FAB Type Research Articles

Endogenous VEGF-C mRNA Expression Increases In Vitro Drug Resistance of Pediatric AML Cells and Is an Independent Prognostic Factor for the Time To Reach Complete Remission in AML.

VEGF-C, has been characterized as a lymphangiogenic and angiogenic growth factor and mediates Acute Myeloid Leukemic (AML) cell proliferation and survival by FLT-4 signaling. Moreover, exogenously added VEGF-C protected 2 AML cell lines and 5 primary AML samples from in vitro chemotherapy-induced apoptosis for 3 drugs via an increased bcl-2/bax ratio (Dias, S. et al. Blood , Mar 15; 99(6):2179–84, 2002). We hypothesized that in vitro as well as in vivo cellular drug resistance of AML cells are increased as a result of downstream effects of endogenous VEGF-C signaling. In primary AML cells from 40 pediatric patients VEGF-C mRNA expression with a quantitative PCR as well as in vitro cellular resistance to 9 drugs with a total cell-kill MTT assay were studied. LC50 values (drug concentration needed to kill 50% of the leukemic cells) were used as a measure of resistance. The best available in vivo equivalent for in vitro drug resistance is the time in days from diagnosis to complete remission (CR). CR is defined as less than 5% leukemic cells in a regenerated bone marrow aspirate. Correlations were calculated using the Spearman rank correlation coefficient. P-values ≤0.05 were considered to be statistically significant. VEGF-C mRNA expression levels varied largely in the samples (median: 0.02, given as arbitrary units, range: 0.004–0.33, n=40). Increased endogenous VEGF-C mRNA expression levels correlated significantly with increased in vitro resistance for 6 of 9 drugs (for cytarabine such a trend was seen), these drugs were all typical AML drugs (Table 1a and 1b). For time to reach CR, cox regression analysis was used to study the effect of VEGF-C, age at diagnosis, sex, treatment protocol, FAB type, cytogenetic risk profile and white blood cell counts on time to reach CR. Interestingly, VEGF-C was in vivo an independent prognostic factor for the time to reach CR (p=0.006, Odds ratio:0.224, 95% confidence interval: 0.077–0.655). LC50 values of the various drugs did not correlate to the time to CR (all p-values were above 0.1). So, high VEGF-C mRNA levels are correlated to a slower decrease of the total number of leukemic blasts in vitro as well as in vivo upon drug exposure. In conclusion, these results suggest for the first time that increased endogenous VEGF-C mRNA expression, probably resulting in increased anti/pro-apoptotic ratios, are correlated with in vitro as well as in vivo drug resistance of pediatric AML cells resulting in a longer time to reach CR.Table 1aCorrelations endogenous VEGF-C mRNA expression and LC50 values.DrugsCytarabineGemcitabineFludarabine6-thioguanineDaunorubicinEtoposideVEGF-Crho0.280.330.310.400.460.43p0.080.040.050.010.0050.008Table 1bDrugsCladibrineVincristineL-asparaginaseVEGF-Crho0.450.280.22p0.0030.120.21Rho indicates spearman rank correlation coefficient. p ≤ 0.05 was considered statistically significant.

A Transcriptional Signature Distinguishing Short and Long-Term Surviving Patients with MDS.

The biology of myelodysplastic syndromes (MDS) is reflected in aberrant transcription levels of the myeloid lineage. It was the purpose of this study to provide potential novel therapeutic targets by identifying molecular alterations that are pertinent to myelodysplastic syndromes.cDNA from bone marrow-derived CD34+ hematopoietic stem and progenitor cells from 6 healthy persons, and 16 patients with MDS (Table 1) was hybridized to cDNA arrays comprising 1185 well-characterized genes.We used the complete cDNA array expression values to build a dendrogram including all samples under investigation.Interestingly, patient survival had a striking impact on our gene signature for it was the parameter that had the strongest statistical association with the segregation of our MDS patients into 2 sub clusters (Figure 1). We examined whether this segregation of MDS patients was associated with age, gender, MDS-type according to FAB, WHO, IPSS, karyotype or survival. We determined correlation coefficients and found “survival” to be the parameter with the strongest statistically significant association with the segregation of MDS patients into 2 sub clusters (Spearman = 0.697, P = 0.003). The WHO type was less significantly associated (Spearman = 0.537, P = 0.032), and the FAB type did not reach statistical significance (Spearman = 0.492, P = 0.053).Transcriptional changes that are associated with short survival may also indicate to potential novel therapeutic targets. Since median age of patients with MDS is 70 at diagnosis, the majority of patients are not suitable for therapies that are not well tolerated. Moreover, some patients have a nearly normal life expectancy. Ideally, new therapeutic agents should, therefore, be tailored for patients with short survival and be well-tolerated like e.g. Bevacizumab and Cetuximab. But, we could not find increased expression of Bevacizumab and Cetuximab related EGFR- or VEGFR-target genes in CD34+ cells from MDS patients with short survival.In sum, there are transcriptional alterations that are strongly associated with short survival, and thus valuable for prognostication of patients with MDS.Table 1Classification, and karyotypes of patients with MDS who were analyzed by means of cDNA array analysisPatient No.Age/ SexFAB (a)IPSS (b)WHO (c)Karyotype161/ FRAint-IRCMD46, XX, 13q-268/ FRAint-I5q-46, XX, 5q-, 12p-362/ MRAint-IRCMD47, XY, +8450/ MRAint-IRCMD46, XY, t(X;1)518/ FRAlowPRA46, XX651/ MRAlowRCMD46, XY751 /FRAint-I5q-46, XX, 5q-, 12p875/ MRAEBhighRAEB II46, XY, 7q-979/ MRAEB-thighsAMLcomplex1068/ MRAEBint-IIRAEB-II46, XY1170/ FRAEB-thighsAML46, XX, 15q-1252/ MRAEB-thighsAMLcomplex1372/ MRAEB-thighsAMLcomplex1475/ MRAEBint-IRAEB-I46, XY1572/ MsAML/MDS-sAMLcomplex1676/ MsAML/MDS-sAML46, XY(a) FAB, French-American-British cooperative group classification, (b) IPSS, international prognostic scoring system; int, intermediate, (c) WHO, World Health Organization classification [Display omitted]

Bi-Allelic Involvement in AML: Examples of 16q Aberrations in Two Cases.

Acute Myeloid Leukaemia is a heterogeneous clonal disorder of the bone marrow, which has characteristic acquired chromosomal abnormalities. Cytogenetic rearrangements of chromosome 16 have a well-established association with AML subtype M4Eo although they have also been described in other AML FAB types. Patients with rearrangements involving CBFb (16p13) and MYH11 (16q22) are associated with a more favourable prognosis. We describe two cases of AML where both homologues of chromosome 16 are involved in separate rearrangements; this to our knowledge has not been reported previously. Further more both patients have had previous treatment and thus the abnormality of the second homologue of chromosome 16 is thought to be treatment related, in-spite of which they have responded well and are both currently in remission. In case 1 (male, AML M5a) it is chromosome16 at band q22 that is involved, inv(16)(p13q22) and t(16;17)(q22;q11), while in case 2 (female, t-AML) chromosome 16 band q24 appears rearranged in der(16)t(8;16)(q1?3;q24) and t(16;21)(q24;q22). Extensive molecular cytogenetic investigations including mapping are presented and candidate gene aberrations discussed.

Tumor-Activated Human NK Cells Specificall Ylyse Autologous and Allogeneic NK-Resistant Tumor Targets.

Pre-incubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias and solid tumors, even when HLA-matched; allogeneic or autologous. The primed NK cells remained non-responsive to HLA-C matched and mismatched normal peripheral blood or bone marrow mononuclear cells from multiple donors and did not affect in vitro colony forming unit assays. NK cells isolated from normal healthy donors and co-incubated with irradiated CTV-1 tumor cells synthesized CD69 within 60 min with maximal expression achieved within 6 hr. The tumor-activated NK cells (T-ANKs) lysed NK resistant RAJI and Daudi cells in a 4-hour assay (Fig 1). The degree of lysis was equivalent to that of matched lymphokine activated killer cells (LAK) after non-specific activation with IL-2. CTV-1 cells are HLA-C type 2 homozygous and express HLA-Bw4 alleles. They thus can ligate KIR2DL1 (CD158a) and KIR3DL1 (CD158e1) on NK cells. Co-cultures of HLA-matched or mis-matched NK with CTV-1 showed there was no significant difference in the generation of T-ANK activity in the presence or absence of KIR ligation. T-ANK cells from allogeneic donors were capable of lysis of primary AML cells of all FAB types, the relatively NK-resistant breast cancer cell line, MCF-7, and primary tumor cells isolated from resected tissue from patients with breast cancer and ascities from patients with ovarian cancer. Autologous T-ANK cells were generated from five AML patients in clinical remission; all of whom had low level or undetectable NK activity to their presentation disease. T-ANK cells from all five donors consistently induced significantly greater lysis of autologous AML blasts than the matched NK cells (p<0.05) (Fig 2). To investigate the tumor-restriction of allogeneic T-ANK cells we isolated NK cells from normal donors and either activated them with CTV-1 cells overnight or maintained them in media. These T-ANK cells were then compared with NK cells from the same donor with respect to lysis of normal autologous and allogeneic PBMC. Neither NK nor T-ANK cells lysed autologous PBMC nor did they lyse PBMC from HLA-C mismatched normal donors. To determine the likelihood of bone marrow suppression by T-ANK cells we established hematopoietic colony forming assays with bone marrow from 5 normal donors and added T-ANK from HLA-C mismatched donors at increasing ratios. CFU-GM, BFU-E and CFU-GEMM were not affected by co-incubation with HLA-mismatched T-ANK even at effector:target ratios of 20:1. We believe that this demonstrates a two stage process for NK activation; priming and triggering, the ligands for which are distinct. Tumor priming of NK primes cells capable of lysing a broad spectrum of NK-resistant tumors which may irrespective of KIR ligation which may represent an “off-the-shelf” immunotherapy product. [Display omitted] [Display omitted]

Gfi1b Association to Human Hematological Abnormalities.

Lineage specific transcription factors play essential roles in regulation of hematopoietic development. Transcription factor abnormalities have been frequently described in acute leukemia, mostly through cytogenetic changes. Nevertheless, point mutations can be easily missed. Recently, mutations in the erythroid and megakaryocyte specific transcription factor GATA1 have been discovered in patients with dyserythropoietic anemia and acute megakaryoblastic leukemia (AML-M7) with Down syndrome. Besides GATA-1, located on the X-chromosome, point mutations have been described in biallelic genes. This is the case of AML1 (RUNX1). PU1 and C/EBPalpha also represent examples of transcription factors in which point mutations are found in leukemia.A new zinc finger transcription factor involved in erythropoiesis is Gfi1b. Gfi1b was recently identified by sequence homology with oncogene Gfi1. Gfi1b knockout has demonstrated that this gene is essential for development of both erythroid and megakaryocytic lineages, and in its absence no enucleated erythrocytes are produced.Several Gfi1b DNA and protein targets (GATA1, Gfi1, AML1, p21WAF1, IL-6 Socs1 and Socs2) have been described that might be involved in malignancy. These findings indicate that Gfi1b is at the centre of hematopoiesis and may be a good candidate gene to be involved in hematological abnormalities.We have searched for Gfi1b point mutations in 122 patients with acute leukemia of all FAB types at diagnosis or relapse and 9 cases of congenital dyserythropoietic anemia. We have amplified Gfi1b promoter, coding and non-coding exons (Nucleic Acids Res 2004; 32:3935–46, MN 004188) by high fidelity PCR and screen for point mutations through dHPLC (Wave, Transgenomic) followed by sequencing of the cases with abnormal pattern. SNIPs in the promoter and exons were further confirmed in at least another PCR, cloned in pGEM-T easy vector system (Promega) and sequenced. Alleles with promoter SNIPs were cloned in pGL3-Enhancer vector (Promega), and transiently cotransfected with pEGFP-C2 (Clontech) to K562 cells. Luciferase activity was determined with Dual-Luciferase Reporter Assay (Promega).We found two promoter SNIPs in sequences conserved from chicken to human, one of them affecting a GATA-1 site, reducing promoter in vitro activity by 60 and 50% respectively. We also discovered a congenital exonic SNIP causing a mammalian conserved serine change to leucine. We excluded these to be frequent polymorphisms by dHPLC analysis of 96 blood donors. Although we cannot at present establish a clear relation between the former SNIPS and leukemia, we will discuss the presence of other milder hematological abnormalities. So far this is the first report of Gfi1b mutations that can be related to human pathology.

Cup-Like AML - New Disease or Morphological Phenomenon?.

Recently Kussick et al. (Leukemia 2004; 18:1591–98) reported cases of AML containing a distinctive, cup-like nuclear indentation in 10% or more of the blasts, which was associated with a loss of HLA-DR expression and FLT3 mutation. To investigate this feature in a larger cohort, we re-evaluated peripheral blood (PB) and bone marrow (BM) smears from 266 patients (male, n=148; female n=118; median age 57 years) from the German AML96 trial for blast cell morphology. Two-hundred blasts in PB and BM each were differentiated to determine the percentage of cells with cup-like nuclear invagination by two consecutive investigators. The slides were seen by two further investigators, who assigned the AML to a cup-like positive or cup-like negative cohort. Both cohorts were analysed for FAB-type, number of PB and BM blasts, CD34 expression, karyotype abnormalities, FLT3 and NPM mutations, as well as survival data. In addition, electron microscopy was performed in cases with high numbers of cup-like blasts.The median number of blasts with cup-like morphology was higher in PB compared to BM (2% vs. 0.5%). With a cut-off value of >=5% of blasts with nuclear invagination in PB and/or BM, 91% (n=48/53) and 94% (n=15/16) of cases were assigned to the cup-like positive cohort, respectively. Therefore we used this cut-off value to define cup-like-positive AML in the further analyses. Age and gender were not different between cup-like-positive (n=55) and -negative cases (n=211). FAB types M1 and M2 represented 62% of patients with cup-like AML, M5A was diagnosed in 20%, and in 11% and 4% the morphology was AML M4 and M4Eo. The median number of CD34+ blasts was 7% in cup-like positive AML compared to 27% in cases without this morphology (p=0.001). FLT3 and NPM aberrations were significantly more frequent in the cup-like positive cohort (FLT3, p=0.001; NPM, p<0.0001; FLT3+NPM, p<0.001) compared to the cup-like negative patients. In line with this, cup-like AML was significantly associated with normal karyotype (p=0.004). The levels of significance were even more pronounced if monocytic leukemias (M4, M4Eo, M5A and M5B) were excluded from the analyses (due to the characteristic cleaved or folded nuclear morphology in these FAB types). Cup-like morphology did not influence response rates (CR after second cycle of induction chemotherapy) and the survival parameters (DFS, OS), neither for the total cohort, nor for subgroups (non-monocytic leukemias, patients <60 years, patients with FLT3 or NPM mutation). Furthermore, multivariate analysis failed to demonstrate cup-like nuclear morphology as an independent risk factor. Electron microscopy revealed that the phenomenon is caused by an accumulation of cytoplasmatic components rather than by primary nuclear changes. The study confirms the hypothesis that cup-like nuclear morphology in AML blasts cells represents a specific morphological phenomenon. Because of the strong association to aberrations in FLT3 and/or NPM genes the feature should be used as a surrogate marker, making a subsequent molecular analysis meaningful. The biological background needs further investigation.

Detection of Activated STAT5 in the Cytoplasm of Neoplastic Cells in Patients with AML, CML, and Systemic Mastocytosis.

The signal transducer and activator of transcription 5 (STAT5) has recently been implicated as essential pro-oncogenic factor in the pathogenesis of myeloid leukemias in mice (Cancer Cell 2005; 7:87–99). More recently, STAT5 activation has also been described to occur in human leukemias. However, so far, little is known about the expression of activated/tyrosine phosphorylated STAT5 (pSTAT5) in various myeloid neoplasms and about the distribution of pSTAT5 in the cellular compartments of the normal and leukemic bone marrow (bm). We have examined the expression of pSTAT5 in the bm in patients with acute myeloid leukemia (AML, FAB M0, n=3, M1, n=6, M2, n=4, M3, n=5, M4, n=5, M5, n=4, M6, n=5, M7, n=4), chronic myeloid leukemia (CML, chronic phase, n=4, accelerated phase, n=5, blast phase, n=5), and systemic mastocytosis (SM, n=30), as well as in the normal bm (n=5). Expression of pSTAT5 was determined on paraffin-embedded bm sections by immunohistochemistry using the pSTAT5-specific antibody AX1. In the normal bm, the antibody AX1 was found to react with megakaryocytes and immature myeloid progenitor cells, whereas erythroid cells and mature granulocytic cells did not stain positive for AX1. In patients with AML and CML, the distribution of pSTAT5 showed a similar pattern. In fact, pSTAT5 was found to be expressed in leukemic blast cells without differences among FAB types as well as megakaryocytic cells, but not in erythroid cells. In patients with SM, neoplastic mast cells were found to be immunoreactive for pSTAT5. Interestingly, in all patients and all cells examined, pSTAT5 was found to be localized in the cytoplasm rather than in the nucleus. The cytoplasmic distribution of pSTAT5 in neoplastic cells was confirmed by immunocytochemical staining experiments performed on primary isolated neoplastic cells (AML, CML, mastocytosis) and respective cell lines (U937, KG1, K562, KU812, HMC-1). In each case, the reactivity of neoplastic cells with the AX1 antibody was abrogated by preincubation of the antibody with a pSTAT5-specific blocking peptide. Moreover, the expression of cytoplasmic pSTAT5 in the leukemic cell lines was demonstrable by flow cytometry. To study the molecular mechanisms underlying STAT5-activation in neoplastic cells, Ba/F3 cells with doxycycline-inducible expression of disease-specific oncoproteins, namely BCR/ABL (CML) and KIT-D816V (SM) were employed. Induction of these oncoproteins in Ba/F3 cells resulted in massive activation of pSTAT5 and DNA binding activity as shown by EMSA and supershift assays. In summary, our data show that neoplastic cells in AML, CML, and SM express cytoplasmic pSTAT5, and that disease-related oncoproteins contribute to STAT5-activation. The particular cytoplasmic localization of pSTAT5 in neoplastic cells suggests that apart from its function as a transcription factor, pSTAT5 may have an additional role as a cytoplasmic regulator in these malignancies.

Relapsed Acute Myeloid Leukemia in Children and Adolescents: Interim Report of the International Randomised Phase III Study Relapsed AML 2001/01.

Relapse occurs in 30–40% of newly diagnosed AML patients, with long-term survival in 20%. Aiming at improved outcome, we initiated a prospective, randomised study for relapsed AML, excluding AML M3 and those >18 years of age at initial diagnosis. FLAG is being used for 2 consecutive courses: fludarabine 30 mg/m2/day x 5, cytarabine 2 g/m2/day x 5, G-CSF 200 μg/m2/dose for 6 days, starting day -1. Liposomal daunorubicin (DaunoXome, DNX) is a new anthracycline with potentially less cardiotoxicity. Therefore, DNX at 60 mg/m2/day on days 1, 3 and 5 was randomly added or not to the first course of FLAG. Main objectives are to determine the efficacy and toxicity of DNX when added to FLAG, and the long-term outcome in a large group of relapsed AML patients. Thirteen groups worldwide are enrolling patients. More than 400 patients were registered by March 2006. This planned 2nd interim analysis with blinded efficacy data concerns 322 eligible and evaluable patients with first relapsed AML, of whom 250 (78%) were actually randomised. Fifty-two percent of patients relapsed early (<1 year from initial diagnosis). The majority (84%) concerned isolated bone marrow relapse, with central nervous system involvement in 6% of all patients. Dominating FAB types are M2 with auer rods, M4 without eosinophils and M5. Poor response to the 1st course of therapy (>20% of blasts in the BM shortly before the 2nd course), was seen in 23% of patients, more often in early relapses (31%) than in late relapses (15%). Early death occurred in 6% of patients. Complete remission (CR) was achieved in 63% of patients after 2 courses, and they have a probability of survival at 3 years (3-yr pSurv.) of 47% compared to 33% for the total group and 8% for patients not achieving CR. Compared to early relapses, patients with late relapse had higher CR rates (76 vs 51%), and higher 3-yr pSurv.: 42% vs 23%. Similarly, patients with either t(8;21) or inv(16) had a significantly better outcome. Death in continuous CR occurred in 8.6% of 322 patients, without excess of deaths in one treatment arm. Nearly all patients in CR have been transplanted, the majority with a matched unrelated donor. There was significant grade III/IV toxicity, but no unexpected toxicity, and no clinically relevant differences between the arms with and without DNX, especially not in cardiotoxicity. In conclusion, it is feasible to perform a large randomised pediatric study in a very international setting. DNX added to FLAG does not result in major additional toxicity, but follow-up of cardiotoxicity should be extended as planned in this protocol. Late relapses do better in terms of CR and overall survival, as well as patients with t(8;21) or inv(16), but early relapses achieving CR have a realistic chance of survival as well with currently 23% of them in continuous CR. The study is ongoing until 360 eligible and fully evaluable patients have been randomised, to answer the question whether liposomal daunorubicin improves outcome in pediatric relapsed AML.

Expression of MAC‐1 (CD11b) in acute myeloid leukemia (AML) is associated with an unfavorable prognosis

There is evidence to suggest, that cellular adhesion molecules and receptors could play a role in leukemia, e.g., through altered adhesive qualities of leukemic blasts. We have studied the expression of the beta2-integrin Mac-1 (CD11b) on mononuclear cells in 48 patients with AML at first diagnosis by flow cytometry using a direct fluorescein-conjugated antibody. A case was defined as positive if more than 20% of the cells expressed Mac-1. Within the FAB types, we observed a high expression rate in cases with M5 (100% MAC-1+ cases, 73% MAC-1+ cells), M4 (75% MAC-1+ cases, 48% MAC-1+ cells) and in cases with FAB-M1 with 71% MAC-1+ cases and 29% MAC-1+ cells. Separating our patients' cohort in cytogenetic risk groups, we could detect significant higher proportions of MAC-1+, cases (88% vs. 27%, P = 0.005) and cells (51% vs. 16%, P = 0.015) with poor cytogenetic risk compared to the favorable risk group. For clinical evaluations only patients treated according to the protocols of the German AML Cooperative Group (AML-CG) were included (n = 29, cases with AML-M3 were excluded). More MAC-1+ cases and cells were found in the "non-responders" group (n = 8) compared to the "responders" group (n = 24). We can conclude that AML cases with high MAC-1 expression are characterized by a worse prognosis. Evaluation of MAC-1 expression in AML might therefore contribute clinically important data with respect to develop new therapies that influence the interactions between integrins like MAC-1 on leukemic cells and endothelial or immunoreactive cells.

Low efficacy of methotrexate in childhood acute myeloid leukemia (AML): Single‐agent therapeutic window study in relapsed AML

The efficacy in pediatric acute myeloid leukemia (AML) of single-agent methotrexate (MTX) at a higher dose than previously applied, 1,000 mg/m2, given as a theoretically beneficial 36-hr continuous infusion, is unknown, but may be beneficial based on preclinical data. We performed a therapeutic window study in children with first relapsed AML treated in four different countries. Based on a comparison between the percentage of leukemic blasts in the bone marrow shortly before and 7-10 days after the MTX infusion, none of the first cohort of nine patients showed a good response, defined as a reduction of blasts of at least 50%. Therefore, the study was closed, concluding that the probability of a good response in this patient-group was most likely to be less than 30%. By that time, another four patients had been enrolled, of which one patient with a late relapsed AML FAB type M7 showed a good response. Toxicity of MTX was limited and tolerable. This study shows that single-agent MTX in the applied regimen in pediatric relapsed AML has limited efficacy. However, it also demonstrates the feasibility of an international and therapeutic window phase II study in pediatric relapsed AML.

Large Interindividual Differences in In Vitro Calicheamicin Sensitivity May Underly Gemtuzumab Ozogamicin Resistance in Acute Myeloid Leukemia (AML).

Gemtuzumab ozogamicin (GO or Mylotarg®) is increasingly being used in the treatment of AML. GO consists of a cytotoxic drug - calicheamicin which is conjugated to an anti-CD33 antibody. Most AML patients highly express CD33 on their blasts. Studies relating CD33 expression to response to GO have failed to show an association. Although 30–50% of patients respond to Mylotarg®, the causes of primary resistance to this drug remain unclear. Previous reports have studied P-gp status and CD33 expression as a cause of resistance to GO. Another factor that might determine response to GO is cellular drug resistance to calicheamicin.In this study we examined in vitro resistance to calicheamicin in 90 initially diagnosed and 32 relapsed pediatric AML samples using the 4 day MTT assay (concentration range 0.000004 – 0.4 μg/ml). The LC50 value, the drug concentration at which 50% of the cells is killed by the drug, is used as a measure of sensitivity. In addition to calicheamicin, some samples were also tested successfully for in vitro sensitivity to etoposide, cytarabine, daunorubicin, idarubicin, mitoxantrone, 6-thioguanine, vincristine and L-asparaginase.The characteristics of the 122 pediatric AML samples included are as follows: 62% boys, median age 9.6 years, median WBC 53.0*109/L, FAB types M0 n=9, M1 n=10, M2 n=18, M3 n=9, M4 n=33, M5 n=24, M7 n=3, unknown n=16. There was a more than 100,000 fold difference in calicheamicin sensitivity between the most sensitive and the most resistant patient samples. FAB M2 samples taken at initial diagnosis (n=13) were significantly more resistant to calicheamicin compared to the other FAB types (Resistance Ratio (RR)=2.5, median LC50 0.033 vs. 0.013 μg/ml, p=0.008). Newly diagnosed AML samples were significantly more sensitive to calicheamicin compared to relapsed AML samples (RR=0.68, median LC50 0.023 vs. 0.034 μg/ml, p=0.042) (although these patients had not been treated with calicheamicin). There was strong cross-resistance between calicheamicin and the anthracyclines idarubicin (Spearmans rho = 0.73, p<0.0001, n=23), daunorubicin (rho=0.61, p<0.0001, n=103) and mitoxantrone (rho=0.52, p=0.039, n=16). In addition, there was moderate cross-resistance with etoposide (rho=0.42, p<0.0001, n=101). No cross-resistance was observed between calicheamicin and cytarabine (rho=0.11, p=0.28, n=106), 6-thioguanine (rho=0.20, p=0.054, n=97), vincristine (rho=0.12, p=0.44, n=46) or L-asparaginase (rho=0.21, p=0.16, n=45). In conclusion, the interpatient differences in calicheamicin sensitivity are the largest differences in in vitro drug sensitivity we have ever observed in pediatric AML. FAB M2 samples are 2.5 fold more resistant to calicheamicin than samples with other FAB types. Initially diagnosed pediatric AML samples are 1.5 fold more sensitive to calicheamicin than relapsed AML samples. There is marked cross-resistance between calicheamicin and the related anthracyclin compounds. Given the large differences in sensitivity to calicheamicin in pediatric AML samples, it is likely that calicheamicin resistance plays a role in resistance to Mylotarg®.

Calcitonin A Genotype Is Associated with Risk of Acute Graft-Versus-Host Disease Following Allogeneic Bone Marrow Transplantation for Children with Acute Myeloid Leukemia in First Remission.

Background: Procalcitonin (PCT) is a 13 kD 116-amino acid prohormone. Elevated serum levels of procalcitonin have been noted in patients with bacterial infections such as septicemia, pneumonia and immune-mediated conditions like graft-versus-host disease (GvHD) and organ rejection. In this study, we sought to examine if a single base pair polymorphism (a T to C transition) at position −624 of the calcitonin A gene (CALCA) was associated with risk of acute GvHD.Subjects: 113 children (median age 10 y; range 0.38 – 19.0 y) with de novo acute myeloid leukemia (AML) treated on recent Children's Oncology Group protocols 2891, 2941 or 2961. All of these patients received matched sibling bone marrow transplant (BMT) in first clinical remission. Ablative conditioning consisted of busulfan and cyclophosphamide. All patients received methotrexate until day 100 for GvHD prophylaxis.Methods: All clinical DNA specimens were subjected to whole genome amplification. Genotyping was performed using the 5′-nuclease (TaqMan) assay on the ABI 7700 DNA analyzer (Applied Biosystems).Results: Observed CALCA -624 T>C genotypes in 113 patients were: TT = 52 (46%), TC = 43 (38%) and CC = 18 (16%); T allele = 73 (65%) and C allele = 40 (35%). There were no significant differences in patient characteristics (age, gender, race, study 2891/2941/2961, white blood count at diagnosis, FAB type, or cytogenetics) between the TT vs. combined TC or CC groups. Overall, acute GvHD grade I–IV (Seattle criteria) was observed in 55/113 patients (49%). Nineteen out of 52 (37%) with TT, while 21 out of 43 (49%) with TC, and 15 out of 18 (83%) with CC CALCA genotypes developed acute GvHD. Alternatively, acute GvHD was observed in 37% patients with common TT genotype and in 59% patients with variant TC or CC genotype (p=0.023). The odds ratio for developing acute GvHD (grade I – IV) for children with TC or CC genotypes was 2.5 (CI 1.17, 5.33) compared with those with TT genotype. The difference in observed acute GvHD remained significant when the analyses were limited to more uniform subgroups: 54 patients on single trial, 2961 only (p=0.028) and 42 whites only on 2961 trial (p=0.028). Severe acute GvHD (grade III – IV) was seen in 6/52 (12%) patients with TT vs. 6/61 (10%) in those with TC or CC genotypes; this difference was not statistically significant (p =0.771). There were no significant differences in day +100 transplant related deaths, engraftment, relapse rate or overall survival. Plans: Association between CALCA genotypes and infectious complications will be also analyzed when clinical data abstraction is completed. CALCA −1752 C>G and 3740 A>G genotyping is in progress and clinical correlative analyses will be repeated at genotype and haplotype levels.

Favourable Prognosis Associated with FLT3 Tyrosine Kinase Domain Mutations in AML in Contrast to the Adverse Outcome Associated with Internal Tandem Duplications.

It is widely accepted that internal tandem duplications (ITDs) of the juxtamembrane domain of FLT3 occur in about one quarter of cases of acute myeloid leukaemia (AML) in young adults and predict for early relapse from complete remission (CR). Constitutive activation of FLT3 can also arise from mutations in the tyrosine kinase domain (TKD) but there is controversy as to the clinical significance of this class of mutation. This is partly due to the small sample size of some studies and the inclusion of elderly patients who have a poor prognosis regardless of their FLT3 status. To definitively resolve this issue we have screened for TKD mutations in AML blast cells from 1339 young adult patients included in the UK MRC AML 10 and 12 trials using a sensitive denaturing HPLC technique (Transgenomic WAVE®). Mutant samples were confirmed by sequencing or specific restriction digest. 161 of 1339 (12%) patients had a TKD mutation which is a higher frequency than previously reported both because of the sensitivity of the technique and the detection of mutations outside the EcoRV digest site. 91 patients (6.8%) were deemed to have high level mutant arbitrarily defined as ≥ 20% of all FLT3 alleles and 70 (5.2%) had a low level mutant. 79 of the 161 (49%) mutants were Asp835Tyr. 8 mutants occurred outside the EcoRV digest site and 3 novel mutations were characterised. 372 (28%) patients in this cohort had an ITD. There was a negative correlation between the presence of an ITD and a TKD mutation with only 2.5% having evidence of both mutations. Furthermore, high levels of both class of mutation in the same patient were not seen so that it is possible that both mutations never arise in the same cell. The demographics of patients with a TKD mutation differed from those with a FLT3 ITD in that the presence of TKD mutations were not correlated with FAB type and were infrequent in patients with secondary AML. Both types of mutation were more frequent in patients with a high white count but were infrequent in patients with adverse cytogenetics. The presence of TKD mutations did not impact on CR rate, the incidence of resistant disease or the induction death rate. In contrast to FLT3 ITDs, TKD mutations were associated with a reduced relapse rate (odds ratio [OR] 0.77, 95% confidence intervals [CI] 0.59–0.99, P.04), improved disease free survival (OR 0.75, CI 0.60–0.93, P.008) and increased overall survival (OR 0.79, CI 0.64–0.97, P.02). In patients with wild type FLT3 the actuarial relapse rate at 5 years was 46%, compared to 57% with an ITD (excluding rare double mutants) and 34% in those with a TKD mutation. The overall survival at 5 years was 44%, 35% and 55% respectively. In multivariate analysis, the presence of a TKD mutation still had an effect on the relapse rate (OR 0.82, CI 0.67–1.01, P.05) and overall survival (OR 0.83, CI 0.70–0.98, P.03). These data suggest that different classes of activating mutation of the same tyrosine kinase receptor can be associated with markedly different clinical outcomes. FLT3 ITDs are associated with a poor prognosis and FLT3 TKD mutations with a relatively good prognosis. This unexpected genotype-phenotype relationship has not previously been described with oncogenic mutations and is significant to the understanding of the pathophysiology of chemoresistance as well as prognostic stratification.

MtDNA Sequence of MDS Patients Treated with CoQ10.

We conducted a pilot study to test the efficacy of coQ10 in improving the cytopenias of MDS patients with low to intermediate risk MDS. Almost every patient experienced a reduction in fatigue and improvement in life quality. Among 25 evaluable patients, 7 responded according to the International Working Group (IWG) criteria. Responses included two trilineage and 1 monolineage response, two cytogenetic responses, and two patients showed a change in FAB type from CMMoL to RA and RARS. CoQ10 is an important coenzyme that has been shown to be involved in protection against oxidative stress, inhibition of apoptosis, and generation of ATP. Mutations in mitochondrial DNA have previously been found in MDS patients. We hypothesized that acquired mutations in the mitochondrial genome may relate to the efficacy of coQ10 treatment. We therefore sequenced the mitochondrial genome (mtDNA) of 9 patients, including 4 responders and 5 nonresponders, and 2 normal samples to determine if differences in the frequency or location of mtDNA mutations could be correlated to response. Whole genomic DNA was isolated from bone marrow monocytes and the mtDNA was PCR amplified in 40 overlapping segments. The PCR products were then sequenced and compared to a mitochondrial genome database (www.mitomap.org ) to identify mutations. This technique will not detect mutations present in only a small subset of DNA copies. However, mutations present in a majority of mtDNA that may directly contribute to the expansion of the MDS clone will be identified. Interestingly, although mutations were observed, no differences were found that distinguished responders from nonresponders. We conclude from this analysis that the clinical benefit to MDS patients is not related to specific mutations in the mitochondrial genome. Clinical response may instead be related to other pleiotropic effects of coQ10, such as inhibition of apoptosis.

Prognosis of Acute Myeloid Leukemia with Translocation (8;21): A Survey of 142 Cases Investigated in the JALSG AML97 Study.

Background: Translocation (8;21) is one of the most common structural aberrations found in AMLand good response rates and a better DFS have been described in pts possessing it. Especially, the DFS rate may exceed 60–70% in younger pts given repetitive courses of HDAra-C. However, it was recently reported that a subtype of AML with t(8;21) showed a poor prognosis and this case also had the KIT mutation. As a result, we retrospectively analyzed the prognosis of AML pts with t(8;21) prospectively enrolled in the JALSG AML97 study.Patients and Methods: Between 1997 and 2001, JALSG AML97 enrolled 809 pts previously untreated for AML and pts with M3 were excluded. From the 789 eligible pts, we selected the subjects with t(8;21) for this study. Induction therapy consisted of cytarabine and idarubicin. All CR pts were randomized to receive either four courses of standard dose consolidation therapy without maintenance therapy or three courses of standard dose consolidation with 6 courses of maintenance therapy. HDAra-C was not administered in the JALSG AML97 study. Peripheral blood and bone marrow smears and karyotypes were reevaluated by the central review committees. Any patients who underwent SCT were censored at the date of SCT. The Kaplan-Meier method was used to estimate OS and DFS. For comparisons of OS or DFS, the log rank test was used.Result: Cytogenetic studies were performed in 783 patients (99.2%) and only 15 studies resulted in failure. One hundred forty-two (18.5%) of those pts showed t(8;21). The median age was 43 yrs (15–64). The male: female ratio was 95: 47. The distribution of FAB types was as follows: AMLM2, 130; M4, 6; M1, 5; and M5, 1. A total of 69 (48.6%) pts had a sole t(8;21), 46 (32.4%) had a loss of a sex chromosome, and 27 (19.0%) had other cytogenetic abnormalities. The CR rate was 90.0%. Thirty-two pts underwent two courses of induction therapy. The early death (< one month) rate was 1.4% (2/142). Only a high WBC count (>=10x109/L) was found to be a marginally significant prognostic factor (p=0.0517). The estimated OS rate at 5yrs was 53.6%, and the DFS of the CR pts was 41.2%. We found no relationship between OS or DFS and the following factors: age, gender, karyotypic abnormality, performance status, the percentage of MPO positive blasts, the presence or absence of Auer rods, the platelet count, the number of induction therapies for CR and the type of postremisson therapy. However, two factors, the WBC count and the percentage of blasts in bone marrow were found to be strongly predictive factors for the outcome. For the pts with a WBC count >=10x109/L, the DFS rate at 5yrs was 30.8%, while for the pts with less than 10x109/L, it was 48.8% (p=0.0215). For the pts with blast >=50% and less than 50%, the DFS rates at 5yrs were 32.2% and 56.7% (p=0.0087), respectively. According to these two factors, a poor risk group (N=30, 21.0%) was thus identified. They had a WBC count >=10x109/L and blast >=50%. The DFS rate at 5yrs was only 22.3 % in the poor risk group while that of the other pts was 48.9% (P=0.0003).Conclusion: This study confirmed that all AML pts with t(8;21) shows a high response rate and a good prognosis, thus indicating that this favorable AML group includes a poor risk group which can be identified based on the WBC count and the blast percent in the bone marrow.

Hematologic Improvement (HI) by TLK199 (Telintra™), a Novel Glutathione Analog, in Myelodysplastic Syndrome: Phase 2 Study Results.

Introduction: Glutathione S-transferase (GST) P1-1 has shown to be an important negative regulator of cellular growth and differentiation. The effect is mediated through binding to Jun kinase (JNK) which causes a decrease in kinase activity. TLK199, a novel analog of glutathione, binds selectively to GSTP1-1 resulting in its dissociation from JNK and subsequent kinase activation. Exposure of hematopoietic progenitor cells to TLK199 led to activation of JNK followed by cellular growth and maturation. TLK199 has shown significant myelostimulant activity in vitro in human bone marrow cell cultures as well as in several in vivo preclinical models of myelopoeisis. In Phase 1, TLK199 treatment resulted in hematologic improvement (HI) in MDS patients at all dose levels.Methods: The objectives of this multicenter Phase 2 study in MDS were to determine the safety (by NCI-CTC) and efficacy (by modified IWG MDS response criteria) of two dose schedules of TLK199 HCl Liposomes for Injection administered at 600 mg/m2 over 60 minutes by constant rate IV infusion daily x 3 or daily x 5 every 3 weeks. Patients (pts) were treated until lack of response or unacceptable toxicity.Results: 52 MDS pts (33 M/19 F),(29 RA, 9 RARS, 8 RAEB, 3 RAEB-t, 1 CMML, 2 UK), median age 69 years (range 22–90), received 244+ cycles (1099+ treatments), median 4 (range 1–13+). Thirty-seven pts (71%) were red cell transfusion dependent and 10 pts (19%) were platelet transfusion dependent prior to entry. Pts had failed a median of 1 prior therapy (range 0–6) including: erythropoietin (27/52%), G-CSF (9/17%), thalidomide (10/19%), azacitidine (7/14%), steroids (6/12%), hormones (2/4%), and other therapies (14/27%). Thirty-nine pts were evaluable for efficacy, 32 pts (82%) experienced HI in one or more blood cell lineages, 14 of 16 pts (88%) with trilineage dysfunction, 8 of 13 pts (62%) with bilineage dysfunction, and all 10 pts (100%) with unilineage dysfunction experienced HI. Lineage response was HI-P (14 of 22/64%), HI-N (9 of 27/33%), and HI-E (22 of 35/63%). Responses were accompanied by clinical symptom improvement, decreases in RBC and platelet transfusion requirements including transfusion independence and improvements in bone marrow maturation, differentiation, M/E ratios, and dysplastic morphology. Most common adverse events were mild to moderate acute infusion related reactions commonly seen with liposomal formulations: back pain (9/17%), nausea (8/15%), chills (8/15%), and bone pain (6/12%).Conclusions: TLK199 is well tolerated and an active agent in all FAB types of MDS. These data support the further clinical development of TLK199 in MDS as well as in other hematologic malignancies characterized by cytopenias.

Relapsed Acute Myeloid Leukemia in Children and Adolescents: Interim Results of the International Randomised Phase III Study “Relapsed AML 2001/01”.

Relapse occurs in 30–40% of newly diagnosed AML patients, with long-term survival in 20% of them. We initiated a study for relapsed AML excluding AML M3 and those >18 years of age. As backbone, FLAG is being used for 2 consecutive courses: fludarabine 30 mg/m2/day x 5, cytarabine 2 g/m2/day x 5, G-CSF 200 μg/m2/dose for 6 days, starting day −1. Liposomal daunorubicin (DaunoXome®, DNX) is a “new” anthracycline with potentially less cardiotoxicity, applicable in the total group of patients, and with the perspective to be useful at initial treatment as well. Therefore, DNX at 60 mg/m2/day on days 1, 3 and 5 was added or not in a 1:1 randomised fashion to the first course of FLAG. The ongoing prospective study must determine the efficacy and toxicity of DNX when added to FLAG, and the long-term outcome in a large group of relapsed AML patients. 13 groups worldwide are enrolling patients. 303 patients were registered as per April 2005. This planned interim analysis with blinded efficacy data concerns 226 eligible and evaluable patients with first relapsed AML, 140 (62%) being male. Median age at relapse was 9.7 (range, 0.7–18) years. 50% of patients relapsed within 1 year from initial diagnosis. The majority concerned isolated bone marrow relapse (85%), with 1% isolated and 5% combined central nervous system involvement. Median WBC at relapse was 4.0 (range, 0.4–293) x 109/l. Dominating FAB types are M2 with auer rods, M4 without eosinophils and M5. The randomisation was actually performed in 81% of patients. A total of 63% of patients was transplanted, the majority with a matched unrelated donor. Poor response to the 1st course of therapy (>20% of blasts in the BM shortly before the 2nd course), was seen in 23% of patients. Early death occurred in 6% of patients. Complete remission (CR) was achieved in 62% of patients, and they have a probability of survival at 2 years (2-yr pSurv.) of 47±6% (compared to 33±5% for the total group), 38±9% for early relapses and 54±7% for late relapses. Patients not achieving CR had a 2-yr pSurv. of 5±4%. Compared to early relapses, patients with late relapse had higher CR rates (74 vs 51%), and higher 2-yr pSurv.: 42±6% vs 21±5%. There was significant grade III/IV toxicity, but no unexpected toxicity, and no clinically relevant differences between the arms with and without DNX, especially not in cardiotoxicity. In conclusion, it is feasible to perform a large randomised study in a very international setting. DNX added to FLAG does not result in major additional toxicity, but follow-up of cardiotoxicity should be extended as planned in this protocol. Late relapses do better in terms of CR and overall survival, but early relapses achieving CR have a realistic chance of survival as well. The study is ongoing until 360 patients have been randomised, to answer the question whether liposomal daunorubicin improves outcome in pediatric relapsed AML. Meanwhile, overall outcome is encouraging, with CR achieved in 62% of patients of whom 47% are survivors at 2 years.

A transgenic mouse model for TEL-JAK2-induced B-cell lymphoma/leukemia

(o1 year of age) the pro-B phenotype was not associated with sensitivity to Ara-C, but the number of infants with pre-B or common phenotypes was small. Thus, Ara-C sensitivity in ALL seems to be associated with age (i.e. o1 year) and immunophenotype, but not with the presence of MLL rearrangements. Possibly, sensitivity to Ara-C as observed in childhood AML M4/M5 cells is associated with the M4/M5 FAB type rather than with young age. In conclusion, the present study does not provide data to support that MLL rearrangements are directly involved in the increased sensitivity to Ara-C as detected in infant ALL and M4/M5 AML.

Serum-free generation and quantification of functionally active Leukemia-derived DC is possible from malignant blasts in acute myeloid leukemia and myelodysplastic syndromes.

Functional dendritic cells (DC) are professional antigen presenting cells (APC) and can be generated in vitro from leukemic cells from acute myeloid leukemia AML patients, giving rise to APC of leukemic origin presenting leukemic antigens (DC(leu)). We have already shown that DC can be successfully generated from AML and myeloplastic syndromes (MDS) cells in serum-free 'standard' medium (X-vivo + GM-CSF + IL-4 +TNFalpha + FL) in 10-14 days. In this study, we present that DC counts generated from mononuclear cells (MNC) varied between 20% (from 55 MDS samples), 34% (from 100 AML samples) and 25% (from 38 healthy MNC samples) medium. Between 53% and 58% of DC are mature CD83+ DC. DC harvests were highest in monocytoid FAB types (AML-M4/M5, MDS-CMML) and independent from cytogenetic risk groups, demonstrating that DC-based strategies can be applied for patients with all cytogenetic risk groups. Proof of the clonal derivation of DC generated was obtained in five AML and four MDS cases with a combined FISH/immunophenotype analysis (FISH-IPA): The clonal numerical chromosome aberrations of the diseases were regularly codetectable with DC markers; however, not with all clonal cells being convertible to leukemia-derived DC(leu) (on average, 53% of blasts in AML or MDS). To the contrary, not all DC generated carried the clonal aberration (on average, 51% of DC). In 41 AML and 13 MDS cases with a suitable antigen expression, we could confirm FISH-IPA data by Flow cytometry: although DC(leu) are regularly detectable, on average only 57% of blasts in AML and 64% of blasts in MDS were converted to DC(leu). After coculture with DC in mixed lymphocyte reactions (MLR), autologous T cells from AML and MDS patients proliferate and upregulate costimulatory receptors. The specific lysis of leukemic cells by autologous T cells could be demonstrated in three cases with AML in a Fluorolysis assay. In six cases with only few DC(leu) or few vital T cells available after the DC/MLR procedure, no lysis of allogeneic or autologous leukemic cells was seen, pointing to the crucial role of both partners in the lysis process. We conclude: (1) the generation of DC is regularly possible in AML and also in MDS under serum-free conditions. (2) Clonal/leukemia-derived DC(leu) can be regularly generated from MDS and AML-MNC; however, not with all blasts being converted to DC(leu) and not all DC generated carrying leukemic markers. We recommend to select DC(leu) for vaccinations or ex vivo T-cell activations to avoid contaminations with non-converted blasts and non-leukemia-derived DC and to improve the harvest of specific, anti-leukemic T cells. DC and DC-primed T cells could provide a practical strategy for the immunotherapy of AML and MDS.

High expression of urokinase plasminogen activator receptor (UPA-R) in acute myeloid leukemia (AML) is associated with worse prognosis

Urokinase-type plasminogen activator receptor (UPA-R; CD87) is a membrane protein responsible for plasmin expression on cells facilitating cellular extravasations and tissue invasions. We studied the expression of the UPA-R on bone marrow (BM) cells of 93 patients with acute myeloid leukemia at first diagnosis and 8 healthy probands as controls by FACS analysis using phycoerythrin (PE)-conjugated antibodies. A case was defined as UPA-R-positive (UPA-R+) if >20% of the gated cells expressed UPA-R. Whereas none of the 8 healthy BM samples was positive for the UPA-R, 32 (34%) of the 93 AML samples were UPA-R+. Expression of UPA-R was heterogeneous in different FAB types, however, with the highest expression rates in monocytic subtypes (FAB M4/M5): 18%/19%/30% of UPA-R+ cases were found in M1/M2 or M3, and 58%/80% of cases with M4 or M5 were UPA-R+. Proportions of UPA-R+ cells varied between 1% and 98% of the mononuclear cell fractions, with the highest proportions in M4/M5 subtypes (on average 27%/40% UPA-R+ cells) and the lowest expression in AML M2 (11% UPA-R+ cells). The density of expressed UPA-R, estimated as mean channel fluorescence activity, was highest in cases with AML M1 (mFI: 124) followed by M4 and M5 (mFI: 78/77) and lowest in AML M2 (mFI: 43). In sAML, higher proportions of UPA-R+ cases (8 of 18; 44%) compared to pAML (24 of 75; 32%) were found as well as higher proportions of UPA-R+ cells (27% vs. 19%). Separating our patients' cohort in cytogenetic risk groups, we could not detect significant differences in the UPA-R expression profiles. For evaluations of the clinical course of AML, only patients treated by the AML-CG protocol (n = 65) were included. In the group of patients who did not respond to AML-CG therapy, significantly higher proportions of UPA-R+ cells (31% vs. 14%, P = 0.0015, t-test) were found. By evaluating a cut-off value for the percentage of positive cells that allows the most significant separation and differentiation between cases with shorter or longer relapse-free survival times, we could show that patients with >26.5% UPA-R-positive cells were characterized by a significantly higher risk for relapse compared to cases with <26.5% positive cells (P = 0.05). In summary, our data show a high expression of the UPA-R in AML, especially in (myelo)monocytoid subtypes. Cases with higher proportions of UPA-R+ cells were characterized by a significant lower remission rate after AML-CG therapy and a higher risk for relapse. Although prospective trials are still lacking, UPA-R is a prognostically relevant factor independent from the karyotype. UPA-R positivity may identify subtypes of AML associated with a more aggressive clinical course. Thus due to lower remission probabilities in UPA-R+ cases, a more intensive induction therapy regimen could be considered.