Multidrug-related protein 1 (MRP1) polymorphisms rs129081, rs212090, and rs212091 predict survival in normal karyotype acute myeloid leukemia.

Resistant disease is still a main obstacle in acute myeloid leukemia (AML) treatment. Therefore, individual genetic variations affecting therapy response are gaining increasing importance. Both SNPs and ABC transporter genes could already be associated with drug resistance. Here, we report allelic variants of MRP1 (ABCC1) SNPs rs129081, rs212090, and rs212091 with significant influences on survival in AML patients. DNA was extracted from bone marrow samples (n = 160) at diagnosis. Genotyping 48 SNPs within seven different ABC transporter genes using real-time PCR revealed rs129081 GG variant with a significant higher OS (p = 0.035) and DFS (p = 0.01). Comparing TT and AA rs212090 variants showed significant influences on DFS (p = 0.021). SNP rs212091 GG expression was associated with worse OS (p = 0.006) and a significant difference in DFS between alleles GG and AA (p = 0.018). The multivariable models confirmed a significant influence on OS for rs212091 (AA HR = 0.296, 95% CI 0.113-0.774, p = 0.013 and GG p = 0.044). Rs129081 variant CG, TT of rs212090, AA, and AG of rs212091 demonstrated significant impact on DFS (p = 0.024, p = 0.029, p = 0.017, and p = 0.042, respectively). This analysis demonstrates a significant influence of MRP1 SNPs on survival in AML. As they were not associated to prognostic characteristics, we suggest these SNPs to be independent prognostic markers for AML.

Therapeutic platelet transfusion versus routine prophylactic transfusion in patients with haematological malignancies: an open-label, multicentre, randomised study.

BACKGROUND: Routine prophylactic platelet transfusion is the standard of care for patients with severe thrombocytopenia. We assessed the effect of a new strategy of therapeutic platelet transfusion on the number of transfusions and safety in patients with hypoproliferative thrombocytopenia. METHODS: We did a multicentre, open-label, randomised parallel-group trial at eight haematology centres in Germany. Patients aged 16-80 years, who were undergoing intensive chemotherapy for acute myeloid leukaemia or autologous haemopoietic stem-cell transplantation for haematological cancers, were randomly assigned via a computer-generated randomisation sequence to receive either platelet transfusion when bleeding occurred (therapeutic strategy) or when morning platelet counts were 10×10(9) per L or lower (prophylactic strategy). Investigators undertaking interventions were not masked to group assignment. The primary endpoint was the number of platelet transfusions. Analysis was by intention to treat. This trial is registered, NCT00521664. FINDINGS: 197 patients were assigned the prophylactic strategy and 199 the therapeutic strategy. Of 391 patients analysed, the therapeutic strategy reduced the mean number of platelet transfusions by 33·5% (95% CI 22·2-43·1; p<0·0001) in all patients (2·44 [2·22-2·67] in prophylactic group vs 1·63 [1·42-1·83] in therapeutic group), 31·6% (18·6-42·6; p<0·0001) in those with acute myeloid leukaemia (2·68 [2·35-3·01] vs 1·83 [1·58-2·10]), and 34·2% (6·6-53·7; p=0·0193) in those who had had autologous transplantation (1·80 [1·45-2·15] vs 1·18 [0·82-1·55]. We noted no increased risk of major haemorrhage in patients who had undergone autologous transplantation. In those with acute myeloid leukaemia, risk of non-fatal grade 4 (mostly CNS) bleeding was increased. We recorded 15 cases of non-fatal haemorrhage: four retinal in each transfusion group, and one vaginal and six cerebral in the therapeutic group. 12 patients died in the study: two from fatal cerebral haemorrhages in the therapeutic group, and ten (five in each treatment group) unrelated to major bleeding. INTERPRETATION: The therapeutic strategy could become a new standard of care after autologous stem-cell transplantation; however, prophylactic platelet transfusion should remain the standard for patients with acute myeloid leukaemia. The new strategy should be used by some haematology centres only if the staff are well educated and experienced in the new approach and can react in a timely way to first signs of CNS bleeding. FUNDING: Deutsche Krebshilfe eV (German Cancer Aid).

Involvement of sirtuins in life-span and aging related diseases.

Since healthy aging remains one of the ideals of modern society, both, the identification of the underlying molecular mechanisms and interventions regarding the aging process are of considerable interest. Among the mechanisms currently being considered, the sirtuin family of histone deacetylases have been implicated to play a crucial role during the aging process both due to their requirement of NAD(+) as a cofactor for enzymatic activity, which determines a crucial link between sirtuins and the energy dependent regulation of gene transcription and their versatile target substrates mainly consisting of key regulators of metabolic, stress and cell cycle control. This chapter summarizes current evidences linking sirtuins to aging and outlines their potential as promising therapeutic targets for the treatment of age-related diseases.

Epigenetic regulation of cellular adhesion in cancer.

Epigenetics describes the development and maintenance of stable heritable gene expression patterns, which allow cells to show different phenotypes despite of a commonly shared genetic code. The increasing knowledge in this field during the last decades reveals its importance for many physiological processes like differentiation, embryogenesis and parental imprinting, but also for some diseases such as cancer. Recent data have shown that the complexity of carcinogenesis can no longer be explained solely on the basis of genetic changes, but epigenomic alterations such as changes of the DNA methylation pattern and/or post-translational histone modifications and changes of microRNA expression need to be equally considered. Such epigenetic alterations may cause permanent changes in gene expression patterns and may therefore essentially contribute to some of the known phenotypic characteristics of cancer cells like the loss of growth control, altered intercellular communication and enhanced motility. The two latter may essentially be associated with the downregulation of cellular adhesion molecules, which may therefore be relevant in the context of cancer invasiveness and prognosis. The targeted modification of the epigenome may therefore open new horizons within the increasingly important field of epigenetic therapeutics-particularly in view of the regulation of cellular adhesion with particular attention to tumor cell invasion and metastasis.

Chromosomal instability correlates with poor outcome in patients with myelodysplastic syndromes irrespectively of the cytogenetic risk group.

Chromosomal instability (CIN), defined by an elevated frequency of the occurrence of novel chromosomal aberrations, is strongly implicated in the generation of aneuploidy, one of the hallmarks of human cancers. As for aneuploidy itself, the role of CIN in the evolution and progression of malignancy is a matter still open to debate. We investigated numerical as well as structural CIN in primary CD34-positive cells by determining the cell-to-cell variability of the chromosome content using fluorescence-in situ-hybridization (FISH). Thereby, CIN was measured in 65 patients with myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML) and control subjects. Among MDS patients, a subgroup with elevated levels of CIN was identified. At a median follow-up of 17.2 months, all patients within this 'high CIN' subgroup had died or progressed to AML, while 80% of MDS patients with normal CIN levels had stable disease (P < 0.001). Notably, there was no statistically significant difference between 'normal CIN' and 'high CIN' MDS patients regarding established risk factors. Hence, elevated CIN levels were associated with poor outcome, and our method provided additional prognostic information beyond conventional cytogenetics. Furthermore, in all three MDS patients for whom serial measurements were available, development of AML was preceded by increasing CIN levels. In conclusion, elevated CIN levels may be valuable as an early indicator of poor prognosis in MDS, hence corroborating the concept of CIN as a driving force in tumour progression.

Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2.

Previous analyses of the sirtuin family of histone deacetylases and its most prominent member SIRT1 have focused primarily on the identification of cellular targets exploring the underlying molecular mechanisms of its implicated function in the control of metabolic homeostasis, differentiation, apoptosis and cell survival. So far, little is known about the regulation of SIRT1 itself. In the study presented herein, we assigned the main region of SIRT1 in vivo phosphorylation to amino acids 643-691 of the unique carboxy-terminal domain. Furthermore, we demonstrate that SIRT1 is a substrate for protein kinase CK2 both in vitro and in vivo. Both, deletion construct analyses and serine-to-alanine mutations identified SIRT1 Ser-659 and Ser-661 as major CK2 phosphorylation sites that are phosphorylated in vivo as well.

Fluorescence in situ hybridization and chromosomal organization of the sirtuin 4 gene (Sirt4) in the mouse.

The sirtuins (SIRT1-7), also being referred to as class III HDACs, exert NAD-dependent deacetylase and/or ADP-ribosyltransferase activities in various cellular compartments including the cell nucleus, the cytoplasm and the mitochondria. The sirtuins play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. SIRT4 is a mitochondrial protein that lacks deacetylase activities but efficiently works as an ADP-ribosyltransferase. We have isolated and characterized the murine Sirt4 genomic sequence, which spans a region of 12kb and which has one single genomic locus. Determination of the exon-intron splice junctions established that SIRT4 is encoded by 6 exons. The 1648bp murine Sirt4 transcript encodes a 418 aa protein with a predictive molecular weight of 47.3kDa. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt4 gene on chromosome 5F and is neighbored by the PLA2G1B and PXN genes.

Chromosomal characterization and localization of the NAD+-dependent histone deacetylase gene sirtuin 1 in the mouse.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the silent information regulator 2 (Sir2) family of histone deacetylases (HDACs). The yeast Sir2 protein and its mammalian derivatives play a central role in epigenetic gene silencing, DNA repair and recombination, the cell cycle, microtubule organization, and in the regulation of aging. We isolated and characterized the murine Sirt1 genomic sequence, which spans 19,910 bp and has one single genomic locus. Determination of the exon-intron splice junctions established that SIRT1 is encoded by 9 exons ranging in size from 80 bp (exon 6) to 1,927 bp (exon 9). Characterization of the 5' flanking genomic region, which precedes the Sirt1 open reading frame, revealed a number of NFkappaB and GATA transcription factor binding sites in addition to a 393-bp CpG island. The 3,882-bp murine Sirt1 transcript has an open reading frame of 2,211 bp and encodes a 737 aa protein with a predicted molecular weight of 80.4 kDa and an isoelectric point of 4.60. Next to this transcript, a shorter, 3,765 bp splice variant with an open reading frame of 2,094 bp, that lacks exon 2 and encodes a 698 aa protein with a predicted molecular weight of 76.0 kDa and an isoelectric point of 4.62 has been reported. Fluorescence in situ hybridization analysis identified a single genomic locus for the murine Sirt1 gene on chromosome 10 B4 and is neighbored by the Herc4 and Dnajc12 genes.

NF-kappaB-dependent control of HIV-1 transcription by the second coding exon of Tat in T cells.

HIV-1 two-exon transactivator protein (Tat) is a 101-aa protein. We investigated the possible contribution of the extreme C terminus of HIV-1 Tat to maximize nuclear transcription factor NF-kappaB activation, long terminal repeat (LTR) transactivation, and viral replication in T cells. C-terminal deletion and substitution mutants made with the infectious clone HIV-89.6 were assayed for their ability to transactivate NF-kappaB-secreted alkaline phosphatase and HIV-1 LTR-luciferase reporter constructs for low concentrations of Tat. A mutant infectious clone of HIV-89.6 engineered by introducing a stop codon at aa 72 in the Tat open-reading frame (HIVDeltatatexon2) replicated at a significantly lower rate than the wild-type HIV-89.6 in phytohemagglutinin-A/IL-2-stimulated primary peripheral blood lymphocytes. Altogether, our results suggest a critical role for the glutamic acids at positions 92, 94, and 96 or lysines at positions 88, 89, and 90, present in the second encoding Tat exon in activating NF-kappaB, transactivating the HIV-1 LTR and enhancing HIV-1 replication in T cells.

Azacytidine causes complex DNA methylation responses in myeloid leukemia.

Aberrant DNA methylation patterns play an important role in the pathogenesis of hematologic malignancies. The DNA methyltransferase inhibitors azacytidine and decitabine have shown significant clinical benefits in the treatment of myelodysplastic syndrome (MDS), but their precise mode of action remains to be established. Both drugs have been shown the ability to deplete DNA methyltransferase enzymes and to induce DNA demethylation and epigenetic reprogramming in vitro. However, drug-induced methylation changes have remained poorly characterized in patients and therapy-related models. We have now analyzed azacytidine-induced demethylation responses in myeloid leukemia cell lines. These cells showed remarkable differences in the drug-induced depletion of DNA methyltransferases that coincided with their demethylation responses. In agreement with these data, DNA methylation analysis of blood and bone marrow samples from MDS patients undergoing azacytidine therapy also revealed substantial differences in the epigenetic responses of individual patients. Significant, transient demethylation could be observed in 3 of 6 patients and affected many hypermethylated loci in a complex pattern. Our results provide important proof-of-mechanism data for the demethylating activity of azacytidine in MDS patients and provide detailed insight into drug-induced demethylation responses.

SIRTUIN 1: regulating the regulator.

Earlier analyses on the sirtuin family of histone deacetylases and its well-known member SIRT1 had their primary focus mostly on the identification of cellular targets exploring molecular mechanisms and functional networks in the control of metabolic homeostasis, differentiation, apoptosis and cell survival. However, only little is known about the regulation of SIRT1 itself, so far. Presently, SIRT1 is gaining increasing importance in the development of innovative treatment strategies for cancer, neurodegenerative disorders and metabolic disease. Based on differences in their catalytic activities, SIRT1 and the sirtuins in general, are insensitive to the classical class I and II HDAC inhibitors which are increasingly becoming part of treatment regimens for solid tumors and hematological malignancies. In this review we outline recent research advances on the regulation of SIRT1 which may provide the basis for the development of therapeutic inhibitors with improved specificity.

Histone deacetylase inhibitor treatment downregulates VLA-4 adhesion in hematopoietic stem cells and acute myeloid leukemia blast cells.

The alpha4beta1 integrin very late activation antigen-4 (VLA-4) is an alpha4 (CD49d)/beta1 (CD29) heterodimer. It plays a key role in the adhesion of both hematopoietic progenitor cells and leukemic blast cells to bone marrow stromal cells which express the vascular cell adhesion molecule-1 (VCAM-1) or produce fibronectin. VLA-4 expression has been associated with bone-marrow minimal residual disease, which causes relapse after chemotherapy in patients with acute myelogenous leukemia. Conversely, the absence of VLA-4 reduces bone marrow retention of both hematopoietic progenitor and leukemic blast cells. We report on the downregulation of VLA-4/CD49d for various acute myelogenous leukemia cells lines, on primary cells from patients with acute myelogenous leukemia, and on hematopoietic stem cells and peripheral blood mononuclear cells from healthy donors on treatment with the histone deacetylase inhibitors suberoylanilide hydroxamic acid and valproic acid, which is associated with decreased adhesion to mesenchymal stromal cells. These findings suggest that HDAC-inhibitor treatment may on the one hand impair stem cell homing, while on the other it may improve peripheral blood stem cell mobilization and significantly help to reduce minimal residual disease from acute myelogenous leukemia.

Aging and anti-aging: unexpected side effects of everyday medication through sirtuin1 modulation.

The sirtuin 1 protein (SIRT1) is a member of the class III NAD+-dependent histone deacetylases, which are also referred to as the 'sirtuins'. The sirtuins and silent information regulator 1 (SIRT1) in particular, are known to play a role in the response to DNA damage, metabolism, longevity and carcinogenesis. SIRT1 regulates different cellular processes such as proliferation, differentiation and apoptosis through deacetylation of important regulatory proteins such as p53, FOXO3a and NFkappaB. A number of different modifiers of SIRT1 expression and activity have been discovered and even food and cosmetic additives (e.g. resveratrol and dihydrocoumarin) have been suggested to either activate or inhibit the activity of human SIRT1. We screened a panel of 18 different drugs which are frequently used in everyday clinical practice with regard to their influence on cell survival and SIRT1 expression in freshly isolated peripheral blood mononuclear cells (PBMCs) from young and healthy volunteers. In this context, we identified L-thyroxin, insulin and sodium nitroprusside to be potent activators of human SIRT1 expression. In addition, treatment of PBMCs with sodium nitroprusside was associated with a significant cellular lifespan extension, while L-thyroxin and insulin were unable to prolong lifespan, suggesting that isolated upregulation of SIRT1 is in fact insufficient to promote longevity. These findings have an important impact on the long-term use of a number of frequently used clinical agents in the treatment of chronic disease with respect to aging and carcinogenesis.

Valproic acid and all-trans retinoic acid: meta-analysis of a palliative treatment regimen in AML and MDS patients.

Currently, no standard treatment is available for elderly patients with de novo/secondary acute myeloid leukemia (AML) who are not eligible for intensive chemotherapy. New, less aggressive therapies are therefore needed. Histone deacetylase inhibitors (HDACi) are known to reduce proliferation and induce differentiation in hematological malignancies. With all-trans retinoic acid (ATRA) these effects have been reported to be even enhanced. Valproic acid (VPA) is an HDACi and has been known as anti-epileptic agent for many years. We treated 21 patients with de novo/secondary AML and 1 patient with myelodysplastic syndrome with ATRA (45 mg/m(2)/day in 2 doses, 14 days, q29 days) and VPA (150 mg/day 1 week, then 300 mg/day, continuously). Treatment was tolerated well with moderate side effects. 4 patients revealed hematological improvement and another 4 patients experienced a reduction in transfusion dependency. The overall response rate was 27%. Our study is presented together with an overview of the literature on the topic.

Fluorescence in situ hybridization and chromosomal organization of the human Sirtuin 7 gene.

Sirtuin 7 (SIRT7) is a member of the sirtuin family of protein deacetylases and is, therefore, a derivative of yeast Silent information regulator 2 (SIR2). SIR2 and its mammalian orthologs play an important role in epigenetic gene silencing, DNA recombination, cellular differentiation and metabolism, and the regulation of aging. In contrast to most sirtuins, SIRT7 does not exert characteristic NAD+-dependent deacetylase activity. We have isolated and characterized the human Sirt7 genomic sequence, which spans a region of 6.2 kb and which has one single genomic locus. Determination of the exon/intron splice junctions found the full-length SIRT7 protein to consist of 10 exons ranging in size from 71 bp (exon 4) to 237 bp (exon 7). The human Sirt7 open reading frame encodes a 400-aa protein with a predictive molecular weight of 44.9 kDa and an isoelectric point of 9.80. Characterization of the 5' flanking genomic region, which precedes the Sirt7 open reading frame, revealed a TATA- and CCAAT-box less promoter that lacks CpG islands. A number of AML-1 and GATA-x transcription factor binding sites were found, which remain to be further evaluated experimentally. Fluorescence in situ hybridization analysis localized the human Sirt7 gene to chromosome 17q25.3; a region which is frequently affected by chromosomal alterations in acute leukemias and lymphomas. Human SIRT7 appears to be most predominantly expressed in the blood and in CD33+ myeloid bone marrow precursor cells, while the lowest levels are found in the ovaries and skeletal muscle. Functional characteristics of SIRT7 are essentially unknown at present and remain to be further elucidated.

Chromosomal organization and fluorescence in situ hybridization of the human Sirtuin 6 gene.

Sirtuin 6 (SIRT6) is a member of the sirtuin deacetylases (sirtuins), which are derivatives of the yeast Silent information regulator 2 (Sir2) protein. SIR2 and its mammalian derivatives play a central role in epigenetic gene silencing, recombination, metabolism, cell differentiation and in the regulation of aging. In contrast to most sirtuins, SIRT6 lacks NAD+-dependent protein deacetylase activity. We have isolated and characterized the human Sirt6 genomic sequence, which spans a region of 8,427 bp and which has one single genomic locus. Determination of the exon-intron splice junctions found the full-length SIRT6 protein to consist of 8 exons ranging in size from 60 bp (exon 4) to 838 bp (exon 8). The human Sirt6 open reading frame encodes a 355-aa protein with a predictive molecular weight of 39.1 kDa and an isoelectric point of 9.12. Characterization of the 5' flanking genomic region, which precedes the Sirt6 open reading frame, revealed a TATA- and CCAAT-box less promoter with an approximately 300-bp long CpG island. A number of AML-1 and GATA-x transcription factor binding sites were found which remain to be further evaluated experimentally. Fluorescence in situ hybridization analysis localized the human Sirt6 gene to chromosome 19p13.3; a region which is frequently affected by chromosomal alterations in acute leukemia. Human SIRT6 appears to be most predominantly expressed in bone cells and in the ovaries while, in the bone marrow, it is practically absent. The functional characteristics of SIRT6 are essentially unknown at present and remain to be elucidated.

Cloning, chromosomal characterization and mapping of the NAD-dependent histone deacetylases gene sirtuin 1.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the silent information regulator 2 (Sir2) family of sirtuin histone deacetylases (HDACs). The yeast Sir2 protein and its mammalian derivatives play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. We have isolated and characterized the human Sirt1 genomic sequence, which spans a region of 33,660 bp and which has one single genomic locus. Determination of the exon-intron splice junctions established that SIRT1 is encoded by 9 exons ranging in size from 80 bp (exon 6) to 2,120 bp (exon 9). Characterization of the 5' flanking genomic region, which precedes the Sirt1 open reading frame, revealed a CCAAT-box and a number of NF-kappaB and GATA transcription factor binding sites in addition to a small 350 bp CpG island. The 4,107 bp human Sirt1 mRNA has an open reading frame of 2,244 bp and encodes a 747 aa protein with a predictive molecular weight of 81.7 kDa and an isoelectric point of 4.55. Fluorescence in situ hybridization analysis localized the human Sirt1 gene to chromosome 10q21.3.

Phase 2 trial of the continuous IV administration of interferon-beta in patients with disseminated malignant melanoma.

BACKGROUND: Interferons have been reported to significantly contribute to tumor suppression via both induction of p53 gene expression and inhibition of angiogenesis. OBJECTIVE: The assessment of treatment toxicity and antitumoral effectiveness of continuous IV administration of interferon-beta based on an overall evaluation of laboratory, radiographic, and clinical parameters observed during the trial. METHODS: The authors treated patients with advanced malignant melanoma with continuous IV infusions of 1 x 10(6) IU interferon-beta daily ( approximately 0.6 x 10(6) IU interferon-beta/m2 daily). RESULTS: Continuous IV administration of interferon-beta had no significant effect on overall patient outcome. Interferon side effects were not a reason for treatment discontinuation in any of the patients observed during this trial. CONCLUSIONS: Continuous IV interferon-beta had no significant effect on overall patient outcome in a group of patients with advanced malignant melanoma. To our knowledge, this is the first report on the continuous IV administration of interferon-beta in patients with advanced malignant melanoma.

Estrogen Receptor Alpha (ESR1) Single-Nucleotide Polymorphisms (SNPs) Affect Malignant Melanoma Susceptibility and Disease Course.

The incidence of malignant melanoma in the developed world is continuously increasing. We conducted a case-control study in order to evaluate the association between each of the four estrogen receptor alpha polymorphisms (ESR1 single-nucleotide polymorphisms [SNPs] +2464C/T, -4576A/C, +1619A/G, and +6362C/T) and malignant melanoma susceptibility and disease course. The study population consisted of 205 Caucasian patients who were diagnosed as having malignant melanoma and 208 healthy Caucasian controls. Through DNA genotyping, we identified a SNP-dependent malignant melanoma susceptibility as well as a SNP-dependent effect on the course of disease and response to therapy.

Trastuzumab in Esophagogastric Cancer: HER2-Testing and Treatment Reality outside Clinical Studies in Germany.

We analysed trends over time in palliative first-line chemotherapy in patients with locally advanced or metastatic esophagogastric cancer. Special focus was on frequency and quality of HER2-testing and trends in drug use in combination with trastuzumab. Earlier published data about patients treated outside clinical studies showed a relatively low rate of HER2-testing and insufficient test quality. A total of 2,808 patients retrospectively documented in Therapiemonitor (®) from 2006 to 2013 were analysed regarding treatment intensity and trends in used drugs. Data on HER2-testing and therapies were analysed in two cohorts documented in 2010 and 2011 (1) compared to 2012 and 2013 (2). Treatment intensity increased: 49.3% of patients received at least a triplet in 2013 compared to 10.1% in 2006. In cohort 2 HER2 expression was tested in 79.1% of the cases. Still, in 26.9% testing was not done as requested by guidelines. Good performance status, multiple metastases, age ≤ 65 years, the objective "to prevent progression," good cognitive capabilities, estimated good compliance, and social integration positively influenced the probability of HER2-testing; comorbidities negatively affected it. Usage of the combination of fluoropyrimidines and cisplatin with trastuzumab declined from 67% in cohort 1 to 50% in cohort 2.