Lorna A. McLintock

A prospective study of real-time panfungal PCR for the early diagnosis of invasive fungal infection in haemato-oncology patients

Summary:A blinded prospective study was performed to determine whether screening of whole blood using a real-time, panfungal polymerase chain reaction (PCR) technique could predict the development of invasive fungal infection (IFI) in immunocompromised haemato-oncology patients. In all, 78 patients (125 treatment episodes) were screened twice weekly by real-time panfungal PCR using LightCycler™ technology. IFI was documented in 19 treatment episodes (five proven, three probable and 11 possible), and in 12, PCR was sequentially positive. PCR positivity occurred in: 4/5 proven; 2/3 probable; 6/11 possible; and 29/106 with no IFI. In 8/12 with IFI and sequentially positive PCR results, PCR positivity occurred before (median 19.5 days) and in 4/12 (median 10.5 days) after the initiation of empirical antifungal therapy. Based on sequential positive results for proven/probable IFI sensitivity, specificity, positive predictive value and negative predictive value were 75, 70, 15 and 98%, respectively. Real-time panfungal PCR is a sensitive tool for the early diagnosis of IFI in immunocompromised haemato-oncology patients. It may be most useful as a screening method in high-risk patients, either to direct early pre-emptive antifungal therapy or to determine when empirical antifungal therapy can be withheld in patients with antibiotic--resistant neutropenic fever. However, these strategies require further assessment in comparative clinical trials.

A pilot study of continuous imatinib vs pulsed imatinib with or without G-CSF in CML patients who have achieved a complete cytogenetic response

A pilot study of continuous imatinib vs pulsed imatinib with or without G-CSF in CML patients who have achieved a complete cytogenetic response

JAK2 V617F and CALR mutations are not mutually exclusive; findings from retrospective analysis of a small patient cohort

Lopes, R.D., Hylek, E.M., Hanna, M., Al-Khalidi, H.R., Ansell, J., Atar, D., Avezum, A., Bahit, M.C., Diaz, R., Easton, J.D., Ezekowitz, J.A., Flaker, G., Garcia, D., Geraldes, M., Gersh, B.J., Golitsyn, S., Goto, S., Hermosillo, A.G., Hohnloser, S.H., Horowitz, J., Mohan, P., Jansky, P., Lewis, B.S., Lopez-Sendon, J.L., Pais, P., Parkhomenko, A., Verheugt, F.W., Zhu, J. & Wallentin, L. (2011) Apixaban versus warfarin in patients with atrial fibrillation. New England Journal of Medicine, 365, 981–992. Keeling, D., Baglin, T., Tait, C., Watson, H., Perry, D., Baglin, C., Kitchen, S. & Makris, M. (2011) Guidelines on oral anticoagulation with warfarin fourth edition. British Journal of Haematology, 154, 311–324. Linkins, L.A., Choi, P.T. & Douketis, J.D. (2003) Clinical impact of bleeding in patients taking oral anticoagulant therapy for venous thromboembolism: a meta-analysis. Annals of Internal Medicine, 139, 893–900. NHS Improvement (2009) Heart and Stroke Improvement. Commissioning for Stroke Prevention in Primary Care. The Role of Atrial Fibrillation. www.improvement.nhs.uk/heart/portals/docu ments2009. Patel, M.R., Mahaffey, K.W., Garg, J., Pan, G., Singer, D.E., Hacke, W., Breithardt, G., Halperin, J.L., Hankey, G.J., Piccini, J.P., Becker, R.C., Nessel, C.C., Paolini, J.F., Berkowitz, S.D., Fox, K.A. & Califf, R.M. (2011) Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. New England Journal of Medicine, 365, 883–891.

Advances in the molecular and serological diagnosis of invasive fungal infection in haemato-oncology patients

Current laboratory diagnostic methods for invasive fungal infection (IFI) in haemato‐oncology patients are insensitive, resulting in late diagnosis and contributing to high mortality. In recent years, progress has been made in the development and evaluation of sensitive sero‐diagnostic assays, including detection of genomic DNA sequences and fungal antigens, which aid in a rapid, early diagnosis of IFI. The sensitivity and specificity of the assays vary considerably between studies, highlighting the need to correlate serological results with conventional laboratory tests and clinical or radiological findings. As part of management protocols, these assays may help to confirm the diagnosis of suspected IFI; however, the impact on mortality from IFI may be greatest when they are used to screen high‐risk patients. Persistently positive screening results could direct early aggressive antifungal therapy, guided further by radiological and microbiological findings combined with regular clinical review, while the excellent negative predictive value may allow treatment to be withheld in patients with antibiotic resistant neutropenic fever but no other signs of IFI. However, this pre‐emptive approach requires evaluation in prospective randomized trials.

Successful haemopoietic stem cell transplantation does not correct mannan-binding lectin deficiency.

Summary:It has been reported that in allogeneic stem cell transplantation, the mannan-binding lectin (MBL) status of the donor has prognostic value for the recipient. Two MBL-deficient patients, with coexisting haematological malignancy, were identified who were treated with bone marrow from donors with normal MBL concentrations. Although both patients engrafted successfully and remain in complete remission, neither seroconverted to the MBL sufficiency status of his donor over a follow-up period exceeding 2 years. This does not support the concept of MBL replacement by stem cell therapy, and does not provide an explanation for high MBL concentrations in stem cell donors protecting recipients from post transplant infections.

Poor performance of galactomannan and mannan sandwich enzyme-linked immunosorbent assays in the diagnosis of invasive fungal infection

The Platelia Aspergillus and Platelia Candida (BioRad Laboratories Ltd, Hertfordshire, UK) are commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits for the detection of galactomannan (GM) and mannan (M) respectively. A number of prospective studies report excellent sensitivities (90–95%) and specificities using the Platelia Aspergillus for the diagnosis of invasive aspergillosis (IA) (McLintock & Jones, 2004). The European Organisation for the Research and Treatment of Cancer and the Mycosis Study Group (EORTC/ MSG) definitions of invasive fungal infection (IFI) include positive GM antigen testing as a microbiological criterion (Ascioglu et al, 2002). Other studies demonstrate poorer sensitivities (0–50%) (McLintock & Jones, 2004) and controversy exits over the most appropriate positive cut-off (Maertens et al, 2004). There are no prospective studies which evaluate the Platelia Candida and retrospective studies report modest sensitivities (30–69%) (McLintock & Jones, 2004). Positive M antigen testing is not included in EORTC/MSG definitions of IFI. A prospective blinded study was performed to assess the value of twice-weekly screening of serum specimens, from haemato-oncology patients at highand intermediate high-risk of IFI (Prentice et al, 2000), with Platelia Aspergillus and Platelia Candida ELISAs. Between December 2000 and December 2001, adult patients receiving allogeneic or autologous stem cell transplantation (SCT) or intensive chemotherapy were recruited. A treatment episode was defined as a single course of chemotherapy, an autologous SCT or an allogeneic SCT until day 100 following the transplant, or a course of corticosteroid therapy for graft-versus-host disease. An investigator blinded to the patient data performed the ELISAs according to the manufacturer’s instructions. Each sample was run in duplicate. Samples were re-tested if the coefficient of variation between duplicates was more than 20%. IFI was classified as proven, probable or possible according to the EORTC/MSG definitions (Ascioglu et al, 2002). As the Platelia Aspergillus ELISA was being evaluated it was excluded from the EORTC/MSG definitions. The Platelia Candida ELISA assay was discontinued after analysis of 82 episodes as 61% of the kits failed the manufacturer’s validation criteria and results were therefore uninterpretable. Additionally, approximately 30% of the available ELISA results were non-reproducible. A total of 1625 Platelia Aspergillus ELISA results, from 125 treatment episodes, involving 78 patients, aged 16–76 years (mean 42, median 44Æ5 years), were analysed. IFI was documented in 19/125 (15Æ2%) treatment episodes: five proven (all candida); three probable (two Candida, one mixed infection with Candida and Aspergillus); and 11 possible (all Aspergillus). The Platelia Aspergillus ELISA results were analysed at three different positive cut-offs: >1Æ5 (manufacturer’s recommended positive cut-off); >1Æ0; and >0Æ5 (Table I). Using a positive cut-off of >1Æ5, sequential positive (‡ 2 consecutive) results were recorded in only one of 125 treatment episodes, during which the patient had proven invasive candidiasis (IC) but no EORTC/MSG evidence of IA. In the 12 cases of EORTC/MSG-defined IA (one probable and

Mixed pulmonary fungal infection with Aspergillus fumigatus and Absidia corymbifera in a patient with relapsed acute myeloid leukaemia.

A 19-year-old male with relapsed acute myeloid leukaemia developed pyrexia, pleuritic chest pain and haemoptysis during re-induction chemotherapy (day 19). Chest computed tomography (CT) scanning demonstrated widespread consolidation. Antibacterial therapy, increasing doses of liposomal amphotericin B (AmBisome , Gilead Sciences International Limited, Cambridge, UK; maximum 8 mg/kg), itraconazole and granulocyte colony-stimulating factor were administered. At neutrophil recovery (day 67), chest X-ray demonstrated cavitation (top left) and CT scan demonstrated halo and air crescent signs (top right) characteristic of invasive aspergillosis. Left upper lobectomy and right wedge resections were performed. Culture demonstrated Aspergillus fumigatus and Absidia corymbifera. Vessel wall histology (silver stain) demonstrated irregular branching septate hyphae consistent with A. fumigatus (bottom left) and lung tissue (haematoxylin and eosin) revealed broad, sparsely septate hyphae consistent with A. corymbifera (bottom right). Invasive fungal infection caused by more than one species is infrequently reported. Absidia corymbifera carries an 80% mortality and is resistant to voriconazole and the ecchinocandins. Amphotericin B is the drug of choice for the treatment of A. corymbifera and other infections caused by the Zygomcytes.

Safety and efficacy of pulsed imatinib with or without G‐CSF versus continuous imatinib in chronic phase chronic myeloid leukaemia patients at 5 years follow‐up

Despite their efficacy in inducing deep and durable responses in chronic phase (CP) chronic myeloid leukaemia (CML) patients, BCR-ABL1 tyrosine kinase inhibitors (TKI) do not eradicate leukaemia stem cells (LSC), as proven by the persistence of BCR-ABL1+ CD34+, colony forming, longterm culture-initiating cells in the bone marrow of patients in sustained molecular response 4 5 (a 4 5-log reduction of BCR-ABL1 transcript levels, MR4 5) following TKI therapy (Chomel et al, 2011). CML LSC survival is independent of BCR-ABL1 kinase activity (Hamilton et al, 2012) and their quiescence is a putative TKI-resistance mechanism causing disease persistence. Moreover TKI exert anti-proliferative rather than pro-apoptotic effects against CML LSCs and might further contribute to disease persistence (Graham et al, 2002). Promoting LSC cellcycle entry using granulocyte-colony stimulating factor (G-CSF) has been shown in vitro to restore their sensitivity to TKI and enhance their eradication (Jorgensen et al, 2006). Based on this evidence, we performed a randomized phase II study (GIMI, EudraCT 2004-000179-33), which compared the safety and efficacy of continuous imatinib (cIM) versus pulsed imatinib (pIM) alone or with G-CSF (pIM+G) therapy administered in 4-week cycles for 48 weeks (12 cycles in total) in CP CML patients with at least a complete cytogenetic response (CCyR) on IM (Drummond et al, 2009) (see reference for study design, primary and secondary endpoints, patient demographics and disease characteristics). The experimental arms were expected to improve CML LSCs eradication by reducing TKI-induced quiescence (pIM) and/or by actively pushing CML LSCs into cell-cycle (pIM+G). At 2 years follow-up no statistically significant differences for the study endpoints were observed, possibly due to the limited numbers (15 patients per arm). However, 6/30 patients across the two experimental arms exhibited either loss of CCyR or major molecular response (MMR) as compared with only 1/15 in the cIM arm, with all but one patient in the experimental arms regaining MMR on restarting continuous IM or nilotinib therapy (Drummond et al, 2009). These results raised some concerns that the experimental schedules might have contributed to the loss of response. Subsequently, a mathematical model of the safety and efficacy of the IM and G-CSF combination suggested that this approach might be detrimental in the shortto mediumterm for patients with persistent disease treated with IM, by increasing the LSCs burden through enhanced proliferation, thus in turn increasing the risk of acquiring a resistance mutation and of disease progression. However, in the longterm (>2500 d, i.e. 6 8 years, from start of treatment), such an approach was predicted to prove beneficial as it would deplete the CML LSCs by increasing their susceptibility to TKI (Foo et al, 2009). Here we report the 5-year follow-up data for the GIMI study. 41/45 patients were available for analysis; four patients had died (one only as a result of CML progression). The median follow-up was 5 67 years. Using an intention to treat analysis, both CCyR and MMR rates were similar among treatment arms with no differences in progression rates. 5/15 patients in the cIM arm compared to 3/15 patients in each experimental arm changed treatment to second generation TKI (Table I).