Improved Clinical Outcomes of High Risk β Thalassemia Major Patients Undergoing a HLA Matched Related Allogeneic Stem Cell Transplant with a Treosulfan Based Conditioning Regimen and Peripheral Blood Stem Cell Grafts

<div><p></p><p>Improving clinical outcomes among high risk Class III β thalassemia major patients (Class IIIHR) receiving an allogeneic SCT remains a challenge. From October, 2009 a treosulfan based regimen (TreoFluT) was used for all consecutive Class III patients (n = 50). The clinical outcomes were compared with the historical conventional busulfan (BuCy) based regimen (n = 139). Use of TreoFluT was associated with a significantly reduced incidence of sinusoidal obstruction syndrome (SOS) among Class IIIHR cases (78% to 30%; P = 0.000) and early TRM (46% to 13%; p = 0.005). There was also a trend towards better engraftment in the Class IIIHR subset (P = 0.055). However, the use of bone marrow (BM) as source of stem cells along with the TreoFluT regimen was associated with 50% early mixed chimerism which reduced to 8.5% with the use of a peripheral blood stem cell graft (PBSC). Use of a PBSC graft was not associated with a significant increase in the incidence of acute or chronic graft versus host disease (GVHD). The overall and event free survival was significantly better among the Class IIIHR subset with the use of TreoFluT Vs. BuCy (86.6±7.3 Vs. 39.4±6.8%; P = 0.002 and 77.8±8.8 Vs. 32.4±6.5%; P = 0.003 respectively). A TreoFluT conditioning regimen with a PBSC graft can significantly improve clinical outcomes of Class IIIHR patients.</p></div

Comparison of Class III patients who received TreoFluTconditioning with either a bone marrow or peripheral blood source of stem cells.

@<p>BM: Bone marrow; #PBSC: Peripheral blood stem cells.</p>θ<p>PRC: Packed red cell units; ^©GVHD: graft versus host disease.</p>∞<p>3 yrs. 3 years Kaplan-Meier estimate±1SE%3.</p

Baseline characteristics, supportive care and clinical outcomes of Class IIIHR (High Risk) group receiving different conditioning regimens.

a<p>BuCy: Busulfan + Cyclophosphamide; *ALT: Alanine aminotransferase.</p>b<p>TreoFluT: Treosulfan, fludarabine and thiotepa; ∧AST: Aspartate aminotransferase.</p>@<p>BM: Bone marrow; #PBSC:Peripheral blood stem cells.</p>θ<p>PRC: Packed red cell units; ^©GVHD: graft versus host disease.</p>∞<p>3 yrs. 3 years Kaplan-Meier estimate±1SE%.</p

Comparison of major contributory cause of early death (death <100 days post transplant) among Class III patients.

a<p>BuCy Busulfan + Cyclophosphamide.</p>b<p>TreoFluT Treosulfan, fludarabine and thiotepa.</p>#<p>DAH Diffuse alveolar hemorrhage.</p>©<p>GVHD Graft versus host disease.</p

Analysis of day 28 chimerism and survival in different subsets.

<p>(A) Day 28 chimerism comparing patients conditioned with aTreoFluT regimen and receivingeither a bone marrow graft (Treo BM) or a peripheral blood stem cell graft (Treo PBSC); NE = not evaluated; indicates cases that had died prior to day 28 and were hence: not evaluated. ▴ Filled triangles indicates cases that had an event defined as either graft rejection or death. △ Empty triangles indicate cases that did not have an event defined as either graft rejection or death (B) Overall survival of all Class III patients conditioned with either a TreoFluT (Treo) or a busulfan based regimen (Bu/Cy) (C) Event free survival of all Class III patients conditioned with either a TreoFluT (Treo) or a busulfan based regimen (Bu/Cy) (D) Overall survival of all Class IIIHR patients conditioned with either a TreoFluT (Treo) or a busulfan based regimen (Bu/Cy) (E) Event free survival of all Class IIIHR patients conditioned with either a TreoFluT (Treo) or a busulfan based regimen (Bu/Cy).</p

Baseline characteristics, supportive care and clinical outcomes of Class III patients receiving different conditioning regimens.

a<p>BuCy: Busulfan + Cyclophosphamide; *ALT: Alanine aminotransferase.</p>b<p>TreoFluT: Treosulfan, fludarabine and thiotepa; ∧AST: Aspartate aminotransferase.</p>@<p>BM: Bone marrow; #PBSC: Peripheral blood stem cells.</p>θ<p>PRC: Packed red cell units; ^©GVHD: graft versus host disease.</p>∞<p>3 yrs. 3 years Kaplan-Meier estimate±1SE%.</p

<i>NRF2</i> overexpression in AML cell lines increases IC50 of ATO & Dnr by up-regulating downstream target expression.

<p>Two AML cell lines (HL-60 and MOLM-13) with relatively low expression of <i>NRF2</i> were made to stably over express <i>NRF2</i>. <b>(A)</b> Overexpression was further confirmed by quantitative real-time PCR (i) and western blot (ii) for Nrf2 <b>(B)</b> Nrf2 downstream target genes (<i>GCLC</i>, <i>GCLM</i>, <i>NQO1</i>, <i>and HMOX-1</i>) were analyzed by real time quantitative PCR. <b>(C)</b> <i>In-vitro</i> cytotoxicity of overexpressed cells to Dnr and ATO was compared with control cells. Overexpression increases resistance to Dnr and ATO.</p

Pharmacological inhibition of Nrf2 with brusatol brings down the IC50 of Ara-C, Dnr & ATO in resistant AML cell lines.

<p>THP1 cells were incubated with Nrf2 inhibitor Brusatol 100nM for 6hrs, followed by increasing concentration of <b>(A)</b> Ara-C (0.1–80μM), <b>(B)</b> Dnr (0.0025–1μM) and <b>(C)</b> ATO (0.1–6μM) for 48hrs. <i>In-vitro</i> cytotoxicity was measured by MTT assay (n = 9). <b>(D)</b> IC50 of THP1 to Dnr and ATO was compared with untreated control cells. <b>(E)</b> THP1 cells were incubate with 100nM of Brusatol followed by treatment with 5μM of Ara-C, 0.25μM of Dnr or 6μM of ATO for 24hrs. Control and treated (5*10³) cells were seeded in methyl cellulose medium (n = 2) and enumerated on day 14. Light microscopic images of colonies were taken on day 14 (10X magnification) (i). Control or treated (5*10³) cells seeded in methyl cellulose medium were stained with methylene blue (ii) and colonies were enumerated (n = 2)(iii). Brusatol alone minimally reduces the colony forming capacity of THP1 cells and considerably reduces in combination with Ara-C, Dnr and ATO.</p

Primary AML cells and cell lines resistant to cytarabine, daunorubicin and arsenic trioxide show increased Nrf2 expression.

<p><b>(A)</b> AML samples categorised based on median IC50 values to Ara-C (6μM), Dnr (0.4μM) and ATO (2.42μM) were analysed for the expression of <i>NRF2</i> by quantitative real time PCR. Y axis denotes relative expression (2^-dCT) of <i>NRF2</i> normalised to <i>GAPDH</i>. <b>(B)</b> <i>NRF2</i> levels in AML cell lines resistant (THP1, U937) or sensitive (HL-60, MOLM-13) to Ara-C, Dnr and ATO were analysed and represented as fold change (2^-ddCT) normalised to Molm13 (with low <i>NRF2</i> RNA)} (n = 5). *Statistical significance were calculated based on Kruskal–Wallis test and p<0.05 is indicated. <b>(C)</b> Flow cytometric analysis of total Nrf2 expression in AML cell lines (n = 4). <b>(D)</b> Immunofluorescence analysis of nuclear Nrf2 expression in AML cell lines {4′,6-diamidino-2-phenylindole (blue) and Nrf2 antibody (red) stained images overlay} (n = 2). <b>(E)</b> Immunoblotting of total Nrf2 in AML cell lines (β-actin was used as loading control) (n = 3). <b>(F)</b> Nrf2 downstream target expression {(<i>HO-1</i>, <i>NQO1</i>, <i>GCLC</i> and <i>GCLM</i>) normalized to <i>GAPDH</i> and expressed relative to that of MOLM-13 cell line (n = 4)}.*Statistical significance were calculated based on Kruskal–Wallis test and p<0.05 is indicated.</p

Brusatol treatment brings down Nrf2 expression and reduces colony forming capacity of THP1 cells.

<p>THP1 cells were treated with 1μM of brusatol reconstituted in DMSO (final concentration of 0.01%) for 6hrs. The expression of Nrf2 after treatment with brusatol was assessed by <b>(A)</b> flow cytometry (n = 2) (i), western blot with β—actin as the loading control (n = 3) (ii) and subcellular expression (iii). <b>(B)</b> THP1 cells were treated with 100nM of Brusatol for 6h and downstream targets of Nrf2 <i>GCLC</i>, <i>GCLM</i>, <i>HO-1</i>, <i>and NQO1</i> was evaluated by quantitative real time PCR (n = 4). RNA expression of all target genes was normalized to <i>GAPDH</i>. *Statistical significance were calculated based on unpaired t test and p<0.05 is indicated. <b>(C)</b> Apoptosis upon incubation with two different concentrations 100nM and 1000nM of Brusatol was assessed by Annexin V-7AAD.</p