Living T9 glioma cells expressing membrane macrophage colony-stimulating factor produce immediate tumor destruction by polymorphonuclear leukocytes and macrophages via a "paraptosis"-induced pathway that promotes systemic immunity against intracranial T9 gliomas

Development of resistance to cytarabine (AraC) is a major problem in the treatment of patients with acute myeloid leukemia (AML). Inactivation of deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We have identified inactive, alternatively spliced dCK forms in leukemic blasts from patients with resistant AML. Because these dCK-spliced variants were only detectable in resistant AML, it was hypothesized that they might play a role in AraC resistance in vivo. In the current study, the biologic role of the alternatively spliced dCK forms in AraC resistance was further investigated by retroviral transductions in rat leukemic cells. Introduction of inactive, alternatively spliced dCK forms into AraC-resistant K7 cells, with no endogenous wild-type (wt) dCK activity, could not restore AraC sensitivity, whereas wt dCK fully restored the AraC-sensitive phenotype. Transfection of alternatively spliced dCK forms into AraC-sensitive KA cells, as well as in human leukemic U937 cells and in phytohemagglutinin-stimulated T cells, did not significantly change sensitivity toward AraC. In addition, cotransduction of wt dCK with alternatively spliced dCK in K7 cells did not result in altered sensitivity to AraC compared with K7 cells only transduced with wt dCK. These data indicate that the alternatively spliced dCK forms cannot act as a dominant-negative inhibitor on dCK wt activity when they are coexpressed in a single cell. However, a cell expressing alternatively spliced dCK forms that has lost wt dCK expression is resistant to the cytotoxic effects of AraC.     

Altered multidrug resistance phenotype caused by anthracycline analogues and cytosine arabinoside in myeloid leukemia.

The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P =.01) and Pgp function (P =.03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P <.05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r =.90, P =.0001) and Pgp-positive blasts (r =.77, P =.0002). This increase in Pgp expression and function was inhibited by the addition of 1 micromol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.     

The prognostic value of cN-II and cN-III enzymes in adult acute myeloid leukemia

We analyzed the expression of deoxycytidine kinase (dCK), UMP/CMP-kinase (UMP/CMP-K), nucleotide diphosphokinase (NDPK-B) and 5'-nucleotidases cN-II, cN-III, cdN and mdN by quantitative polymerase chain reaction at diagnosis in leukemic blasts from 96 patients with acute myeloid leukemia (AML) treated with ara-C. Our results show that high mRNA levels of cN-II and low mRNA levels of cN-III are correlated with a worse clinical outcome and suggest that these enzymes may have a role in sensitivity to ara-C in AML patients.     

Deoxycytidine kinase and cN-II nucleotidase expression in blast cells predict survival in acute myeloid leukaemia patients treated with cytarabine

Summary. The cytotoxic activity of cytarabine (ara-C) in leukaemic blasts depends on activating enzymes such as deoxycytidine kinase (dCK) and inactivating enzymes such as the 5'-nucleotidases. We have analysed dCK and 'high-Km' 5'-nucleotidase (cN-II) mRNA expression by the quantitative real-time polymerase chain reaction at diagnosis in leukaemic blasts from 115 acute myeloid leukaemia (AML) patients treated with ara-C. The prognostic value of these parameters as well as that of the cN-II/dCK ratio was determined. In univariate analyses: (1) low levels of dCK, high levels of cN-II and a high cN-II/dCK ratio predicted shorter disease-free survival (DFS); (2) low levels of dCK and cN-II/dCK ratio also predicted shorter overall survival (OS). In a multivariate analysis taking into account other clinical and laboratory variables: (1) high cN-II expression, a high cN-II/dCK ratio, age ≥ 60 years and an unfavourable karyotype were independent prognostic factors for DFS; and (2) a high cN-II/dCK ratio, age ≥ 60 years and an unfavourable karyotype predicted shorter OS. Age, karyotype and cN-II/dCK ratio were used to define a prognostic score that permitted the identification of high- and low-risk groups. Our results suggest that dCK and cN-II mRNA expression in leukaemic blasts at diagnosis is correlated with clinical outcome and may play a functional role in the resistance to ara-C in patients with AML.     

Structural and functional analysis of the cytidine deaminase gene in patients with acute myeloid leukaemia

Gene transfer of the cytidine deaminase (CDD) cDNA has recently been shown to induce cellular resistance to cytarabine (AraC) in vitro . To investigate the role for CDD in acute myeloid leukaemia (AML) we analysed the CDD activity and CDD gene structure in blast material from well-defined patients with untreated and AraC refractory (RF) AML. Median CDD activity in previously untreated AML was significantly lower than in RF-AML blasts ( P  = 0.015) and was significantly lower in patients with complete remission than with blast persistence following induction chemotherapy ( P  = 0.043). Structural investigation of the CDD gene by Southern analyses and RT-PCR showed no detectable aberrations. Sequence analysis of the CDD cDNA from nine RF-AML patients showed inconsistent aberrations in three patients. Semiquantitative assessment of CDD mRNA expression revealed a significant correlation with CDD activity. In conclusion, concordant with another recent study our data suggest a correlation of pretherapeutic CDD activity with induction treatment response. Besides the previously described prognostic impact of mdr1 expression, this result could be useful for the development of risk-adapted AML treatment strategies and warrants further studies of CDD activity in well-defined cohorts of AML patients and of the mechanisms involved in the regulation of CDD activity.     

The pharmacodynamic basis for the increased antileukaemic efficacy of cytosine arabinoside-based treatment regimens in acute myeloid leukaemia with a high proliferative activity

The current study was initiated to explore the mechanisms underlying the previously demonstrated association between the proliferative activity of leukaemic blasts and the response to cytosine arabinoside (AraC)-based therapy in de novo acute myeloid leukaemia (AML). The activity of key enzymes of AraC metabolism-deoxycytidine kinase (DCK), cytidine deaminase (DCD) and polymerase alpha (PolyA) were determined in blast cells from 33 patients. In addition, formation and retention of intracellular levels of AraC triphosphate (AraCTP) and DNA incorporation of AraC were measured, as was the proliferative activity of leukaemic blasts by [3H]-TdR incorporation before and after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) for 48 h. AraC incorporation into the DNA (median 0.60 pmol/105 cells) was significantly related to the proliferative activity of AML blasts (r = 0.74, P < 0.001). Similarly, priming with GM-CSF or G-CSF increased both the proliferative activity of AML blasts by a median of 1.84- and 1.64-fold, respectively, and the incorporation of AraC into the DNA (1.29- and 1.40-fold respectively). In contrast, no relationship was found between the endogenous proliferative activity (EPA) and enzyme activities regulating AraC activation (DCK; median 4.70 pmol/min/mg protein), inactivation (DCD; median 2.92 pmol/min/mg protein) or inhibitory effects (PolyA; median 1.50 pmol/min/mg protein), nor the formation or retention of AraCTP (median 306.1 ng/107 cell and 1.6 h respectively). When samples were grouped according to EPA (more than or less than the median), slowly proliferating specimens had a higher response to cytokine priming for proliferative activity and incorporation of AraC into DNA. Clinical data of 15 patients were available. Although all eight patients with a high endogenous proliferative activity reached complete remission, only four out of seven patients with a low proliferative activity responded, whereas the other three patients were non-responders (P = 0.077).     

5'-(3')-nucleotidase mRNA levels in blast cells are a prognostic factor in acute myeloid leukemia patients treated with cytarabine

We analyzed cytosolic 5'-(3')-nucleotidase (dNT-1) mRNA expression by quantitative polymerase chain reaction at diagnosis in leukemic blasts from 114 patients with acute myeloid leukemia (AML) treated with ara-C. Our results show that low dNT-1 mRNA expression in leukemic blasts at diagnosis is correlated with a worse clinical outcome and suggest that this enzyme may have a role in sensitivity to ara-C in AML patients.     

A phase 1/2 study of chemosensitization with the CXCR4 antagonist plerixafor in relapsed or refractory acute myeloid leukemia

The interaction of acute myeloid leukemia (AML) blasts with the leukemic microenvironment is postulated to be an important mediator of resistance to chemotherapy and disease relapse. We hypothesized that inhibition of the CXCR4/CXCL12 axis by the small molecule inhibitor, plerixafor, would disrupt the interaction of leukemic blasts with the environment and increase the sensitivity of AML blasts to chemotherapy. In this phase 1/2 study, 52 patients with relapsed or refractory AML were treated with plerixafor in combination with mitoxantrone, etoposide, and cytarabine. In phase 1, plerixafor was escalated to a maximum of 0.24 mg/kg/d without any dose-limiting toxicities. In phase 2, 46 patients were treated with plerixafor 0.24 mg/kg/d in combination with chemotherapy with an overall complete remission and complete remission with incomplete blood count recovery rate (CR + CRi) of 46%. Correlative studies demonstrated a 2-fold mobilization in leukemic blasts into the peripheral circulation. No evidence of symptomatic hyperleukocytosis or delayed count recovery was observed with the addition of plerixafor. We conclude that the addition of plerixafor to cytotoxic chemotherapy is feasible in AML, and results in encouraging rates of remission with correlative studies demonstrating in vivo evidence of disruption of the CXCR4/CXCL12 axis.     

Quantitative real-time RT-PCR detects elevated Wilms tumor gene (WT1) expression in autologous blood stem cell preparations (PBSCs) from acute myeloid leukemia (AML) patients indicating contamination with leukemic blasts

High-dose chemotherapy with subsequent autologous stem cell transplantation is believed to be of therapeutic benefit in patients with acute myeloid leukemia (AML), especially when no allogeneic bone marrow donor is available. One of the main risks is contamination of the stem cell preparations with leukemic blasts, which may account for a higher relapse rate compared to allogeneic bone marrow transplantation. Since overexpression of WT1 is common in leukemic blasts, we investigated, whether PBSCs from AML patients express WT1 at a higher level as compared to patients with solid cancers. PBSCs of seven patients with AML and of five patients with solid cancers were investigated for WT1 expression. Total WT1 copy count was determined in a standardized quantitative real time RT-PCR. WT1 expression was found in all AML PBSCs with an average copy number of 49.99 +/- 61.09. In solid cancers WT1 expression was statistically significantly lower with a copy number of 3.51 +/- 1.92. In AML patients with sustained complete remission we found a nearly significantly lower WT1 expression than in patients who relapsed within the first year after stem cell transplantation. Our data show a higher WT1 expression in PBSCs of AML patients compared to patients with solid cancers. This finding might indicate a contamination with leukemic blasts. Quantification of WT1 in PBSCs might therefore be useful to estimate the risk of relapse after autologous stem cell transplantation in AML patients.     

Triptolide cooperates with chemotherapy to induce apoptosis in acute myeloid leukemia cells

OBJECTIVE: Triptolide has shown antitumor activity in a broad range of solid tumors and on leukemic cells in vitro. MATERIALS AND METHODS: The THP1 cell line and primary acute myeloid leukemia (AML) cells were cultured with triptolide alone or in association with AraC or idarubicin in increasing concentrations. Apoptosis was measured by flow cytometry using DiOC6(3) for the cell line and fluorescein isothiocyanateAnnexin-V and CD45 labeling for fresh blast cells. Protein expression was measured by Western blot. Cell cycle distribution of apoptotic cells was measured by flow cytometry. RESULTS: A synergistic effect was observed when triptolide was added to idarubicin or to AraC to induce apoptosis of THP-1 leukemic cells. The triptolide/AraC association was also investigated in vitro on primary blast cells from 25 AML patients. This combination induced significantly higher percentages of apoptosis vs treatment with each drug separately (p<0.005). The IkappaB and X-linked inhibitor of apoptosis protein contents, which were altered by triptolide in idarubicin-treated cells, were not modified in AraC-treated cells. The association of AraC with triptolide increased the number of cells blocked in the S phase and most underwent apoptosis. CONCLUSION: These results suggest that, by modifying the cell cycle kinetics, AraC sensitizes AML cells to apoptosis induced by low concentration triptolide. The in vitro proapoptotic effect of triptolide associated with the antiproliferative activity of AraC warrants further clinical investigation for treatment of AML patients, especially elderly patients for whom low-dose AraC treatment could be improved by the addition of triptolide.     

Serum soluble c-kit receptor and expression of c-kit protein and mRNA in acute myeloid leukemia

Abstract: To investigate the clinical role of the soluble form of c-kit receptor (s-kit) in patients with acute myeloid leukemia (AML), we determined the levels of serum s-kit and expression of c-kit antigens and mRNA in leukemic cells. The serum s-kit level was measured using ELISA assay in 30 AML patients and 20 normal controls. C-kit antigens of leukemic blasts were stained immunohistologically, and c-kit mRNA was detected by RT-PCR. The serum s-kit level in M1 and M2 were significantly increased (p<0.01) and that in M4 or M5 was significantly decreased (p<0.05) compared to that in the controls. In the comparisons among subtypes of FAB classification, M1 and M2 showed significantly higher levels than M4 or M5 (p<0.05 and p<0.01, respectively). Both expression of c-kit antigens and mRNA were observed in M0 (1/4), M1 (2/4) and M2 (6/8), but neither was observed in M4 or M5. The serum s-kit levels were correlated with the absolute number of AML blasts in peripheral blood (r=0.564, p<0.05). These results indicate that the serum s-kit level is related to the stage of differentiation of AML blasts in accordance with the expression of c-kit protein and mRNA in AML blasts, and is useful for assessment of leukemic cell burden.     

P-glycoprotein (P-170) expression in acute leukemias

Multidrug resistance (MDR) is still a major obstacle to chemotherapy success in acute myeloid leukemia (AML) and to a less extent acute lymphoblastic leukemia (ALL). Recent studies have shown that the expression of certain gene products mediate the development of resistance to chemotherapeutic agents. The most well characterized of these genes is the multidrug resistance gene MDR-1. This study was planned to study the expression of P-glycoprotein/170 in patients with acute leukemia and the effect of Cyclosporin A (CSA) as a modulator of P-glycoprotein functional activity. The study was carried out on 20 patients with acute leukemia (14 AML cases and 6 ALL cases). In addition, 6 normal individuals served as a reference group. Flow cytometric analysis of P-gp/170 surface expression was performed using UIC-2 MoAb together with the functional assay using Rhodamine 123 (Rh 123) and Cyclosporin A as a modulator.P-gp/170 was expressed on the leukemic cells of 37.5% of relapsed patients (40.0% of AML and 33.3% of ALL cases), whereas 27.2% of de novo patients expressed P-gp/170 (33.3% of AML cases and 0% of ALL cases). The functional activity of MDR-1 gp was 71.4% in AML and 33.3% in ALL patients compared with16.6% in normal lymphocytes. From this study, it is clear that P-gp/170 is expressed to a higher degree in leukemic cells and this is greater in relapsed compared to de novo cases and more in AML than ALL blasts. Functional activity is a more sensitive predictor of chemoresistance than P-gp/170 surface expression.     

Safety and efficacy of gemtuzumab ozogamicin in pediatric patients with advanced CD33+ acute myeloid leukemia

This open-label, dose-escalation study evaluated the safety and efficacy of single-agent gemtuzumab ozogamicin, a humanized anti-CD33 antibody-targeted chemotherapeutic agent, for pediatric patients with multiple relapsed or primary refractory acute myeloid leukemia (AML). Twenty-nine children 1 to 16 years of age (relapsed disease, 19; refractory disease, 10) received gemtuzumab ozogamicin ranging from 6 to 9 mg/m2 per dose for 2 doses (separated by 2 weeks) infused over 2 hours. All patients had anticipated myelosuppression. Other toxicities included grade 3/4 hyperbilirubinemia (7%) and elevated hepatic transaminase levels (21%); the incidence of grade 3/4 mucositis (3%) or sepsis (24%) was relatively low. One patient treated at 9 mg/m2 developed veno-occlusive disease (VOD) of the liver and defined the dose-limiting toxicity. Thirteen patients underwent hematopoietic stem-cell transplantation less than 3.5 months after the last dose of gemtuzumab ozogamicin; 6 (40%) developed VOD. Eight of 29 (28%) patients achieved overall remission. Remissions were comparable in patients with refractory (30%) and relapsed (26%) disease. Mean multidrug resistance-protein-mediated drug efflux was significantly lower in the leukemic blasts of patients achieving remission (P < .005). Gemtuzumab ozogamicin was relatively well tolerated at 6 mg/m2 for 2 doses and was equally effective in patients with refractory and relapsed disease. Further studies in combination with standard induction therapy for childhood AML are warranted.     

Low-dose cytarabine and aclarubicin in combination with granulocyte colony-stimulating factor (CAG regimen) for previously treated patients with relapsed or primary resistant acute myelogenous leukemia (AML) and previously untreated elderly patients with AML, secondary AML, and refractory anemia with excess blasts in transformation

We used the CAG regimen (low-dose cytarabine [10 mg/m2 per 12 hours, days 1-14], aclarubicin [14 mg/m2 per day, days 1-4], and granulocyte colony-stimulating factor [200 micrograms/m2 per day, days 1-14]) for the treatment of patients with primary resistant acute myelogenous leukemia (AML) and previously untreated elderly patients with AML, secondary AML, and refractory anemia with excess blasts in transformation (RAEB-T) in addition to relapsed AML. Forty-three of 69 (62%) patients achieved complete remission (CR), including 29 of 35 (83%) patients with relapsed AML, 1 of 8 patients with primary resistant AML, 5 of 8 elderly patients with previously untreated AML, and 8 of 18 patients with previously untreated secondary AML or RAEB-T. Ten of 22 (45%) patients > or = 65 years old achieved CR. The patients who achieved CR received at least 1 course of modified CAG therapy as the first consolidation therapy, followed by various second consolidation and intensification therapies. The median disease-free survival and overall survival were 8 and 15 months, respectively, for relapsed AML; 11 and 8 months for the elderly patients; and 8 and 17 months for secondary AML and RAEB-T. Myelosuppression was mild to moderate, and other than fever, severe nonhematologic toxicity was rare. CAG as the induction therapy seems promising for the treatment of various categories of poor-prognosis AML.     

Cellular drug resistance in childhood acute myeloid leukemia is related to chromosomal abnormalities

Specific cytogenetic abnormalities predict prognosis in childhood acute myeloid leukemia (AML). However, it is unknown why they are predictive and whether this is related to drug resistance. We previously reported that Down syndrome (DS) AML was associated with favorable resistance profiles. Here, we successfully analyzed drug resistance and (cyto-) genetic abnormalities of 109 untreated childhood AML samples using the 4-day total cell-kill methyl-thiazolyl tetrazolium (MTT) assay. Patients were classified according to the genetic abnormalities in the leukemic cells: t(8;21), inv(16), t(15;17), t(9;11), other 11q23 translocations, abnormalities of chromosome 5/7, trisomy 8 alone, normal karyotype, single random, and multiple (defined as 2 or more) abnormalities. The DS AML samples were excluded from the subgroup analysis. Samples with chromosome 5/7 abnormalities were median 3.9-fold (P =.01) more resistant to cytarabine than other AML samples. The t(9;11) samples were more sensitive to cytarabine (median 2.9-fold, P =.002), etoposide (13.1-fold, P =.001), the anthracyclines (2.9- to 8.0-fold, P <.01), and 2-chlorodeoxyadenosine (10.0-fold, P =.002) than other AML samples. The trisomy 8 and t(15;17) groups were too small for meaningful analysis. All other genetic subgroups did not show specific resistance profiles. Overall, we found no differences in drug resistance in samples taken at diagnosis between patients remaining in continuous complete remission (CCR) versus the refractory/relapsed patients. Within several genetic subgroups, however, relapsed/refractory patients were more cytarabine resistant when compared with patients remaining in CCR, but numbers were small and the results were not significant. We conclude that some, but not all, cytogenetic subgroups in childhood AML display specific drug-resistance profiles.     

In vitro efficacy of forodesine and nelarabine (ara-G) in pediatric leukemia

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)-ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G-sensitive than -resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.     

Dasatinib response in acute myeloid leukemia is correlated with FLT3/ITD,PTPN11 mutations and a unique gene expression signature

Novel targeted therapies improve the survival of specific subgroups (defined by genetic variants) of patients with acute myeloid leukemia (AML), validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in highthroughput, in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells. Our study aimed to predict in vitro response to targeted therapies, based on molecular markers, with subsequent validation in leukemic stem cells. We performed ex vivo screening of sensitivity to 46 drugs on 29 primary AML samples at diagnosis or relapse. Using unsupervised hierarchical clustering analysis we identified a group with sensitivity to several tyrosine kinase inhibitors, including the multi-tyrosine kinase inhibitor, dasatinib, and searched for correlations between the response to dasatinib, exome sequencing and gene expression in our dataset and in the Beat AML dataset. Unsupervised hierarchical clustering analysis of gene expression resulted in clustering of dasatinib responders and non-responders. In vitro response to dasatinib could be predicted based on gene expression (area under the curve=0.78). Furthermore, mutations in FLT3/ITD and PTPN11 were enriched in the dasatinib-sensitive samples as opposed to mutations in TP53 which were enriched in resistant samples. Based on these results, we selected FLT3/ITD AML samples and injected them into NSG-SGM3 mice. Our results demonstrate that in a subgroup of FLT3/ITD AML (4 out of 9) dasatinib significantly inhibited leukemic stem cell engraftment. In summary we show that dasatinib has an anti-leukemic effect both on bulk blasts and, more importantly, on leukemic stem cells from a subset of AML patients that can be identified based on mutational and expression profiles. Our data provide a rational basis for clinical trials of dasatinib in a molecularly selected subset of AML patients.