Peter Mollee

Sequential treatment with rituximab followed by CHOP chemotherapy in adult B-cell post-transplant lymphoproliferative disorder (PTLD): the prospective international multicentre phase 2 PTLD-1 trial.

BACKGROUND Post-transplantation lymphoproliferative disorder (PTLD) develops in 1-10% of transplant recipients and can be Epstein-Barr virus (EBV) associated. To improve long-term efficacy after rituximab monotherapy and to avoid the toxic effects of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy seen in first-line treatment, we initiated a phase 2 trial to test whether the subsequent use of rituximab and CHOP would improve the outcome of patients with PTLD. METHODS In this international multicentre open-label phase 2 trial, treatment-naive adult solid-organ transplant recipients diagnosed with CD20-positive PTLD who had failed to respond to upfront immunosuppression reduction received four courses of rituximab (375 mg/m(2) intravenously) once a week followed by 4 weeks without treatment and four cycles of CHOP every 3 weeks. In case of disease progression during rituximab monotherapy, CHOP was started immediately. Supportive therapy with granulocyte-colony stimulating factor after chemotherapy was mandatory and antibiotic prophylaxis was recommended. The primary endpoint was treatment efficacy measured as response rates in all patients who completed treatment with rituximab and CHOP, per protocol, and response duration, in all patients who completed all planned therapy and responded. Secondary endpoints were frequency of infections, treatment-related mortality, and overall survival. This study is registered at ClinicalTrials.gov, number NCT01458548. FINDINGS 74 patients were enrolled between Dec 12, 2002 and May 5, 2008, of whom 70 patients were eligible to receive treatment. PTLD was of late type in 53 (76%) of 70 patients, monomorphic in 67 (96%) of 70, and histologically EBV associated in 29 (44%) of 66 cases. Four of 70 patients did not receive CHOP. 53 of 59 patients had a complete or partial response (90%, 95% CI 79-96), of which 40 (68%, 55-78) were complete responses. At data cutoff (June 1, 2011) median response duration in the 53 patients who had responded to treatment had not yet been reached (>79·1 months). The main adverse events were grade 3-4 leucopenia in 42 of 62 patients (68%, 55-78) and infections of grade 3-4 in 26 of 64 patients (41%, 29-53). Seven of 66 patients (11%, 5-21) had CHOP-associated treatment-related mortality. Median overall survival was 6·6 years (95% CI 2·8-10·4; n=70). INTERPRETATION Our results support the use of sequential immunochemotherapy with rituximab and CHOP in PTLD. FUNDING F Hoffmann-La Roche, Amgen Germany, Chugaï France.

Plasma Epstein-Barr Virus (EBV) DNA Is a Biomarker for EBV-Positive Hodgkin's Lymphoma

Purpose: Latent Epstein-Barr virus (EBV) genomes are found in the malignant cells of approximately one-third of Hodgkins lymphoma (HL) cases. Detection and quantitation of EBV viral DNA could potentially be used as a biomarker of disease activity. Experimental Design: Initially, EBV-DNA viral load was prospectively monitored from peripheral blood mononuclear cells (PBMC) in patients with HL. Subsequently, we analyzed viral load in plasma from a second cohort of patients. A total of 58 patients with HL (31 newly diagnosed, 6 relapsed, and 21 in long-term remission) were tested. Using real-time PCR, 43 PBMC and 52 plasma samples were analyzed. Results: EBV-DNA was detectable in the plasma of all EBV-positive patients with HL prior to therapy. However, viral DNA was undetectable following therapy in responding patients (P = 0.0156), EBV-positive HL patients in long-term remission (P = 0.0011), and in all patients with EBV-negative HL (P = 0.0238). Conversely, there was no association seen for the EBV-DNA load measured from PBMC in patients with active EBV-positive HL patients as compared with EBV-negative HL, or patients in long-term remission. EBV-DNA load in matched plasma/PBMC samples were not correlated. Conclusions: We show that free plasma EBV-DNA has excellent sensitivity and specificity, and can be used as a noninvasive biomarker for EBV-positive HL and that serial monitoring could predict response to therapy. Additional prospective studies are required to further evaluate the use of free plasma EBV-DNA as a biomarker for monitoring response to treatment in patients with EBV-positive HL.

Catheter-associated bloodstream infection incidence and risk factors in adults with cancer: a prospective cohort study

Central venous catheter-associated bloodstream infections (CABSIs) cause considerable morbidity in patients with cancer. We determined the incidence and risk factors for CABSI by performing a prospective observational cohort study of all adult patients requiring a central venous access device (CVAD) in a haematology-oncology unit. All CVADs were inserted under ultrasound guidance by trained operators in a dedicated interventional radiology facility. A total of 1127 CVADs were assessed in 727 patients over 51,514 line-days. The rate of CABSI per 1000 line-days was 2.50. Factors associated with CABSI included: type of CVAD, greatest for non-tunnelled lines [hazard ratio (HR): 3.50; P < 0.0001] and tunnelled lines (HR: 1.77; P = 0.011) compared to peripherally inserted central venous catheter (PICC) lines; patient diagnosis, greatest for aggressive haematological malignancies (HR: 3.17; P = 0.0007) and least for oesophageal, colon and rectal cancers (HR: 0.29; P = 0.019) compared to other solid tumours; side of insertion, greatest for right-sided lines (HR: 1.60; P = 0.027); and number of prior line insertions (HR: 1.20; P = 0.022). In patients with aggressive haematological malignancies there was significantly more CABSI with non-tunnelled lines (HR: 3.9; P < 0.001) and a trend to more CABSI with tunnelled lines (HR: 1.43; P = 0.12) compared to patients with PICC lines, as well as increased CABSI for right-sided insertions (HR: 1.62; P = 0.047). This study highlights the utility of a standardised CABSI surveillance strategy in adult patients with cancer, provides further data to support the use of PICC lines in such patient populations, and suggests that the side of line insertion may influence risk of CABSI.

Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome.

Therapeutic options are limited for elderly patients with acute myeloid leukemia (AML). A phase Ib/II study was undertaken to evaluate the maximum-tolerated dose (MTD) and preliminary efficacy of the pan-histone deacetylase inhibitor panobinostat (LBH589) in combination with azacitidine in patients with AML or high-risk myelodysplastic syndrome (MDS) naïve to intensive chemotherapy. Thirty-nine patients (AML=29, MDS=10) received azacitidine 75 mg/m2 subcutaneously (days 1–5) and oral panobinostat (starting on day 5, thrice weekly for seven doses) in 28-day cycles until toxicity or disease progression. Dose-limiting toxicities during the phase Ib stage were observed in 0/4 patients receiving 10 mg panobinostat, in 1/7 patients (fatigue) receiving 20 mg, in 1/6 patients (fatigue) receiving 30 mg and in 4/5 patients (fatigue, syncope, hyponatremia and somnolence) receiving 40 mg. In phase II, an additional 17 patients received panobinostat at a MTD of 30 mg. The overall response rate (ORR=CR+CRi+PR) in patients with AML was 31% (9/29) and that in patients with MDS was 50% (5/10). After a median follow-up of 13 months, the median overall survival was 8 and 16 months in patients with AML and MDS, respectively. Increased histone H3 and H4 acetylation was a useful early biomarker of clinical response. Combining panobinostat with azacitidine was tolerable and clinically active in high-risk MDS/AML patients, warranting further exploration.

Pure red cell aplasia due to parvovirus following treatment with CHOP and rituximab for B-cell lymphoma*

Summary.  A 26‐year‐old woman, diagnosed with diffuse large B‐cell lymphoma, was treated with CHOP (cyclophosphamide, hydroxydaunomycin, oncovin, prednisone), rituximab and radiotherapy. She developed transfusion‐dependant anaemia, which persisted following chemotherapy. Bone marrow aspirate and biopsy were consistent with pure red cell aplasia and parvovirus infection. Serology was negative for previous or acute infection but parvovirus DNA was detected by polymerase chain reaction. Administration of intravenous immunoglobulin (1 g/kg) resulted in reticulocytosis and recovery of her haemoglobin. We hypothesize that rituximab caused depletion of her normal B cells, resulting in an inability to mount a primary immune response to parvovirus infection.

Immunity, homing and efficacy of allogeneic adoptive immunotherapy for posttransplant lymphoproliferative disorders

Adoptive immunotherapy using autologous Epstein‐Barr virus (EBV)‐specific cytotoxic T‐lymphocytes (auto‐CTL) can regress posttransplant lymphoproliferative disorders (PTLD). Widespread applicability of auto‐CTL remains constrained. Generation is time‐consuming, and auto‐CTL cannot be established in patients treated with the B‐cell depleting antibody rituximab. By contrast, pregenerated allogeneic CTL (allo‐CTL) offers immediate accessibility. Allo‐CTL has previously shown efficacy in “early” polyclonal‐ PTLD. We treated three patients with aggressive, advanced monoclonal‐PTLD following solid‐organ transplantation. All were refractory to at least three prior therapies. Despite HLA disparity, there was negligible toxicity, with early in vivo antiviral efficacy and reconstitution of EBV peptide‐specific immunity. Two patients attained complete remission (CR). One remains in CR 17 months following therapy, coincident with persistence of donor‐derived tumor targeted EBV‐specific CTL; the other died of non‐PTLD related pathology. In the third patient, autopsy demonstrated homing of allo‐CTL at the tumor site. Larger prospective studies of EBV‐specific allo‐CTL in PTLD are warranted.

CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro

Abstract Chronic lymphocytic leukemia (CLL) is predominantly a disease of accumulation rather than rapid proliferation. To date, no cell lines exist, as CLL cells undergo rapid apoptosis when cultured in vitro, suggesting that a favorable in vivo microenvironment is required. To identify survival signals we cultured primary CLL peripheral blood mononuclear cells (PBMCs) at high density, which has previously been shown to dramatically improve survival. Using antibody arrays we measured the level of 42 cytokines in culture supernatants and showed that inerleukin-6 (IL-6), IL-8, CXCL2 and CCL2 were highly up-regulated in culture. This is the first report to describe a role for CCL2 and CXCL2 in CLL cell survival. Importantly, CXCL2, IL-6 and IL-8 were significantly up-regulated in primary patient plasma. The addition of either CXCL2 or CCL2 enhanced CLL cell survival, while antibodies blocking these chemokines reduced survival. Co-culture of CLL cells and PBMC accessory cells separated by transwells provided a similar degree of survival protection compared to normal culture, whereas CLL cells cultured alone died rapidly. Interestingly, CCL2 and CXCL2 appeared to be produced by CLL cells but only when co-cultured with accessory cells. Thus, we speculate that accessory cells release soluble factors that promote the production of these pro-survival chemokines from CLL cells and physical interactions are not required. Our data support the concept that the CLL microenvironment is critical, and suggests that soluble factors are more important than physical interactions.

Cardiac Amyloid Imaging with 18F-Florbetaben PET: A Pilot Study

Our aim was to determine the feasibility of 18F-florbetaben PET in diagnosing cardiac amyloidosis. Methods: 18F-florbetaben PET was performed on 14 patients: 5 amyloid light chain, 5 amyloid transthyretin, and 4 control with hypertensive heart disease. Qualitative and quantitative assessments of 18F-florbetaben activity were performed using the SUVmean of the left ventricular myocardium and blood pool and calculation of target-to-background SUV ratio. Myocardial 18F-forbetaben retention was also calculated as the percentage mean myocardial SUV change between 0 and 5 min and 15 and 20 min after radiotracer injection. Global left ventricular longitudinal and right ventricular free wall longitudinal strain were calculated using 2-dimensional speckle-tracking echocardiography. Results: Target-to-background SUV ratio and percentage myocardial 18F-forbetaben retention were higher in amyloid patients than in hypertensive controls. A cutoff of 40% was able to differentiate between cardiac amyloid patients and hypertensive controls. Percentage myocardial 18F-forbetaben retention was an independent determinant of both global left ventricular longitudinal and right ventricular free wall longitudinal strain via an inverse curve relationship. Conclusion: 18F-florbetaben PET imaging can accurately identify and differentiate between cardiac amyloidosis and hypertensive heart disease. Percentage myocardial 18F-florbetaben retention was an independent determinant of myocardial dysfunction in cardiac amyloidosis.

A ≥1 log rise in RQ-PCR transcript levels defines molecular relapse in core binding factor acute myeloid leukemia and predicts subsequent morphologic relapse

Core binding factor acute myeloid leukemia (CBF AML), with t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and the associated fusion gene transcripts AML1/ETO or CBFβ/MYH11, has a favourable clinical prognosis although significant numbers of patients still suffer relapse. We examined the prognostic utility of serial bone marrow minimal residual disease (MRD) monitoring by RQ-PCR in a cohort of patients with CBF AML with long term clinical follow-up. Twenty-nine patients were evaluated with a median follow of 34 months. Twelve relapses occurred at a median of 11 months (range 4 – 17) from diagnosis. RQ-PCR levels at diagnosis, post-induction chemotherapy and post-consolidation were not predictive of outcome. However, a ≥1 log10 rise at any stage in transcript level relative to the level from a remission bone marrow sample correlated with inferior leukemia free survival (LFS) and imminent morphologic relapse (hazard ratio 8.6). Relapses occurred a median of 60 days (range 45 – 272) after a log10 rise. A ≥1 log10 rise in transcript levels strongly predicts subsequent morphologic relapse in CBF AML and therefore defines molecular relapse. Our data support a simple RQ-PCR model for prediction of impending relapse which has the potential for widespread clinical applicability. Prospective identification of high risk patients will enable clinical trials to assess the efficacy of treatment initiated at molecular relapse.

Validation of whole blood impedance aggregometry as a new diagnostic tool for HIT: results of a large Australian study

Heparin-induced thrombocytopenia (HIT) remains a challenge, with diagnosis confirmed only by functional assays. The gold standard 14C-serotonin release assay (SRA) is highly sensitive but technically challenging and unsuitable for routine use. We conducted a large study to validate whole blood impedance aggregometry (WBIA) as a suitable diagnostic tool for HIT. WBIA and SRA were used to test 181 samples positive for H-PF4 antibodies by PaGIA or ELISA. Using the same high responder donor, 77 samples were positive by WBIA (aggregation with low-dose but not high-dose heparin). Using the strict definition for SRA positivity, 72 samples were true HIT. In nine samples, serotonin release with high-dose heparin dropped by > 50% but was still >20%; these were retested after a one-half dilution and 8/9 became positive. Ten other samples were discrepant between the two assays: one strongly positive (89% release) and six weakly positive samples by SRA (average release 56%) were WBIA negative. When these samples were retested using a random donor, only two remained SRA positive. Three samples were strongly WBIA positive but SRA negative; two were retested by SRA with 0.5IU/ml heparin and one became positive. Under controlled conditions, using the same selected high-responder donor, WBIA and SRA performed similarly with slightly increased sensitivity of the WBIA when using the strict definition of SRA positivity. WBIA is easy to perform with rapid turn-around time and warrants a multi-laboratory trial to complete its validation as a confirmatory assay for platelet-activating HIT antibodies.