Highly Sensitive and Accurate Assessment of Minimal Residual Disease in Chronic Lymphocytic Leukemia Using the Novel CD160-ROR1 Assay.

Undetectable minimal residual disease (MRD) in Chronic Lymphocytic Leukemia (CLL) has a favorable prognostic outcome compared with MRD that can be detected. This study investigated a flow cytometric assay (CD160-ROR1FCA) targeting the tumor-specific antigens CD160 and receptor tyrosine kinase-like orphan receptor 1 (ROR1), along with CD2, CD5, CD19, CD45. CD160-ROR1FCA was compared with the originally published 8-colour European Research Initiative for CLL (ERIC) gold-standard assay for CLL MRD detection. CD160-ROR1FCA had a limit of detection of 0.001% and showed strong correlation with ERIC (R = 0.98, p < 0.01) with negligible differences in MRD detection (bias -0.3152 95%CI 5.586 to -6.216). Using CD160-ROR1FCA, increased expression of both CD160 and ROR1 was found in Monoclonal B cell Lymphocytosis (MBL) compared to low-level polyclonal B-cell expansions (p < 0.01). Patients in CR and with undetectable MRD had a longer EFS (not reached) than those in CR but with detectable MRD (756 days, p < 0.01) versus 113 days in patients with partial remission (p < 0.01). Patients with MRD levels of >0.01 to 0.1% had a longer EFS (2,333 days), versus levels between 0.1 to 1% (1,049 days). CD160-ROR1FCA is a novel assay for routine CLL MRD measurement and for MBL detection. MRD status assessed by CD160-ROR1FCA after CLL treatment correlated with EFS.

National mycology laboratory diagnostic capacity for invasive fungal diseases in 2017: Evidence of sub-optimal practice.

A survey of laboratory testing capabilities for systemic fungal pathogens was undertaken in the UK, to identify where improved compliance with published standards and guidelines is required and to inform antifungal stewardship (AFS). The survey captured information from laboratories in the UK on diagnostic capacity for invasive fungal diseases (IFD), including identification, serology, molecular diagnostics and susceptibility testing. The survey was circulated in March 2017 through key networks. Of 154 laboratories providing diagnostic mycology services in the UK, 80 (52%) responded to the survey. Results indicated that 85% of respondents identified fungal isolates from high risk patients to species level, and that many laboratories (78%) could access local susceptibility testing for yeasts, whereas 17% could for Aspergillus species. However, direct microscopy was only used in 49% as a first line investigation on samples where it would be appropriate. A low number of respondents identified yeasts cultured from intravascular line tips to species level (63%) and even fewer fully identified urine isolates from critically ill patients (42%) or the immunocompromised (39%). Less than half of respondents advised therapeutic drug monitoring (TDM) for flucytosine. Few laboratories had access to local ß-glucan (4%) or galactomannan (20%) testing. The survey highlights that the current level of fungal diagnostics in the UK is below accepted best practice with an urgent need to improve across many diagnostic areas including the timely accessibility of fungal biomarkers, susceptibility testing and provision of TDM testing. Improvements are important to facilitate the delivery of diagnostic driven AFS strategies as well as appropriate management of IFD.

Increased autocrine interleukin-6 production is significantly associated with worse clinical outcome in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) remains incurable with current standard therapy. We have previously reported that an increased expression of interleukin-6 (IL-6) receptor CD126 leads to resistance of CLL cells to chemotherapy and worse prognosis for patients with CLL. In this study, we determine whether autocrine IL-6 production by CLL B cells is associated with poor clinical outcome and explore IL-6-mediated survival mechanism in primary CLL cells. Our results demonstrate that higher levels of autocrine IL-6 are significantly associated with shorter absolute lymphocyte doubling time, patients received treatment, without complete remission, advanced Binet stages, 17p/11q deletion, and shorter time to first time treatment and progression-free survival. IL-6 activated both STAT3 and nuclear factor kappa B (NF-κB) in primary CLL cells. Blocking IL-6 receptor and JAK2 inhibited IL-6-mediated activation of STAT3 and NF-κB. Our study demonstrates that an increased autocrine IL-6 production by CLL B-cells are associated with worse clinical outcome for patients with CLL. IL-6 promotes CLL cell survival by activating both STAT3 and NF-κB through diverse signaling cascades. Neutralizing IL-6 or blocking IL-6 receptor might contribute overcoming the resistance of CLL cells to chemotherapy. We propose that the measurement of autocrine IL-6 could be a useful approach to predict clinical outcome.

An investigation of antifungal stewardship programmes in England.

PURPOSE: We sought to explore the current status of antifungal stewardship (AFS) initiatives across National Health Service (NHS) Trusts within England, the challenges and barriers, as well as ways to improve current AFS programmes. METHODOLOGY: An electronic survey was sent to all 155 acute NHS Trusts in England. A total of 47 Trusts, corresponding to 30 % of English acute Trusts, responded to the the survey; 46 Trusts (98 %) had an antimicrobial stewardship (AMS) programme but only 5 (11 %) had a dedicated AFS programme. Overall, 20 (43 %) Trusts said they included AFS as part of their AMS programmes. From those conducting AFS programmes, 7 (28 %) have an AFS/management team, 16 (64 %) monitor and report on antifungal usage, 5 (20 %) have dedicated AFS ward rounds and 12 (48 %) are directly involved in the management of invasive fungal infections.Results/Key findings. Altogether, 13 acute Trusts (52 %) started their AFS programme to manage costs, whilst 12 (48 %) commenced the programme due to clinical need; 27 (73 %) declared that they would increase their AFS initiatives if they could. Of those without an AFS programme, 14 (67 %) responded that this was due to lack of resources/staff time. Overall, 12 Trusts (57 %) responded that the availability of rapid diagnostics and clinical support would enable them to conduct AFS activities. CONCLUSION: Although a minority of Trusts conduct dedicated AFS programmes, nearly half include AFS as part of routine AMS activities. Cost issues are the main driver for AFS, followed by clinical need. The availability of rapid diagnostics and clinical support could help increase AFS initiatives.

Randomized comparison of liposomal amphotericin B versus placebo to prevent invasive mycoses in acute lymphoblastic leukaemia.

Objectives: To prevent invasive fungal disease (IFD) in adult patients undergoing remission-induction chemotherapy for newly diagnosed acute lymphoblastic leukaemia (ALL). Patients and methods: In a double-blind multicentre Phase 3 study, patients received prophylactic liposomal amphotericin B (L-AMB) at 5 mg/kg intravenously or placebo twice weekly in a 2:1 random allocation during remission-induction treatment. The primary endpoint was the development of proven or probable IFD. Secondary endpoints included those focused on the safety and tolerability of prophylactic L-AMB. Results: Three hundred and fifty-five patients from 86 centres in Europe and South America received at least one dose of L-AMB ( n = 237) or placebo ( n = 118). Rates of proven and probable IFD assessed independently were 7.9% (18/228) in the L-AMB group and 11.7% (13/111) in the placebo group ( P = 0.24). Rates of possible IFD were 4.8% (11/228) in the L-AMB and 5.4% (6/111) in the placebo group ( P = 0.82). The remission-induction phase was a median of 22 days for both groups. Overall mortality was similar between the groups: 7.2% (17/237) for L-AMB and 6.8% (8/118) for placebo ( P = 1.00). Hypokalaemia and creatinine increase were significantly more frequent with L-AMB. Conclusions: The IFD rate among adult patients undergoing remission-induction chemotherapy for newly diagnosed ALL was 11.7% in the placebo group, and was not significantly different in patients receiving L-AMB, suggesting that the L-AMB regimen studied is not effective as prophylaxis against IFD. The IFD rate appears higher than previously reported, warranting further investigation. Tolerability of L-AMB was what might be expected. Further studies are needed to determine the optimal antifungal strategy during remission-induction chemotherapy of ALL.

A CONSORT analysis of randomised controlled trials for the treatment of invasive aspergillosis.

There is no assessment of the reporting quality of antifungal randomized, controlled trials (RCT), upon which guidelines for the treatment of invasive aspergillosis (IA) in patients with hematological malignancy are based. Trial reports were identified through Trip, Cochrane, Medline, and Embase database searches. Report quality was assessed using the 25-item CONSORT checklist and a rating scale of 1 (strongly disagree) to 4 (strongly agree). The primary endpoint was quality as assessed by mean group-scores among papers published at the time of the most recent IA treatment guidelines. Seven RCTs were identified for analysis. Overall mean group-score for all seven papers was 2.44 (out of a total of four). There were significant differences between publications regarding overall reporting quality (P < .001) and specifically for the Methods and Results (P = .004 and P = .010, respectively), which best reflect data quality. The Cornely trial report achieved the highest mean group-score overall (3.15 ± 0.93; 95% CI, 2.82, 3.47), as well as for Methods (3.36) and Results (3.40). Mean group scores also showed that it was of significantly higher overall quality than the other six publications (P-value range; .012 to <.001), and of higher quality for Methods than five publications (P-value range; .013 to <.001). Incorporating this CONSORT analysis into the evidence-based grading systems in North American (IDSA), European (ECIL and ESCMID) IA guidelines could alter the value placed on these RCTs, thereby impacting on clinical recommendations.

STAT3 and NF-κB cooperatively control in vitro spontaneous apoptosis and poor chemo-responsiveness in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an adult disease characterized by in vivo accumulation of mature CD5/CD19/CD23 triple positive B cells and is currently incurable. CLL cells undergo spontaneous apoptosis in response to in vitro cell culture condition but the underlying mechanism is unclear. We hypothesize that the sensitivity of CLL cells to spontaneous apoptosis may be associated with the constitutive activities of transcription factors STAT3 and/or NF-κB. We now show that the sensitivity of fresh CLL cells to spontaneous apoptosis is highly variable among different patients during 48 hours' cell culture and inversely correlated with in vivo constitutively activated STAT3 and NF-κB (p < 0.001). Both activated STAT3 and NF-κB maintain the levels of anti-apoptotic protein Mcl-1/Bcl-xL and autocrine IL-6 production. CLL cells with higher susceptibility to in vitro spontaneous apoptosis show the greatest chemosensitivity (p < 0.001), which is reflected clinically as achieving a complete response (CR) (p < 0.001), longer lymphocyte doubling times (p < 0.01), time to first treatment (p < 0.01), and progression free survival (p < 0.05). Our data suggest that the sensitivity of CLL cells to in vitro spontaneous apoptosis is co-regulated by constitutively activated STAT3 and NF-κB and reflects the in vivo chemo-responsiveness and clinical outcomes.

CD126 and Targeted Therapy with Tocilizumab in Chronic Lymphocytic Leukemia.

PURPOSE: IL6 promotes tumor growth and signal transduction via both its membrane-bound (CD126) and soluble receptors (sCD126). We aimed to study whether the levels of CD126 expression in chronic lymphocytic leukemic (CLL) cells can predict in vitro and in vivo treatment response. EXPERIMENTAL DESIGN: The levels of membrane-bound CD126 expression were determined on freshly isolated CLL B cells (n = 58) using flow cytometry. These CLL cells were treated with chlorambucil or fludarabine with or without anti-CD126 antibody tocilizumab for 24 hours and IL6-mediated STAT3 transcriptional activity and cell-cycle alteration were evaluated. RESULTS: CD126 surface expression was found in all cases and positively correlated with the levels of in vivo constitutive STAT3 activity. The levels of CD126 expression were significantly and positively correlated with the resistance of CLL cells to in vitro treatment with chlorambucil or fludarabine and poor in vivo treatment response of CLL patients. Blocking IL6 signaling with the anti-CD126 antibody, tocilizumab, had profound effects on STAT3-mediated survival and growth signals: decreased Mcl-1 and Bcl-xL, favoring an apoptotic profile; and decreased p27 with increased cyclin E and CDK2 expression, leading to cell-cycle shift from G0-G1 These tocilizumab-mediated changes induced chemosensitization in resistant CLL cells, with the greatest effect seen in cells with higher CD126 expression (P < 0.001). CONCLUSIONS: CLL cells with higher CD126 expression are more resistant to treatment in vivo and in vitro via IL6-CD126-STAT3 axis. Blocking CD126 using tocilizumab sensitizes CLL cells to chemotherapy. Clin Cancer Res; 22(10); 2462-9. ©2015 AACR.

Dynamin-related protein Drp1 is required for Bax translocation to mitochondria in response to irradiation-induced apoptosis.

Translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria is a crucial step in DNA damage-mediated apoptosis, and is also found to be involved in mitochondrial fragmentation. Irradiation-induced cytochrome c release and apoptosis was associated with Bax activation, but not mitochondrial fragmentation. Both Bax and Drp1 translocated from the cytosol to the mitochondria in response to irradiation. However, Drp1 mitochondrial translocation and oligomerization did not require Bax, and failed to induce apoptosis in Bax deficient diffuse large B-cell lymphoma (DLBCL) cells. Using fluorescent microscopy and the intensity correlation analysis, we demonstrated that Bax and Drp1 were colocalized and the levels of colocalization were increased by UV irradiation. Using co-immuno-precipitation, we confirmed that Bax and Drp1 were binding partners. Irradiation induced a time-associated increase in the interaction between active Bax and Drp1. Knocking down Drp1 using siRNA blocked UV irradiation-mediated Bax mitochondrial translocation. In conclusion, our findings demonstrate for the first time, that Drp1 is required for Bax mitochondrial translocation, but Drp1-induced mitochondrial fragmentation alone is not sufficient to induce apoptosis in DLBCL cells.

Aspergillus-Specific Lateral-Flow Device and Real-Time PCR Testing of Bronchoalveolar Lavage Fluid: a Combination Biomarker Approach for Clinical Diagnosis of Invasive Pulmonary Aspergillosis.

Clinical experience with the impact of serum biomarkers for invasive fungal disease (IFD) varies markedly in hemato-oncology. Invasive pulmonary aspergillosis (IPA) is the most common manifestation, so we evaluated biomarkers in bronchoalveolar lavage (BAL) fluid. An Aspergillus-specific lateral-flow device (LFD), quantitative real-time PCR (qPCR), and the galactomannan (GM) test were used with 32 BAL fluid samples from 32 patients at risk of IPA. Eight patients had proven IPA, 3 had probable IPA, 6 had possible IPA, and 15 patients had no IPA by European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group/Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (EORTC/MSG) criteria. The diagnostic accuracies of the tests were evaluated, and pairwise agreement between biomarkers was calculated. The diagnostic performance of the EORTC/MSG criteria was evaluated against the test(s) identified to be the most useful for IPA diagnosis. Using the EORTC/MSG criteria, the sensitivities of qPCR and LFD were 100% and the sensitivity of the GM test was 87.5% (GM test index cutoff, >0.8), with the tests having specificities of between 66.7 and 86.7%. The agreement between the results of qPCR and LFD was almost perfect (Cohen's kappa coefficient = 0.93, 95% confidence interval, 0.81 to 1.00). LFD and qPCR combined had a sensitivity of 100% and a specificity of 85.7%. Calcofluor staining and culture of all BAL fluid samples were negative for fungal infection. The median time from the start of mold-active antifungal therapy to the time of collection of BAL fluid was 6 days. Reversing roles and using dual testing by LFD and qPCR to classify cases, the EORTC/MSG criteria had a sensitivity of 83.3%. All three tests are useful for the diagnosis of IPA in BAL fluid samples. Despite the significant delays between the start of antifungal therapy and bronchoscopy, unlike microscopy and culture, the biomarkers remained informative. In particular, the combination of LFD and qPCR allows the sensitive and specific detection of IPA.

Use of biosimilar filgrastim compared with lenograstim in autologous haematopoietic stem-cell transplant and in sibling allogeneic transplant.

OBJECTIVES: Biosimilar filgrastim was compared with lenograstim for autologous haematopoietic stem-cell transplant (HSCT) in patients with haematological malignancies. Data from a separate group of sibling donors who underwent allogeneic HSCT are also reported. METHODS: Patients with lymphoma or multiple myeloma (MM) who underwent autologous HSCT with biosimilar filgrastim were compared with a historical control group of patients who received lenograstim. Peripheral blood (PB) cells counts were monitored after 7-8 consecutive days of granulocyte-colony stimulating factor (G-CSF) injection and apheresis was performed on day 8 if PB CD34+ cell count was ⩾10 cells/µl. The target PB CD34+ cell doses were ⩾2.0 × 10(6)/kg (lymphoma), ⩾4.0 × 10(6)/kg (MM ⩾60 years old) or ⩾8.0 × 10(6)/kg (MM <60 years old). RESULTS: A total of 259 patients were included in the autologous HSCT comparison (biosimilar filgrastim, n = 104; lenograstim, n = 155). In patients with lymphoma and older MM patients (⩾60 years old), no significant differences were observed between groups with regard to stem-cell mobilization parameters. However, in MM patients <60 years old, all parameters were significantly superior in the biosimilar filgrastim group, including the need for 1 rather than 2 apheresis procedures. No significant differences were observed between groups in median number of days to absolute neutrophil count (ANC) or platelet recovery. In the allogeneic setting, 47 sibling donors received biosimilar filgrastim. Mean CD34+ count at the first apheresis was 6.1 × 10(6)/kg. A total of 13 donors needed a second apheresis and 4 required a third. Among recipients, median days to ANC recovery was 16 (10-28) and to platelet recovery was 13 (9-54). CONCLUSIONS: Biosimilar filgrastim is as effective as lenograstim for autologous HSCT in patients with lymphoma or MM patients ⩾60 years old. However, mobilization with biosimilar filgrastim appeared to be superior to that with lenograstim in younger MM patients.

Significant decline in Galactomannan Signal during storage of clinical serum samples.

Galactomannan (GM) is widely used for detection of invasive aspergillosis in high-risk haemato-oncology patients. Recent publications have reported a lack of repeatability of GM detection. The objective of this retrospective study was to assess the repeatability of GM levels during storage of clinical samples. In a GM screening strategy, positive sera were repeat tested as per manufacturer's recommendations. Short-term (ST) storage of samples was at +4 °C while long-term (LT) storage was at -80 °C. Bronchoalveolar (BAL) fluid was also repeating tested after ST storage and LT storage. Wilcoxon Signed Ranks Test was employed to assess the repeatability of GM levels. In a subset of 14 GM positive sera, repeat testing was performed on both the original serum and ethylenediaminetetraacetic acid (EDTA) pre-treated sample. There was a significant reduction in GM signals on repeat testing following ST storage (median GM index: 0.65 vs. 0.19; p < 0.001) and LT storage (median GM index: 0.56 vs. 0.10; p < 0.001) of serum samples. Of samples that were initially GM positive, an average GM index reduction of 50% was seen, with approximately two-thirds becoming GM negative on repeat testing of the same sample. In contrast, GM signal loss was not seen on repeat testing of BAL fluid following ST or LT storage. When GM positive serum samples were repeat tested using EDTA pre-treated serum from the first step of the testing protocol, all samples remained GM positive. In contrast, when the same samples were repeat tested from the original collected serum, 9 samples (64%) became GM negative. The significant reduction in GM signals during ST and LT storage of serum samples has implications for clinical management. Although the reasons for GM decline are unknown, they occur prior to the EDTA pre-treatment stage, indicating that the time from phlebotomy to testing should be minimized. BAL fluid GM index values remain stable.

A MIQE-compliant real-time PCR assay for Aspergillus detection.

The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.

Impact of the revised (2008) EORTC/MSG definitions for invasive fungal disease on the rates of diagnosis of invasive aspergillosis.

Diagnosis of invasive aspergillosis (IA) remains a challenge as the clinical manifestations are not specific, and a histological diagnosis is often unfeasible. The 2002 European Organization for Research and Treatment of Cancer (EORTC) and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (MSG) criteria for classification of cases into possible, probable or proven were revised in 2008. Our objective was to analyze the impact of these revisions on the diagnosis of IA. A retrospective analysis of 589 high risk patient-episodes revealed that 125 of 155 'possible' (81%) and 12 of 16 'probable' (75%) cases of IA should be changed to 'non-classifiable' when the new criteria were applied. We concluded, as expected, that the 2008 EORTC/MSG revised definitions reduced the number of cases classified as 'possible' IA, but additionally, there has been a dramatic reduction in 'probable' cases. These changes have significant implications on the interpretation of clinical trial data based on EORTC/MSG classifications.

Differential and tumor-specific expression of CD160 in B-cell malignancies.

CD160 is a human natural killer (NK)-cell-activating receptor that is also expressed on T-cell subsets. In the present study, we examined 811 consecutive cases of B-cell lymphoproliferative disorders (B-LPDs), and demonstrated CD160 expression in 98% (590 of 600) of chronic lymphocytic leukemia (CLL) cases, 100% (32 of 32) of hairy cell leukemia (HCL) cases, 15% (5 of 34) of mantle cell lymphoma (MCL) in the leukemic phase, and 16% (23 of 145) of other B-LPD cases. CD160 transcript and protein were absent in the normal B-cell hierarchy, from stem cells, B-cell precursors, maturing B cells in the germinal center, and circulating B cells, including CD5(+)CD19(+) B1 cells in umbilical cord. CD160 positivity was significantly higher in CLL and HCL in terms of percentage (65.9% and 67.8%, respectively, P < .0001) and median fluorescence intensity (552 and 857, respectively, P < .0001) compared with all other B-LPD cases. Lymph node CLL samples were also CD160(+). Using the disease-specific expression of CD5, CD23, and CD160, a score of 3 characterized CLL (diagnostic odds ratio, 1430); a score of 0 excluded CLL, MCL, and HCL; and the CD23/CD5 ratio differentiated CLL from leukemic CD23(+) MCL. In the B-cell lineage, CD160 is a tumor-specific antigen known to mediate cellular activation signals in CLL, and is a novel target for therapeutic manipulation and monitoring of minimal residual disease.