors affecting outcome of autologous peripheral hematopoietic cell transplantation in patients with acute myeloid leukemia in first remission.

A total of 22 patients with acute myeloid leukemia (AML) in first complete remission receiving autologous blood stem cell transplantation (ABSCT) were investigated in order to determine factors affecting outcome. All but two patients had a normal karyotype and received the same high-dose chemotherapy followed by G-CSF-mobilized peripheral blood stem cells after the second (n=5) or third (n=17) course of induction and post-remission chemotherapy, respectively. With a median follow-up of 30 months, the median disease-free survival is 24.1 months. Univariate analysis showed that three chemotherapy cycles before ABSCT were associated with a significant better disease-free survival (P=0.0018) and overall survival (P=0.0033), whereas the presence of an FLT3-mutation (n=6) showed no impact. The number of megakaryocytic progenitors (CFU-MK) infused tended to correlate with primary platelet engraftment (P=0.07) and were predictive for neutrophil (P=0.011) and platelet counts (P=0.009) 180 days after transplantation. Patients receiving a higher amount of CFU-MK had a better event-free survival (P=0.02). Our data suggest that the content of CFU-MK within the graft predicts the quality of hematological recovery and long-term disease control. Additionally, a minimum of three chemotherapy cycles before ABSCT seems to be associated with an improved outcome.

Comparison of spectral karyotyping and conventional cytogenetics in 39 patients with acute myeloid leukemia and myelodysplastic syndrome.

Spectral karyotyping (SKY) was performed in patients with acute myeloid leukemia (AML; n = 25), secondary AML (s-AML; n = 7), myelodysplastic syndrome (MDS; n = 6) and s-MDS (n = 1) to complement conventional cytogenetic investigations. According to the results of conventional cytogenetics the patients were subdivided into three groups: group 1, normal karyotype, n = 19 cases, median age = 64 years; group 2, patients displaying either one or two single aberrations, n = 10 cases, median age = 54 years; group 3, patients with > or =3 independent aberrations, n = 10 cases, median age = 61.5 years. SKY identified no abnormal metaphases in group 1. In one patient of group 2 a hidden translocation t(7;14)(q3?1;q2?2) could be revealed with SKY. Conventional cytogenetics had only shown trisomy 8. A similar t(7;14) was also detected in one patient of group 3. SKY was helpful for the delineation of marker chromosomes and additional material. Furthermore, SKY could distinguish between partial and total monosomies or real existing and apparent deletions. The combination of G-banding, FISH and SKY was found very useful for the precise delineation of the karyotype. As a result of our study we recommend SKY investigation as an important additional tool for accurate chromosome analysis. The detected t(7;14) might represent a novel recurrent translocation in acute myeloid leukemias.

P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate.

Imatinib (Glivec), STI571) is an intracellular acting drug that demonstrates high activity against BCR-ABL-positive chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). However, many patients, especially with advanced disease, develop drug resistance. Here, we show by a novel high-performance liquid chromatography-based method that intracellular levels of imatinib decrease in P-glycoprotein (Pgp)-positive leukemic cells. In a model of K562 cells with gradually increasing Pgp expression, a Pgp-dependent decline of intracellular imatinib levels was observed. Decreased imatinib levels were associated with a retained phosphorylation pattern of the Bcr-Abl target Crkl and loss of effect of imatinib on cellular proliferation and apoptosis. The modulation of Pgp by cyclosporin A (CSA) readily restored imatinib cytotoxicity in these cells. Finally, we provide first data showing a biological effect of Pgp modulation in the imatinib treatment of a patient with BCR-ABL-positive ALL. MDR1 overexpression must therefore be considered as an important clinical mechanism in the diversity of resistance development to imatinib treatment.

Hematopoietic cell transplantation in patients with intermediate and high-risk AML: results from the randomized Study Alliance Leukemia (SAL) AML 2003 trial.

The optimal timing of allogeneic hematopoietic stem cell transplantation (HCT) in acute myeloid leukemia (AML) is controversial. We report on 1179 patients with a median age of 48 years who were randomized upfront. In the control arm, sibling HCT was scheduled in the first complete remission for intermediate-risk or high-risk AML and matched unrelated HCT in complex karyotype AML. In the experimental arm, matched unrelated HCT in first remission was offered also to patients with an FLT3-ITD (FMS-like tyrosine kinase 3-internal tandem duplication) allelic ratio >0.8, poor day +15 marrow blast clearance and adverse karyotypes. Further, allogeneic HCT was recommended in high-risk AML to be performed in aplasia after induction chemotherapy. In the intent-to-treat (ITT) analysis, superiority of the experimental transplant strategy could not be shown with respect to overall survival (OS) or event-free survival. As-treated analyses suggest a profound effect of allogeneic HCT on OS (HR 0.73; P=0.002) and event-free survival (HR 0.67; P<0.001). In high-risk patients, OS was significantly improved after allogeneic HCT in aplasia (HR 0.64; P=0.046) and after HCT in remission (HR 0.74; P=0.03). Although superiority of one study arm could not be demonstrated in the ITT analysis, secondary analyses suggest that early allogeneic HCT is a promising strategy for patients with high-risk AML.

A new PCR MIMIC strategy to quantify low mdr1 mRNA levels in drug resistant cell lines and AML blast samples.

Determination of the MDR-phenotype in patients suffering from AML is an important hallmark of treatment outcome but is often complicated by technical problems in P-gp assessment. A PCR-MIMIC strategy was employed to construct PCR-fragments for a competitive and quantitative mdr1 reverse transcription-PCR-assay. Using K562 cells, which had been selected for drug resistance to the epipodophyllotoxin VP16, a stepwise increase of mdr1 levels depending on the concentration of VP16 was shown with the MIMIC technique. Comparison of mdr1 levels in drug selected K562 cells with the corresponding levels for P-gp and functional data indicated a mRNA threshold that has to be exceeded for the full expression of the MDR-phenotype. Subsequently mdr1 levels of 34 samples of de novo acute myeloid leukemia were determined with the PCR-MIMIC strategy. Ten patient samples could be identified with elevated mdr1 levels which were substantially lower than the levels observed in the MDR-cell line K 562 0.7 microM VP16. Outcome analysis revealed that eight of the ten patients had an unfavourable prognosis and did not achieve CR after induction chemotherapy. Coexpression of mdr1 and CD 34 was not associated with CR in all examined cases. Moreover all these patients had unfavourable cytogenetic aberrations. These data indicate a sensitive technique with applicability in patient material.

Double lumen port access in patients receiving allogeneic blood stem cell transplantation.

We performed a prospective trial investigating the feasibility of a double lumen port access in 26 patients with hematological malignancies or solid tumors receiving either standard conditioning (n = 9, median age 49 years (range 19-65)) or dose-reduced conditioning (n = 17, median age 56 years (range 35-66)) followed by allogeneic blood stem cell transplantation. The port system was implanted within 3 months (n = 20, range 7-91 days) before transplantation or as indicated at different time points after transplantation (n = 6, range 28-680 days). Most infusions, including the graft itself and all blood drawings, were performed via the port. Over a cumulative duration of 5622 days (1310 days after standard conditioning (range 56-349) and 4431 days after dose-reduced conditioning (range 49-489)) two port systems of patients receiving standard conditioning were removed due to early postimplantation pocket infection on day 6 and 8 after insertion, respectively. In the dose-reduced conditioning group only one late removal (day 287) of a port was required. Most of the patients in both groups reported less pain and a higher degree of comfort compared to peripheral or central venipuncture. The use of double lumen port access during conditioning and in an outpatient setting after allogeneic hemopoietic stem cell transplantation is feasible and advantageous for both patient and medical staff. Implantation several weeks before the start of conditioning might help in avoiding early infectious complications after conventional myeloablative conditioning.

Complex cytogenetic and immunophenotypic aberrations in a patient with Sezary syndrome.

Sezary syndrome is defined as the leukemic variation of cutaneous T-cell lymphomas. Here we describe the cytogenetic pattern of peripheral T-cells of a 50-year-old male patient suffering from this disease. We used Giemsa-banding (G-banding) technique and a fluorescence in situ hybridization (FISH) assay to determine cytogenetic changes affecting 15 different chromosomes. The cells displayed an abnormal hypodiploid karyotype with a prominent insertion located at the short arm of chromosome 1. Unbalanced translocations were observed involving chromosomes 4 and 14. Besides other abnormalities we detected a 6q- deletion. These multiple genetic changes may reflect the high aggressivity of the neoplastically transformed T-cell population and the poor response to chemotherapeutic treatment.