Jamie Cavenagh

Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells

Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukemia-initiating cells. One method commonly used to isolate these rare cells is to sort cells stained with fluorochrome-conjugated antibodies into fractions, then transplant the different fractions into immunodeficient mice to test their repopulating ability. The antibodies are generally treated as being neutral in terms of their effects on the experiment. Human repopulating cells are thought to express CD34 and lack CD38. Here we present evidence that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukemia cells. We show that this effect is Fc-mediated and can be overcome by treating mice with immunosuppressive antibodies. When this inhibitory effect is prevented, we demonstrate that the CD34(+)CD38(+) fraction of certain acute myeloid leukemia samples contains all, or at least most, leukemia-initiating cell capacity. This study highlights the potential pitfall of antibody-mediated clearance of repopulating cells and is important for any groups working with this model. More importantly, the work suggests that there is greater variation in the phenotypes of leukemia-initiating cells than previously suggested.

Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia

The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

Guidelines for the diagnosis and management of aplastic anaemia

King’s College Hospital, St Mary’s Hospital, Barts and The London Hospital, London, Birmingham Children’s Hospital, Birmingham, Barts and The London School of Medicine and Dentistry, St George’s Hospital, London, Queen Elizabeth Hospital, King’s Lynn, Norfolk, Ashford Hospital, Middlesex, London, Patient representative, St Helier Hospital, Carshalton, Surrey, Royal Bournemouth Hospital, Dorset, Chesterfield Royal Hospital, Derbyshire, and Manchester Royal Infirmary, Manchester, UK.

Lenalidomide and Dexamethasone in Transplant-Ineligible Patients with Myeloma

BACKGROUND The combination melphalan-prednisone-thalidomide (MPT) is considered a standard therapy for patients with myeloma who are ineligible for stem-cell transplantation. However, emerging data on the use of lenalidomide and low-dose dexamethasone warrant a prospective comparison of the two approaches. METHODS We randomly assigned 1623 patients to lenalidomide and dexamethasone in 28-day cycles until disease progression (535 patients), to the same combination for 72 weeks (18 cycles; 541 patients), or to MPT for 72 weeks (547 patients). The primary end point was progression-free survival with continuous lenalidomide-dexamethasone versus MPT. RESULTS The median progression-free survival was 25.5 months with continuous lenalidomide-dexamethasone, 20.7 months with 18 cycles of lenalidomide-dexamethasone, and 21.2 months with MPT (hazard ratio for the risk of progression or death, 0.72 for continuous lenalidomide-dexamethasone vs. MPT and 0.70 for continuous lenalidomide-dexamethasone vs. 18 cycles of lenalidomide-dexamethasone; P<0.001 for both comparisons). Continuous lenalidomide-dexamethasone was superior to MPT for all secondary efficacy end points, including overall survival (at the interim analysis). Overall survival at 4 years was 59% with continuous lenalidomide-dexamethasone, 56% with 18 cycles of lenalidomide-dexamethasone, and 51% with MPT. Grade 3 or 4 adverse events were somewhat less frequent with continuous lenalidomide-dexamethasone than with MPT (70% vs. 78%). As compared with MPT, continuous lenalidomide-dexamethasone was associated with fewer hematologic and neurologic toxic events, a moderate increase in infections, and fewer second primary hematologic cancers. CONCLUSIONS As compared with MPT, continuous lenalidomide-dexamethasone given until disease progression was associated with a significant improvement in progression-free survival, with an overall survival benefit at the interim analysis, among patients with newly diagnosed multiple myeloma who were ineligible for stem-cell transplantation. (Funded by Intergroupe, Francophone du Myélome and Celgene; FIRST ClinicalTrials.gov number, NCT00689936; European Union Drug Regulating Authorities Clinical Trials number, 2007-004823-39.).

PAD combination therapy (PS‐341/bortezomib, doxorubicin and dexamethasone) for previously untreated patients with multiple myeloma

Bortezomib (formerly PS‐341) has significant activity in patients with relapsed multiple myeloma (MM), its efficacy is increased with the addition of dexamethasone and it demonstrates synergy with doxorubicin, thus providing the rationale for combination therapy with bortezomib, doxorubicin and dexamethasone (PAD). Patients with untreated MM received four 21‐d cycles of PAD, comprising bortezomib 1·3 mg/m2 on days 1, 4, 8 and 11, along with dexamethasone 40 mg on days 1–4, 8–11 and 15–18 during cycle 1 and days 1–4 during cycles 2–4. During days 1–4, patients also received 0, 4·5 or 9 mg/m2 of doxorubicin at dose levels 1, 2, and 3 respectively. Following peripheral blood stem cell (PBSC) collection, patients received high‐dose melphalan (MEL200) with PBSC transplantation (PBSCT). After PAD induction alone, 20 of 21 patients (95%) achieved at least a partial response (PR), including complete response (CR) in five patients (24%). Twenty of 21 had PBSC mobilized, and 18 of 20 received MEL200/PBSCT. In an intention‐to‐treat analysis, response rates were: CR 43%, near CR 14%, very good PR 24%, PR 14% and stable disease 5%. PAD was effective, did not prejudice subsequent PBSC collection, and should be further evaluated in prospective randomized trials.

Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34− fraction

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

Reversibility of symptomatic peripheral neuropathy with bortezomib in the phase III APEX trial in relapsed multiple myeloma: Impact of a dose-modification guideline

The frequency, characteristics and reversibility of bortezomib‐associated peripheral neuropathy were evaluated in the phase III APEX (Assessment of Proteasome Inhibition for Extending Remissions) trial in patients with relapsed myeloma, and the impact of a dose‐modification guideline on peripheral neuropathy severity and reversibility was assessed. Patients received bortezomib 1·3 mg/m2 (days 1, 4, 8, 11, eight 21‐d cycles, then days 1, 8, 15, 22, three 35‐d cycles); bortezomib was held, dose‐reduced or discontinued depending on peripheral neuropathy severity, according to a protocol‐specified dose‐modification guideline. Overall, 124/331 patients (37%) had treatment‐emergent peripheral neuropathy, including 30 (9%) with grade ≥3; incidence and severity were not affected by age, number/type of prior therapies, baseline glycosylated haemoglobin level, or diabetes history. Grade ≥3 incidence appeared lower versus phase II trials (13%) that did not specifically provide dose‐modification guidelines. Of patients with grade ≥2 peripheral neuropathy, 58/91 (64%) experienced improvement or resolution to baseline at a median of 110 d, including 49/72 (68%) who had dose modification versus 9/19 (47%) who did not. Efficacy did not appear adversely affected by dose modification for grade ≥2 peripheral neuropathy. Bortezomib‐associated peripheral neuropathy is manageable and reversible in most patients with relapsed myeloma. Dose modification using a specific guideline improves peripheral neuropathy management without adversely affecting outcome.

Guidelines for the diagnosis and management of multiple myeloma 2011.

653. Spencer, A., Prince, H.M., Roberts, A.W., Prosser, I.W., Bradstock, K.F., Coyle, L., Gill, D.S., Horvath, N., Reynolds, J. & Kennedy, N. (2009) Consolidation therapy with low-dose thalido- mide and prednisolone prolongs the survival of multiple myeloma patients undergoing a single autologous stem-cell transplantation procedure. Journal of Clinical Oncology, 27, 1788–1793. Srkalovic, G., Cameron, M.G., Rybicki, L., Deitcher, S.R., Kattke-Marchant, K. & Hussein, M.A. (2004) Monoclonal gammopathy of undeter- mined significance and multiple myeloma are associated with an increased incidence of venothromboembolic disease. Cancer, 101, 558– 666. Stadtmauer, E.A., Weber, D.M., Niesvizky, R., Belch, A., Prince, M.H., San Miguel, J.F., Facon, T., Olesnyckyj, M., Yu, Z., Zeldis, J.B., Knight, R.D. Guideline 74 a 2011 Blackwell Publishing Ltd, British Journal of Haematology, 154, 32–75 & Dimopoulos, M.A. (2009) Lenalidomide in combination with dexamethasone at first relapse in comparison with its use as later salvage therapy in relapsed or refractory multiple myeloma. European Journal of Haematology, 82, 426–432. Stewart, A.K., Vescio, R., Schiller, G., Ballester, O., Noga, S., Rugo, H., Freytes, C., Stadtmauer, E., Tarantolo, S., Sahebi, F., Stiff, P., Meharchard, J., Schlossman, R., Brown, R., Tully, H., Benyunes, M., Jacobs, C., Berenson, R., White, M., DiPersio, J., Anderson, K.C. & Berenson, J. (2001) Purging of autologous peripheral-blood stem cells using CD34 selection does not improve overall or progression-free survival after high-dose chemo- therapy for multiple myeloma: results of a multicenter randomized controlled trial. Journal of Clinical Oncology, 19, 3771–3779. Stewart, A.K., Chen, C.I., Howson-Jan, K., White, D., Roy, J., Kovacs, M.J., Shustik, C., Sadura, A., Shepherd, L., Ding, K., Meyer, R.M. & Belch, A.R. (2004) Results of a multicenter randomized phase II trial of thalidomide and prednisone mainte- nance therapy for multiple myeloma after autol- ogous stem cell transplant. Clinical Cancer Research, 10, 8170–8176. Stewart, A.K., Bergsagel, P.L., Greipp, P.R., Dis- penzieri, A., Gertz, M.A., Hayman, S.R., Kumar, S., Lacy, M.Q., Lust, J.A., Russell, S.J., Witzig, T.E., Zeldenrust, S.R., Dingli, D., Reeder, C.B., Roy, V., Kyle, R.A., Rajkumar, S.V. & Fonseca, R. (2007) A practical guide to defining high-risk myeloma for clinical trials, patient counseling and choice of therapy. Leukemia, 21, 529–534. Terpos, E. & Dimopoulos, M.A. (2005) Myeloma bone disease: pathophysiology and management. Annals of Oncology, 16, 1223–1231. Terpos, E. & Rahemtulla, A. (2004) Bisphosphonate treatment for multiple myeloma. Drugs of today (Barcelona, Spain: 1998), 40, 29–40. Terpos, E., Sezer, O., Croucher, P.I., Garcia-Sanz, R., Boccadoro, M., San Miguel, J., Ashcroft, J., Blade, J., Cavo, M., Delforge, M., Dimopoulos, M.A., Facon, T., Macro, M., Waage, A. & Son- neveld, P. (2009) The use of bisphosphonates in multiple myeloma: recommendations of an ex- pert panel on behalf of the European Myeloma Network. Annals of Oncology, 20, 1303–1317. Tosi, P., Zamagni, E., Cellini, C., Cangini, D., Tac- chetti, P., Tura, S., Baccarani, M. & Cavo, M. (2004) Thalidomide alone or in combination with dexamethasone in patients with advanced, relapsed or refractory multiple myeloma and renal failure. European Journal of Haematology, 73, 98–103. Tosi, P., Zamagni, E., Cellini, C., Plasmati, R., Cangini, D., Tacchetti, P., Perrone, G., Pastorelli, F., Tura, S., Baccarani, M. & Cavo, M. (2005) Neurological toxicity of long-term (>1 yr) thalidomide therapy in patients with multiple myeloma. European Journal of Haematology, 74, 212–216. Vela-Ojeda, J., Garcia-Ruiz-Esparza, M.A., Padilla- Gonzalez, Y., Gomez-Almaguer, D., Gutierrez- Aguirre, C.H., Gomez-Rangel, D., Morales- Toquero, A., Ruiz-Delgado, G.J., Delgado-Lamas, J.L. & Ruiz-Arguelles, G.J. (2007) Autologous peripheral blood stem cell transplantation in multiple myeloma using oral versus I.V. melphalan. Annals of Hematology, 86, 277–282. van de Velde, H.J., Liu, X., Chen, G., Cakana, A., Deraedt, W. & Bayssas, M. (2007) Complete response correlates with long-term survival and progression-free survival in high-dose therapy in multiple myeloma. Haematologica, 92, 1399–1406. Vigneau, C., Ardiet, C., Bret, M., Laville, M., Fiere, D., Tranchand, B. & Fouque, D. (2002) Inter- mediate-dose (25 mg/m) IV melphalan for multiple myeloma with renal failure. Journal of Nephrology, 15, 684–689. Vogel, C.L., Yanagihara, R.H., Wood, A.J., Schnell, F.M., Henderson, C., Kaplan, B.H., Purdy, M.H., Orlowski, R., Decker, J.L., Lacerna, L. & Hohneker, J.A. (2004) Safety and pain palliation of zoledronic acid in patients with breast cancer, prostate cancer, or multiple myeloma who previously received bisphosphonate therapy. Oncologist, 9, 687–695. Waage, A., Gimsing, P., Juliusson, G., Turesson, I., Gulbrandsen, N., Eriksson, T., Hjorth, M., Niel- sen, J.L., Lenhoff, S., Westin, J. & Wisloff, F. (2004) Early response predicts thalidomide efficiency in patients with advanced multiple myeloma. British Journal of Haematology, 125, 149–155. Wang, M., Knight, R., Dimopoulos, M., Siegel, D., Rajkumar, S.V., Facon, T., Alexanian, R., Yu, Z., Zeldis, J., Olesnyckyj, M. & Weber, D. (2006) Lenalidomide in combination with dexametha- sone was more effective than dexamethasone in patients who have received prior thalidomide for relapsed or refractory multiple myeloma. Blood (ASH Annual Meeting Abstracts), 108, Abstract 3553. Weber, D.M., Gavino, M., Delasalle, K., Rankin, K., Giralt, S. & Alexanian, R. (1999) Thalidomide alone or with dexamethasone for multiple mye- loma. Blood, 94(Suppl. I), 604a. Weber, D., Wang, M., Chen, C., Belch, A., Stadt- mauer, E.A., Niesvisky, R., Yu, Z., Olesnyckyj, M., Zeldis, J., Knight, R.D. & Dimopoulos, M. (2006) Lenalidomide Plus High-Dose Dexamethasone Provides Improved Overall Survival Compared to High-Dose Dexamethasone Alone for Relapsed or Refractory Multiple Myeloma (MM): Results of 2 Phase III Studies (MM-009, MM-010) and Subgroup Analysis of Patients with Impaired Renal Function. Blood (ASH Annual Meeting Abstracts), 108, 3547. Weber, D.M., Chen, C., Niesvizky, R., Wang, M., Belch, A., Stadtmauer, E.A., Siegel, D., Borrello, I., Rajkumar, S.V., Chanan-Khan, A.A., Lonial, S., Yu, Z., Patin, J., Olesnyckyj, M., Zeldis, J.B. & Knight, R.D. (2007) Lenalidomide plus dexa- methasone for relapsed multiple myeloma in North America. New England Journal of Medicine, 357, 2133–2142. Wechalekar, A., Amato, D., Chen, C., Stewart K., A. & Reece, D. (2005) IgD multiple myeloma–a clinical profile and outcome with chemotherapy and autologous stem cell transplantation. Annals of Hematology, 84, 115–117. Wijermans, P., Schaafsma, M., Norden, Y.v., Ammerlaan, R., Wittebol, S., Sinnige, H., Zweegman, S., Kooi, M.v.M., Griend, R.V.d., Lokhorst, H. & Sonneveld, P. (2008) Melphalan/ prednisone versus melphalan/prednisone/thalid- omide in induction therapy for multiple myelo- ma in elderly patients: final analysis of the dutch cooperative group HOVON 49 study. Blood (ASH Annual Meeting Abstracts), 112, Abstract 649. Zervas, K., Mihou, D., Katodritou, E., Pouli, A., Mitsouli, C.H., Anagnostopoulos, A., Delibasi, S., Kyrtsonis, M.C., Anagnostopoulos, N., Terpos, E., Zikos, P., Maniatis, A. & Dimopoulos, M.A. (2007) VAD-doxil versus VAD-doxil plus tha- lidomide as initial treatment for multiple mye- loma: results of a multicenter randomized trial of the Greek Myeloma Study Group. Annals of Oncology, 18, 1369–1375. Zucchelli, P., Pasquali, S., Cagnoli, L. & Ferrari, G. (1988) Controlled plasma exchange trial in acute renal failure due to multiple myeloma. Kidney International, 33, 1175–1180. Guideline a 2011 Blackwell Publishing Ltd, British Journal of Haematology, 154, 32–75 75

A phase 2 study of the safety and efficacy of rituximab with plasma exchange in acute acquired thrombotic thrombocytopenic purpura.

The safety and efficacy of weekly rituximab 375 mg/m(2) (×4), given within 3 days of acute TTP admission, with standard therapy (PEX and steroids) was evaluated. Clinical outcomes were compared to historical controls (n = 40) who had not received rituximab. Within the trial group, 15 of 40 required ICU admission and 15% of all cases with the highest troponin T levels on admission were ventilated. Before the second rituximab infusion, 68% of cases had a platelet count > 50 × 10(9)/L and 38% > 150 × 10(9)/L. Fewer PEX were required in whites compared to nonwhite in the rituximab group (mean 14 vs 21, P = .0095). Inpatient stay was reduced by 7 days in the non-ICU trial cases compared to historical controls (P = .04), especially in whites, with a mean reduction of 7 days (P = .05). Ten percent of trial cases relapsed, median, 27 months (17-31 months), compared to 57% in historical controls, median 18 months (3-60 months; P = .0011). There were no excess infections or serious adverse events with rituximab. In conclusion, rituximab appears a safe and effective therapy. Inpatient stay and relapse are significantly reduced in the rituximab cohort. Rituximab should be considered in conjunction with standard therapy on acute presentation of TTP. This study was registered at www.clinicaltrials.gov as NCT009-3713.

Remission in acute refractory and relapsing thrombotic thrombocytopenic purpura following rituximab is associated with a reduction in IgG antibodies to ADAMTS-13

Thrombotic thrombocytopenic purpura (TTP) is a life‐threatening disorder and plasma exchange (PEX) remains the primary treatment modality. Twenty‐five patients with acute refractory/relapsing idiopathic TTP received rituximab in conjunction with PEX because of progressive clinical disease or deterioration in laboratory parameters, despite intensive standard therapy. In relapsing TTP, rituximab was started if antibody to ADAMTS‐13 (a disintegrin and metalloproteinase with thrombospondin motif‐13) was demonstrated during previous episodes. All 25 patients attained complete clinical and laboratory remission in a median of 11 d after initiating rituximab. In 21 cases, ADAMTS‐13 activity was within the normal range following rituximab. Inhibitors were detected in 24/25 patients by mixing studies and/or immunoglobulin G (IgG) antibodies to ADAMTS‐13 pre‐rituximab. There was no evidence of inhibitors and/or IgG activity <10% in 23/25 patients following rituximab. In acute refractory cases, the median number of PEX pre‐rituximab and following the first rituximab infusion was 13 and 9, respectively. There have been no infectious complications, despite low CD 19 levels and no relapses. In patients with acute refractory/relapsing idiopathic TTP, rituximab appears to be a safe, effective, targeted therapy with a significant reduction in the requirement for PEX.