Histopathologic Characterization of Vexas Syndrome

Abstract Introduction: VEXAS syndrome (vacuoles, E1 ubiquitin ligase, X-linked, autoinflammatory, somatic) is a newly recognized inflammatory disorder caused by somatic mutations in the UBA1 gene. Bone marrows from these patients reveal a range of morphological changes in hematopoietic precursor cells. In this study, we aim to assess the laboratory indices and morphologic spectrum of bone marrow pathology in VEXAS syndrome. Methods: We identified 16 cases of VEXAS syndrome. All cases had confirmed UBA1 mutation. We reviewed bone marrow biopsies corresponding to the date of diagnosis. This study was approved by the Mayo Clinic Institutional Review Board. Results: All patients were male with a median age of 73 years - associated autoimmune disorders included Sweet syndrome, inflammatory arthritis, relapsing polychondritis and granulomatosis with polyangiitis. 14/16 patients had anemia with median hemoglobin of 10.4 (Range: 6.7- 14.1 g/dL). 15/16 had macrocytosis with median MCV 110.4 (Range: 94.8- 123.1 /fL). 5/16 had thrombocytopenia with median platelet count 174 (Range: 20- 500 x10^9/L). 7/16 had leukopenia with median WBC 3.65 (Range: 2.4- 11.6 x10^9/ L). The ESR and CRP medians were 61.0 mm/hr and 81.5 mg/L, respectively. Karyotype was performed in 12 patients of which 11 were normal and the remaining case showed a complex karyotype. An NGS panel targeting the most frequent myeloid disorder associated gene mutations was negative in 10/15 cases. GS for myeloid mutations revealed pathogenic mutations in 5 patients, involving genes TET2 (2/5), DNMT3A (2/5), and TP53 (1/5). Conclusions: Bone marrow findings in VEXAS syndrome, in this series of 16 patients, are individually non-specific, yet when taken altogether in the overtly abnormal cases, are very suggestive when the clinical index of suspicion is high. In such scenarios, the combined clinical and bone marrow findings should prompt discussion and consideration for UBA1 mutation testing given the significant clinical implications for patient management and prognosis. Disclosures Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding. Warrington: Eli Lilly: Research Funding; Kiniksa: Research Funding.

Download Full-text

Correlation of Hemophagocytosis with Clinical Criteria of Hemophagocytic Lymphohistiocytosis and Recommendations for Screening Bone Marrow Samples in Adult Patients

Blood ◽

10.1182/blood-2020-139246 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-38

Author(s):

Caroline Wilson ◽

Wei-i Lee ◽

Matthew Cook ◽

Lillian Smyth ◽

Dipti Talaulikar

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Hemophagocytic Lymphohistiocytosis ◽

Research Funding ◽

Interobserver Variability ◽

Laboratory Data ◽

Adult Patients ◽

Advisory Committees ◽

Bone Marrow Biopsies ◽

Immunohistochemical Stain

Introduction Hemophagocytic lymphohistiocytosis (HLH) is a rare condition resulting from a dysregulated inflammatory response. It can prove difficult to diagnose and portends a poor prognosis. Bone marrow (BM) biopsy is an easily accessible test that is often used to identify the presence of hemophagocytosis and assess for underlying malignancy. Currently there are no evidence-based guidelines on the reporting of hemophagocytosis on BM biopsy and no reports of a correlation between hemophagocytosis with the clinical diagnostic criteria for HLH. We therefore aimed to assess if the amount of hemophagocytosis identified in the BM biopsy correlates with HLH-2004 criteria. Secondary aims were to evaluate inter-observer variability in reporting hemophagocytosis, and to formulate recommendations for screening in BM specimens. Method A retrospective review of bone marrow biopsies from adult patients under investigation for HLH was undertaken independently by 2 hematopathologists who were blinded to the original biopsy report. Relevant clinical and laboratory data was extracted from medical records. The average number of actively hemophagocytic cells in each slide prepared from BM aspirates were quantified into 0, 1, 2-4 and ≥5. On trephine samples, hemophagocytosis was reported as either 'present' or 'absent', with the assistance of the CD68 immunohistochemical stain. Cases with discordance pertaining to the degree of hemophagocytosis were reviewed by both assessors to reach a consensus. Results Sixty-two specimens from 59 patients were available for assessment. An underlying hematological condition was identified in 34 cases (58%). The most common underlying hematological condition was lymphoma, found in 15 cases (25%). There was a significant association between the amount of hemophagocytosis identified on the aspirate samples and the number of HLH-2004 criteria met (p<0.05). In patients where hemophagocytosis was present (n=31), there was a significant correlation between the amount of hemophagocytosis and ferritin levels (p<0.05). Interobserver variability was present in 63% of cases. Based on our review, we make the following recommendations for reporting of hemophagocytosis in the BM samples:> 1. Count only macrophages ingesting intact hemopoietic cells. W2. Quantify the average number of active histiocytes per aspirate slide. W3. Count histiocytes away from particles where the cellular outline is clear. W4. Avoid counting conglomerates of histiocytes where the cellular margins are indistinct W5. On the aspirate specimen, assess for hemophagocytosis on both the trail and squash preparations. W6. Delineating hemophagocytosis on trephine samples is difficult without the use of a CD68 immunohistochemical stain. Interestingly, a study by Ho et al found no association between the BM histologic findings and the probability of hemophagocytosis (Ho et al, American Journal of Clinical Pathology, 2014). This difference highlights the need for standardised reporting of BM specimens. Conclusion Our findings indicate that the amount of hemophagocytosis present on BM samples correlates with the number of HLH-2004 criteria met. We found marked interobserver variability which we anticipate can be rectified with our recommendations on the reporting of hemophagocytosis. Disclosures Talaulikar: Takeda: Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Download Full-text

Detection and Monitoring of BRAF and NRAS Mutant Clones in Myeloma Patients By Digital PCR of Circulating DNA

Blood ◽

10.1182/blood.v126.23.4196.4196 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4196-4196 ◽

Cited By ~ 1

Author(s):

Even Holth Rustad ◽

Hong Yan Dai ◽

Eivind Coward ◽

Kristine Misund ◽

Anders Sundan ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Plasma Cells ◽

Digital Pcr ◽

Braf V600e ◽

M Protein ◽

Bone Marrow Biopsies ◽

Blood Samples ◽

Sequential Samples ◽

Clonal Development

Abstract Introduction Targeted mutation specific therapy is a promising approach in cancer therapy. However, an obstacle for this approach is the vast heterogeneity of the clonal composition and development. Tumor biopsies represent only a snapshot of the situation. Furthermore, monitoring of the clonal development is difficult because biopsies may not be representative for the whole tumor and availability of repeat biopsies is limited. To meet these difficulties we have established and optimized a method based on Digital PCR (dPCR) for analyses of circulating cell free (cf)DNA from sequential samples of serum and plasma from patients with multiple myeloma. Methods We investigated 19 patients for the BRAF V600E mutation. Nine were previously confirmed as mutation positive in bone marrow biopsies/purfied plasma cells by two independent methods (PCR/immunohistochemistry/whole exome sequencing) whereas 10 were mutation negative (Rustad et al Blood Cancer J 2015). Two patients with NRAS Q61K mutation detected in serial bone marrow samples were also included. In total, 67 serum and 21 EDTA-plasma samples were analyzed. Blood samples were taken, processed and frozen at -800 C within 1,5 hour. The samples were stored for a median of 5 years (range 0-23) before DNA isolation and analysis. Mutation detection by dPCR was performed using a droplet-based system and validated primer/probe-sets (BioRad). In-house validation and optimization of the assay was carried out using cancer cell lines OH2 and HT29 with NRAS Q61 and BRAF V600E mutations respectively. The limit of detection was 1-3 copies of mutated DNA per reaction and no false positives were detected. The threshold of positivity was set to 1 droplet per sample. Experiments were performed in accordance with the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines (Huggett et al Clin Chem 2013). Results BRAF or NRAS mutated cfDNA was detected in all patients with a confirmed mutation in tumor tissue, and in none of the mutation-negative controls (p = 0.000003, Fisher's exact test). When looking only at tumor tissue and blood samples obtained at the same time, mutation positivity was confirmed in the blood of 9/10 patients (p = 0.00012). Furthermore, there was a positive correlation between the percentage of mutated plasma cells in bone marrow biopsies and the concentration of mutated cfDNA (Spearman correlation R = 0.63, p = 0.025). Serial samples were analyzed from 5 patients and provided information about 3 different aspects: 1. Patients 1 (figure), 2 and 3, had large clones (50-100 %) of BRAF or NRAS mutated cells in diagnostic and relapse bone marrow samples. Mutated cfDNA correlated closely to M-protein levels in these patients as demonstrated in the figure. A corollary of the figure is that the BRAF mutated clone produces M protein and is sensitive to MP. 2. Patient 4 developed a pelvic extra medullary plasmacytoma with 75-100% BRAF mutation positive cells (immunohistochemistry), however, time-matched serum samples showed only a modest peak with 23 mutated copies/ml. 3. Patient 5 had a moderately sized BRAF V600E mutated clone of 50-75 % at diagnosis, which, according to serum levels, persisted through the disease course. However, two months prior to death, the patient rapidly deteriorated and became refractory to treatment. BM aspirate showed 95 % plasma cells with plasmablastic morphology. A serum sample contained > 600 ng/ml of cfDNA, 10-100 fold more than any other sample in our study, and was highly positive for BRAF V600E mutation (59 000 copies/ml). The patient clearly had expansion of an aggressive BRAF mutated clone that could easily be detected by serum analysis. Conclusions This study demonstrates that mutations such as BRAF V600E and NRAS Q61K can be reliably detected and monitored in sequential serum or plasma samples from myeloma patients. Quantitative mutation analysis compared to M protein in sequential samples provided significant information with clinical relevance. The great advantage of this approach is the easy access to blood samples compared to bone marrow aspirate/biopsy. This will facilitate studies of clonal development during treatment and detection of druggable mutations. Figure 1. Co-variation of M-protein and circulating BRAF V600E mutated DNA in patient 1. Figure 1. Co-variation of M-protein and circulating BRAF V600E mutated DNA in patient 1. Disclosures Waage: Celgene: Research Funding; Amgen: Research Funding; Janssen: Research Funding.

Download Full-text

Loss of Abelson Interactor-1 Is Linked to Inflammatory Hematopoiesis and Accelerated Aging of Hematopoietic System

Blood ◽

10.1182/blood-2018-99-118393 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1285-1285

Author(s):

Anna Dorota Chorzalska ◽

John Morgan ◽

Max Petersen ◽

Diana O Treaba ◽

Adam J Olszewski ◽

...

Keyword(s):

Bone Marrow ◽

Animal Models ◽

Research Funding ◽

Molecular Mechanisms ◽

Bone Marrow Failure ◽

Accelerated Aging ◽

Fold Increase ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Oncology Research

Abstract Background: Hematopoietic stem cells (HSC) ensure homeostasis and lifelong maintenance of hematopoietic system, but with age, they gradually lose quiescence, self-renewal potential, and system restoration capacity. HSC aging results in a differentiation shift towards myeloid lineage, anemia, thrombocytosis, decrease in T and B cells, imbalance in macrophage function, and increased osteoclast activity. Mechanisms involved in HSC aging include increased mTOR activity and ROS production, impaired autophagy, epigenetic reprograming, and cumulative DNA damage. Intriguingly, cellular and molecular similarities between aging and inflammation have led to a novel concept of "inflammation-associated aging of hematopoiesis". Understanding the molecular mechanisms responsible for this process may impact strategies targeting age-related diseases, including neoplasms. However, to date only few primary animal models of inflammation have shown bone marrow failure, so new animal models need to be established to provide mechanistic insight into the long-term implications of chronic inflammation on the hematopoietic system. We have previously shown that bone marrow-specific deletion of an adapter protein Abelson interactor-1 (Abi1) leads to a myeloproliferative neoplasm (MPN)-like disease in 35-56-week-old mice, mechanistically associated with increased activity of Src Family Kinases (SFKs), STAT3 and NF-κB. At both transcript and protein levels, Abi-1 is also significantly reduced in HSCs and granulocytes from patients with primary myelofibrosis (PMF), and Abi-1-deficient HSC in human PMF show increased SFK-STAT3-NF-κB signaling (Chorzalska, ASH 2017). Methods: Myeloid/lymphoid, stem/progenitor populations profiling by FACS, bone marrow transplantation assays, transcriptomics and proteomics analyses as well pro-inflammatory cytokine profiling and histopathology analyses were performed on the transgenic Abi-1KO mice carrying bone marrow-selective knockout at 4 weeks post-recombination, upon confirming both inducible inactivation of the Abi1flox allele and loss of Abi-1 protein in the marrow (Fig.1A, B). Results: To better understand initial systemic events that lead to the development of MPN-like disease in aged Abi-1KO mice we have now characterized early changes within the hematopoietic system associated with loss of Abi-1. Blood count analysis indicated leukocytosis, anemia and thrombocytosis, and an increase in the fraction of myeloid (CD11b+/Gr-1+) as well as macrophage/monocyte (F4/80+) cells at the expense of lymphoid (B220+) cells in Abi-1KO relative to Abi-1WT mice (Fig.1C). Previously reported 2.6-fold increase in Abi-1KO LT-HSCs (Chorzalska, ASH 2017) was now shown to be associated with 30% increase in number of LT-HSCs is in the S/G2/M phases of the cell cycle relative to Abi-1WT LT-HSCs (Fig. 1D). Lethally irradiated recipient C57BL/6 wild-type mice transplanted with bone marrow cells from Abi-1KO relative to Abi-1WT mice (in the absence of competitor cells) showed progressive loss of chimerism in primary and secondary recipients (Fig. E). Genome-wide gene expression analysis of Abi-1WT vs. Abi-1KO LSK cells showed significant overexpression of genes regulated by or involved in regulation of the NF-κB pathway (Fig. 1F). Plasma cytokine levels showed 2-fold increase in IL-1B, IL-12, IL-17, IL-23, IL-27, and MCP-1 and nearly 10-fold increase in INFγ (Fig. 1G). Label-free, intensity-based quantitative proteomic analysis of bone marrow from 20-week-old Abi-1KO and Abi-1WT mice showed abundance of peptides derived from Mac-1, myeloperoxidase, STAT1, STAT3, and SFKs - Hck and Fgr, confirming not only activation of SFKs and STAT3 signaling, but also increase in proteins associated with myeloid lineages (Fig. 1H). Loss of bone density (Fig. 1I) and a significant decrease in thymus size (Fig. 1J) were observed in Abi-1KO mice relative to Abi-1WT mice. Conclusions: In sum, phenotypic analyses performed 4-10-weeks post Abi1 gene inactivation indicate changes consistent with accelerated aging of hematopoietic system that are mechanistically linked to inflammatory SFK-STAT3-NF-κB signaling. To our knowledge this is the first animal model linking accelerated inflammation-driven aging of hematopoietic system to development of an MPN in aged mice. Disclosures Olszewski: Genentech: Research Funding; TG Therapeutics: Research Funding; Spectrum Pharmaceuticals: Consultancy, Research Funding. Reagan:Pfizer: Research Funding; Alexion: Honoraria; Takeda Oncology: Research Funding.

Download Full-text

ANKRD26 Coding Variants Presenting with Giant Platelets and a Predisposition to Myeloid Neoplasia

Blood ◽

10.1182/blood-2019-130520 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4233-4233

Author(s):

Teodora Kuzmanovic ◽

Bhumika J. Patel ◽

Jibran Durrani ◽

Hassan Awada ◽

Cassandra M Kerr ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Adaptor Protein ◽

Cytoskeletal Proteins ◽

Advisory Committees ◽

Giant Platelets ◽

Monosomy 7 ◽

Oncology Research ◽

Coding Variants

Mutations (MT) in the 5' untranslated region (UTR) of ANKRD26 (A26) are implicated in ANKRD26- related thrombocytopenia (A26-RT), an autosomal dominant disorder of mild to moderate thrombocytopenia (TP) often presenting in adulthood, although severe and pediatric cases are reported. Erythrocyte and leukocyte counts are normal to increased, with unremarkable morphology. Platelet (plt) size is usually normal, as with ETV6- and RUNX1-mutated TP. Together, A26, ETV6, and RUNX1 germline (GL) MT comprise a separate 2016 WHO category of myeloid neoplasms (MN) with GL predisposition and preexisting platelet disorders. Normal plt size separates A26-RT from other familial TPs with giant plts. No consistent morphological plt aberrations have been reported. Bleeding history is absent or mild, and while TP is not life threating, 8-10% of patients (pts) develop a MN, including a 30-fold increased risk for AML relative to the general population. A26 is an inner membrane adaptor protein with 2 major domains: ankyrin repeats (ANKR) and coil-coil (C-C), both of which interact with signaling and cytoskeletal proteins. A26-RT MT are almost exclusively in the 5'UTR, however, rare A26 coding variants (A26-CV) are reported to segregate with familial TP. Still, some studies on A26-RT limit sequencing to the 5'UTR. As such, A26-CV are not well represented or described. We performed whole-exome sequencing (WES) on 195 pts with MN seen at Cleveland Clinic between 2004 and 2012, and downloaded WES .bam files from online sources, totaling 653 MN cases. Using a standard pipeline for discovery of rare GL variants (<1% population frequency), we identified 22 pts with A26-CV (6 MDS, 3 MDS/MPN, 13 AML) with 13 unique variants (2 benign/likely benign, 1 variant of unknown significance, 10 not found) and a median (med) VAF of 47%. Half of the variants were within either ANKR or C-C domains. The occurrence of these variants in our cohort was 3% vs.1% of the healthy population (p=0.0001). Complete clinical description was available for 8 A26-CV pts, all diagnosed with MDS or MDS/MPN at med age of 64. Two pts had antecedent bruising 1 year prior to diagnosis (dx). No prior bleeding was noted. Three pts had prior TP, two of whom had bicytopenia and pancytopenia. First degree family history (FH) was positive for cancer in 6 pts (75%), including 2 pts with FH of hematologic neoplasms. One pt had a 2nd-degree relative with a non-malignant hematologic condition requiring transfusions. At dx, cytogenetics were normal and complex in 2 pts each (25%). Deletions in chromosomes 5, 17, 20, and Y were observed in 1 pt each, and monosomy 7 in 2 pts. On bone marrow aspirate, 6 pts (75%) had dysmegakaryopoiesis, found in interstitial patterns and clusters. Mild to moderate dyserythropoiesis was observed in 5 pts (63%), and 5 pts had dysgranulopoiesis. The med blast percentage was 1.5% (range 0-8%), with 5 pts having hypercellular marrow, med 65% (range 40-95%). On corresponding CBC, pts were anemic (med hemoglobin of 9.5 g/dL), with the majority (n=5) showing signs of erythroid dysplasia, and also thrombocytopenic (med plt of 99 k/μL), with giant plts observed in 3 pts. Two pts had both giant plts and abnormal erythroid morphology. Hypo and monolobate, hyposegmented, and hypogranular forms were observed in the 3 lineages, as well as detached nuclear lobes, budding, segmentation, and nuclear-cytoplasmic dyssynchrony. On next generation sequencing, co-occurring SRSF2 and ASXL1 MT were observed in 3 and 2 pts, respectively. In sum, we have identified 22 A26-CV in MN, suggesting a role in predisposition as with A26-RT. We have seen in our cohort that A26-CV pts present differently from those with A26-RT. They have a variable past medical history and limited FH of TP, are anemic, with multilineage dysplasia observed not just in bone marrow, but also on peripheral blood smear, especially in megakaryocytes and erythrocytes. This is not surprising, as A26 is expressed in both lineages. The presence of giant plts is noteworthy. The mechanism for hypomorphic A26-CV may differ from that of the A26 5' UTR, which increase A26 levels in late-stage megakaryopoiesis by abrogating RUNX1/FLI1 binding, leading to aberrant proplatelet formation. Given the plt size and presence of nuclear phenotypes, altered interactions with signaling and cytoskeletal proteins could be involved, and may represent a novel A26 phenotype. Further investigation and association of A26-CV with MN ontogeny is under way. Disclosures Mukherjee: Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau. Advani:Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Research Funding. Nazha:Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Gerds:Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Roche: Research Funding; Incyte: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Download Full-text

Establishment of a Highly Sensitive Allele Specific Wild Type Blocker Polymerase Chain Reaction Followed by Pyrosequencing for Detection of the p.D816V Point Mutation.

Blood ◽

10.1182/blood.v114.22.4975.4975 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4975-4975

Author(s):

Udo Siebolts ◽

Haifa Kathrin Al-Ali ◽

Dietger Niederwieser ◽

Claudia Wickenhauser

Keyword(s):

Bone Marrow ◽

Point Mutation ◽

Dna Sequences ◽

Research Funding ◽

High Sensitivity ◽

Wild Type ◽

Bone Marrow Biopsies ◽

Allele Specific ◽

Sensitive Allele ◽

Formalin Fixed

Abstract Abstract 4975 Background The vast majority of mastocytosis patients carry the p.D816V activating point mutation in exon 17 leading to imatinib resistance. In addition, several less common activating kit mutations, at least partially accessible to imatinib, have been described in a minority of systemic and cutaneous mastocytosis. Therefore, exact molecular identification of patients with mastocytosis of uncertain dignitiy is a precondition for diagnostic and therapeutic purposes. However, atypical mast cell infiltrates within the bone marrow biopsies often are very small and microdissection may be hampered by the inconspicuousness of the atypical mast cells. Hence, an appropriate molecular test should exhibit an exceedingly high sensitivity. Methods In an attempt to combine high sensitivity and robustness we created an approach where we adapted the principle of wild-type blocker PCR employing LNA-substituted oligonucleotides and pyrosequencing technique. First, the unique properties of LNA substituted oligonucleotides were employed. As a consequence of their 2′-O, 4′-C methylene bridge and their characteristic bicyclic structure binding affinity is significantly increased, resulting in high melting points. One base mismatch between an LNA oligonucleotide and the complementary strand can lower the melting point 20-30°C, thereby allowing a LNA oligonucleotide to discriminate a one base pair difference between templates and, in contrary, a single base pair mismatch in the normal DNA octomer decreases Tm only by 10°C. In order to allow LNA-substituted oligonucleotides to block amplification of wild-type a mutated form of Taq polymerase, termed the Stoffel fragment was applied. Stoffel fragment is a modified form of AmpliTaq® DNA polymerase lacking intrinsic 5' to 3' exonuclease activity. Employing this methodological setting including the Stoffel fragment, the LNA oligonucleotides and appropriate primer 20 rounds of PCR were performed and an amplicon of 185 bp was received. As demonstrated employing mixtures of assembled DNA sequences keeping the WT or the p.D816V point mutation the WT/D816V ratio turned in favour of the mutated region performing the LNA – Stoffel PCR. Unique in our approach was a second step in which the suchlike amplified PCR product then was adopted in a pyrosequencing assay (Pyromark 24, Qiagen, Hilden, Germany). Results First of all, employing WT DNA sequences and the L1236 cell line, the ability of the blocking LNA oligonucleotides to prevent primer extension on WT-DNA by AmpliTaq® DNA polymerase was excluded. To determine the minimum concentration of blocker necessary to prevent amplification of WT oligonucleotides, we used serial dilutions of the blocking oligonucleotides and revealed that a minimum of 50 mM of blocker was necessary to prevent a highly competitive WT-amplicon. We then analyzed the sensitivity of the assay and spiced assembled DNA sequences keeping the WT or the D816V point mutation. Performing this experiment, 1:105 (0,001%) mutant DNA sequences were definitely detectable performing this assay but not performing conventional pyrosequencing without specific preamplification. In none of the control experiments employing WT DNA a false positive amplicon of the exon 16 D816V region was seen indicating an excellent specifity of the assay. In the pyrograms only light emission peaks >10% were evaluated. To determine the utility of our technique for detecting minority mutations in bone marrow specimen containing few cells or mixed cell populations, we performed the here presented assay on genomic DNA extracted from formalin-fixed, EDTA decalcified paraffin-embedded trephine biopsies. In all 20 trephine biopsies under study, comprising a morphologically established atypical mast cell density from 1 – 15% a clear cut decision of the genomic status was possible. Conclusions Wild-type blocker PCR employing nonextendable LNA oligonucleotides represents an exceedingly sensitive, allele-specific method. In combination with the pyrosequencing technique the here presented assay is highly reliable and reproducible, simple and fast to perform (3 h), facile to interpret and should be equally applicable to other single-base mutations. In bone marrow biopsies mRNA fragments often are highly degradated. Therefore, in contrast to PCR assays performed on cDNA, the here presented assay is ideal suited for the molecular diagnosis of systemic mastocytosis on formalin-fixed tissues. Disclosures Siebolts: Novartis: Research Funding. Al-Ali:Novartis:. Niederwieser:Novartis: Research Funding. Wickenhauser:Novartis: Research Funding.

Download Full-text

Phase II Trial of Single Agent Panobinostat (LBH589) In Relapsed or Relapsed/Refractory Waldenstrom Macroglobulinemia

Blood ◽

10.1182/blood.v116.21.3952.3952 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3952-3952 ◽

Cited By ~ 7

Author(s):

Irene Ghobrial ◽

Tiffany Poon ◽

Meghan Rourke ◽

Stacey Chuma ◽

Janet Kunsman ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Stable Disease ◽

Research Funding ◽

Bone Marrow Involvement ◽

Ct Scans ◽

Advisory Committees ◽

Bone Marrow Biopsies ◽

Grade 3

Abstract Abstract 3952 Introduction: This study aimed to determine the safety and activity of panobinostat (LBH589) in patients with relapsed or relapsed/refractory Waldenstrom Macroglobulinemia (WM). This was based on our preclinical studies showing that panobinostat induces significant activity in cell lines and patient samples. Methods: Eligibility criteria include: 1) patients with relapsed or relapsed/refractory WM with any prior lines of therapy, 2) measurable disease and symptomatic disease, 3) off prior chemotherapy> 3 weeks, or biological/novel therapy for WM > 2 weeks. Patients received panobinostat at 30 mg three times a week (Mondays, Wed and Fridays). Patients were assessed after every cycle for the first 6 cycles and then every 3 months thereafter. Subjects who had response or stable disease were allowed to continue on therapy until disease progression or unacceptable toxicity. A planned restaging was performed at the end of cycle 6 including CT scans and bone marrow biopsies. Results: Twenty-seven patients have been enrolled to date. The median age is 62 years (47-80), the median lines of prior therapy is 3 (range, 1–7). All of the patients received prior rituximab. The median hemoglobin at screening is 10.3 g/dL (range 8.2–14.3), the median IgM M-spike by protein electropheresis at study entry is 1.9 g/dL (range, 0.63–5.1), and median serum IgM at baseline is 3610 mg/dL (range, 804- 10, 300). The median bone marrow involvement at enrollment was high for patients with WM, 50%, range (5-95%), with more than 10 patients having 70% or higher bone marrow involvement at baseline. The median number of cycles on therapy is 4 (range 1 – 12). 4 of the patients came off due to toxicity. Minimal response (MR) or better has been achieved in 15 (60%) of patients, with 6 (24%) PR, 9 (36%) MR. In addition, 9 (36%) patients achieved stable disease and 1 (4%) showed progression. The median decrease in IgM is 1020 mg/dL (0- 3970 decrease in IgM) with a median % decrease of 37.13%. Responses were prompt. The median time to first response was 2 cycles (range, 2–4). Bone marrow biopsies at the end of study (or at 6 months follow up) are available on 7 patients, of which 3 showed a significant decrease in bone marrow involvement and 4 showed stable involvement. The 4 patients who had stable bone marrow disease showed 1 PR and 3 MR responses by paraprotein level. Grade 3 and 4 toxicities include 4 (15%) cases of anemia including 1 case of hemolytic anemia, 1 (3%) case of grade 4 leucopenia (but the patient had grade 3 leucopenia at baseline), 7 (26%) of neutropenia, 14 (52%) of thrombocytopenia, 1 (4%) grade 3 GI bleed due to thrombocytopenia, 1 (3%) Grade 4 hyperglycemia and 1 (3%) grade 3 syncope and 3 (27%) grade 3 fatigue. The most common grade 2 toxicities were thrombocytopenia, anemia, and fatigue. There were 5 (20%) cases of asymptomatic pulmonary infiltrates of ground glass opacity observed on routine CT scans in follow up. Of these, 3 came off study for other reasons not related to the pulmonary infiltrates, 1 received a course of corticosteroids and had improvement of infiltrates, and 1 had dose reduction of therapy. All patients except for 2 have been dose reduced due to thrombocytopenia, fatigue, diarrhea, or anemia. Dose reductions include 25 mg three times a week, 20 mg three times a week and 20 mg three times every other week. The protocol was amended to allow a starting dose of 25 mg three times a week, which is better tolerated than 30 mg in this patient population. Conclusions: Panobinostat is an active therapeutic agent in patients with relapsed or refractory WM, with an overall response rate of 60% in patients with relapsed or refractory WM. The dose schedule of 25 mg three times a week is better tolerated in this patient population. Further studies to include this agent in combination with rituximab or bortezomib are being evaluated. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Richardson:Keryx Biopharmaceuticals: Honoraria. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding.

Download Full-text

AL Amyloidosis and Concomitant Myeloma: Time to Reconsider Assumptions

Blood ◽

10.1182/blood.v116.21.4044.4044 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4044-4044

Author(s):

Wesley Witteles ◽

Ronald Witteles ◽

Michaela Liedtke ◽

Sally Arai ◽

Richard Lafayette ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Plasma Cell ◽

Research Funding ◽

Plasma Cells ◽

Renal Involvement ◽

World Health ◽

Bone Lesions ◽

Al Amyloidosis ◽

Bone Marrow Biopsies

Abstract Abstract 4044 Background: Conventionally, multiple myeloma is believed to coexist in approximately 10% of AL amyloidosis patients. However, it is unclear whether this figure is too low based on current World Health Organization criteria. These criteria, mainly created to differentiate myeloma from monoclonal gammopathy of undetermined significance, include the presence of ≥ 10% plasma cells on a bone marrow biopsy or aspirate as being diagnostic of myeloma. Aims: To define the frequency and relevance of a concomitant diagnosis of myeloma in patients with AL amyloidosis. Methods: Records from consecutive patients with biopsy-proven AL amyloidosis treated at the Stanford University Amyloid Center were reviewed. Plasma cell percentages were determined by manual counts from bone marrow aspirate smears and by CD138 immunohistochemistry (IHC) performed on bone marrow core biopsies. Results: A total of 41 patients (median age 61 years, 32% female) were evaluated. The median number of organs involved with amyloidosis was 2 (range 1–4), with 28 patients (68%) having cardiac involvement, 22 patients (54%) having renal involvement, 15 patients (37%) having gastrointestinal involvement, 12 patients (29%) having soft tissue involvement, and 10 patients (24%) having nervous system involvement. All patients had bone marrow biopsies and aspirates performed at the time of amyloid diagnosis, with most undergoing both manual counts of plasma cells from aspirates and IHC from core biopsies. Based on conventional criteria, manual aspirate counts defined 15/28 (54%) patients as having myeloma, and IHC defined 26/31 (84%) patients as having myeloma (p=0.01). Only nine patients had a detectable serum paraprotein on immunofixation (median 1.1 g/dl, range 0.4–2.6). 81% of patients had an elevated serum free light chain (85% lambda), with a median level of 37.3 mg/dl (range 8.6–256 mg/dl). Compared to the frequency of elevated plasma cells, the prevalence of anemia (29%), hypercalcemia (14%), impaired kidney function (21%), and lytic lesions (7%) was low. After a median follow-up of 13 months (range 1–127 months), the one-year overall survival (74% vs. 58%) and three-year overall survival (50% vs. 50%) was not significantly different between patients with ≥10% plasma cells and patients with <10% plasma cells (p=NS). Discussion: As defined by bone marrow plasma cell involvement, a strikingly high percentage (84%) of AL amyloidosis patients would be considered to have concurrent myeloma. This figure is much higher than has been traditionally quoted in the literature, likely due to the utilization of newer methods of counting plasma cells. There was a low prevalence of myeloma-associated end-organ effects (hypercalcemia, anemia, renal insufficiency, lytic bone lesions), and a myeloma diagnosis had no impact on survival. Conclusion: In this cohort of AL amyloid patients, concomitant myeloma was present in the vast majority of patients using modern diagnostic techniques. The significance of this diagnosis appears to be minimal – calling into question whether the diagnostic criteria for myeloma should be redefined in this population. Disclosures: Witteles: Celgene: Research Funding. Liedtke:Celgene: Lecture fee, Research Funding. Schrier:Celgene: Research Funding.

Download Full-text

High Cereblon Protein Expression Correlates with Improved Response and Survival in Myeloma Patients Treated with Lenalidomide

Blood ◽

10.1182/blood.v120.21.931.931 ◽

2012 ◽

Vol 120 (21) ◽

pp. 931-931 ◽

Cited By ~ 6

Author(s):

Alexander Klimowicz ◽

Paola Neri ◽

Andrew Belch ◽

Michelle Dean ◽

Li Ren ◽

...

Keyword(s):

Bone Marrow ◽

Regression Analysis ◽

Protein Expression ◽

Research Funding ◽

Cox Regression ◽

Survival Outcomes ◽

Newly Diagnosed ◽

Bone Marrow Biopsies ◽

Kaplan Meier ◽

Z Scores

Abstract Abstract 931 Background: Cereblon (CRBN), an adaptor protein of the Cul4A-DDB1-ROC1 ubiquitin E3 ligase complex was recently identified as a primary target of thalidomide teratogenicity and as essential requirement for IMiDs mediated cytotoxicity in multiple myeloma (MM) cells in vitro (Zhu YX et al, 2011). We have undertaken the current study to confirm the association between cereblon protein expression and the clinical response to lenalidomide. Methods: We constructed tissue microarrays (TMA) using bone marrow biopsies collected immediately prior to initiating therapy with lenalidomide. Fluorescence immunohistochemistry was performed using a polyclonal anti-CRBN antibody (HDA045910, Sigma-Aldrich) at a dilution of 1:4000, with 3 minutes of antigen retrieval at 121°C in a decloaking chamber in a pH=9 Tris/EDTA-based buffer (S2367, Dako). Tissue microarray slides were scanned on a HistoRx PM-2000 and digital images where analyzed with AQUA analysis software to determine the CRBN AQUA scores (protein expression = AQUA scores defined as the average CRBN pixel intensity within CD138 positive cells). CRBN AQUA scores where standardized on the Z-distribution (Z= X-μ/σ). The clinical parameters, response criteria and survival outcomes (PFS and OS) of these patients were defined according to the international uniform response criteria. The Kaplan-Meier method was used to estimate OS and PFS. Multivariate analysis was performed using the Cox regression method. Results: 42 patients with newly diagnosed (71.4%) or relapsed / refractory (28.6%) MM patients treated with lenalidomide and dexamethasone (MM009, MM016 or MM020 trials) and available pre-treatment bone marrow biopsies were included in this analysis. In this cohort, median age was 68 (range 46–88), median Hb 114 g/L (range 77–145), median Calcium 2.35 mmol/L (range 1.62–2.82), median creatinine 91.5 μmoles/L (range 44–500), high LDH in 21.4%, median albumin 35.5 g/L (range 23–47), β2 microglobulin 4.66 mg/L (range 1.2–35.19), ISS I 19%, II 35.7% and III 45.3% and high-risk cytogenetics (del17p13, t(4;14) by FISH) in 26.6%. Response CR/nCR was observed in 13/42 (31 %), PR in 21/42 (50%), MR in 4/42 (9.5%) and PD in 4/42 (9.5%). With a median follow-up of 22.4 months (range 0.72–65.6), 28/42 (67%) progressed with mPFS 19.53 months (95% CI 8.57–30.496) and mOS 28.733 months (95% CI 24.061–33.406). Cereblon expression or AQUA normalized Z scores ranged from -1.419 to 3.895. Kaplan Meier log-rank survival analysis were generated based on CRBN normalized AQUA Z scores with the bottom (Q4) and top quartiles (Q1-3) defined as CRBN-low or CRBN-high groups respectively. PFS was significantly shorter in CRBN-low (5.633 months) versus CRBN-high (19.733 months; p= 0.008). Similarly, OS was also reduced in CRBN-low patients (11.4 versus 30.467 months; p=0.033). In univariate Cox regression analysis, cereblon protein expression was significantly associated with PFS (HR 0.322; 95% CI 0.133–0.780; p=0.012) and OS (HR 0.323; 95% CI 0.108–0.970; p=0.044). Cereblon expression remained an independent predictor of PFS (HR 0.161; p=0.01) but not for OS when ISS and cytogenetics were included in multivariate regression analysis. In the CRBN-high group only 5/31 patients (16.1%), compared to 54.5% (6/11) in the CRBN-low group, failed to respond (≤MR) to lenalidomide. Similar to the protein tissue array analysis, low CRBN mRNA was also significantly associated with shorter PFS (p=0.008) in a chip microarray analysis of CRBN expression (Affymetrix probe 222533_at) in a cohort of 32 MM patients treated with lenalidomide and dexamethasone. Cereblon protein expression (AQUA normalized Z score) significantly correlated with CRBN mRNA microarray values (Affymetrix probe 222533_at) in 17 patients with matching protein and mRNA samples (Spearman's rho 0.417; p= 0.048). In contrast, and confirming the specificity of cereblon for response to IMiDs, no association between cereblon protein expression and response to therapy or survival outcomes (PFS/OS) was observed in a independent cohort of newly diagnosed MM patients (n=37) treated with bortezomib induction therapy and ASCT. Conclusion: Using an automated, observer-independent and fully quantitative approach, our studies confirm the association between cereblon protein expression and response to lenalidomide in MM. Disclosures: Neri: Johnson ans Johnson: Research Funding. Bahlis:Johnson and Johnson: Honoraria, Research Funding; Celgene: Honoraria.

Download Full-text

Plerixafor, G-CSF and Azacitidine For The Treatment Of MDS: Results Of a Phase I Trial

Blood ◽

10.1182/blood.v122.21.2816.2816 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2816-2816

Author(s):

Mark A. Schroeder ◽

Marcus Grillot ◽

Teresa Reineck ◽

Meagan A Jacoby ◽

Rizwan Romee ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Phase I ◽

Research Funding ◽

Phase I Trial ◽

Response Rates ◽

Response To Treatment ◽

Hematologic Toxicity ◽

Oncology Research ◽

Grade 3

Abstract Azacitidine has been shown to prolong survival and delay progression to leukemia in intermediate-2 and high risk myelodysplastic syndrome (MDS) in a randomized study compared to best supportive care. The combination of G-CSF and plerixafor is synergistic in increasing the release of stem and progenitor cells from the bone marrow through disruption of critical bone marrow stromal interactions including the CXCR4 / CXCL12 axis. The interaction of bone marrow stromal cells with the MDS tumor clone may play a roll in pathogenesis and response to treatment. We hypothesized that resistance of MDS to azacitidine may be related to MDS tumor and BM-stromal cell interactions, and disruption of these interactions by treatment with plerixafor + G-CSF could enhance sensitivity to azacitidine, thus improving complete and partial response rates. We conducted a phase I trial to investigate the safety and tolerability of plerixafor + G-CSF in combination with azacitidine in adult (18 years or older) MDS patients. Secondary objectives included response rates and biologic correlates evaluating: kinetics, phenotype, cell cycle status and kinetics of mobilization of MDS blasts compared to normal stem cells in select patients with informative cytogenetics. Major inclusion criteria included MDS defined by WHO criteria, 5 – 20% blasts on bone marrow aspirate, and at least one cytopenia in one cell lineage. Subjects receiving prior hypomethylating therapy were allowed. A standard 3+3 trial with 3 cohorts (320, 440, and 560 mcg/kg/day SC) was conducted. Dose limiting toxicity was defined as grade 3 or higher non-hematologic toxicity and hematologic toxicity of leukostasis or tumor lysis. Myelosuppression, infection, grade III nausea, fatigue, weight loss and electrolyte abnormalities were not considered dose limiting. Subjects initially received G-CSF 10 mcg/kg subcutaneous (SC) daily D1 – D8, plerixafor SC daily D3 – D8 and azacitidine 75mg/m2 SC D3 – D8, 4 hours after plerixafor administration. The trial was amended after the first 3 subjects to reduce G-CSF dose and administration to 5 days. Results Two of the first three subjects enrolled in cohort 1 (320 mcg/kg/d plerixafor) had leukocytosis. The trial was amended to reduce the G-CSF dose (10 mcg/kg to 5 mcg/kg) and duration (8 days to 5 days) because of this. One subject had symptoms of leukostasis with a WBC reaching nearly 100K/uL and a subsequent subject developed hyperleukocytosis (WBC = 80K/uL) without leukostasis. The trial was amended to reduce the G-CSF to 5 days concurrent with plerixafor and azacitidine along with defining dose holding parameters for G-CSF and plerixafor if the peripheral blood WBC exceeded 40K/uL or if absolute blast count exceeded 10K/uL. Since amendment of the trial, 64 subjects have been screened and 20 subjects have been enrolled and are evaluable. Subjects included 65% males, median age 67, and MDS diagnosis at study entry including 6/18 (33%) RAEB-1 and 12/18 (67%) RAEB-2. 5/18 subjects (28%) had plerixafor and G-CSF held during treatment because of leukocytosis. 9/18 subjects (50%) had received no prior treatment for their MDS. DLTs were experienced in Cohort 1 related to thrombocytosis (n =1) and in Cohort 2 related to atrial fibrillation (n = 1) with near syncope. Major non-hematologic grade 3 or 4 adverse events included epistaxis, hypocalcemia, GI bleed, headache, dyspnea, infection with neutropenia, and bone pain. The MTD was determined to be 560mcg/kg plerixafor SC with no subjects (n = 6) in this cohort experiencing a DLT. The median number of cycles completed was 3. Reasons for stopping treatment included progression to leukemia (n = 6), physician choice (n = 2), withdrawal of consent (n = 1), adverse event (n = 2). Best response in those evaluable after completing 2 cycles of treatment (n = 14) showed marrow CR in 5/14 (36%, 3 in those not previously treated for MDS), stable disease in 5/14 (36%) and progressive disease 4/14 (29%). Conclusion Plerixafor plus G-CSF in combination with azacitidine was well tolerated in the studied MDS patients when given over 5 days and may be associated with encouraging response rates. Correlative studies are ongoing to evaluate changes in cell cycle, apoptosis and preferential mobilization of blasts using this regimen. We are currently enrolling an expanded cohort of 7 subjects at the MTD dose to evaluate preferential mobilization of blasts with plerixafor alone in cases using informative cytogenetics. Disclosures: Schroeder: Celgene: Research Funding; Sanofi Oncology: Research Funding. Off Label Use: Plerixafor and G-CSF for the treatment of MDS. Welch:Eisai: Research Funding. Stockerl-Goldstein:Millennium: Speakers Bureau; Celgene : Speakers Bureau.

Download Full-text

KB004, a Novel Non-Fucosylated Humaneered® Antibody, Targeting EphA3, Is Active and Well Tolerated in a Phase I/II Study of Advanced Hematologic Malignancies

Blood ◽

10.1182/blood.v124.21.3756.3756 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3756-3756 ◽

Cited By ~ 3

Author(s):

Ronan T Swords ◽

Andrew H Wei ◽

Simon Durrant ◽

Anjali S. Advani ◽

Mark S Hertzberg ◽

...

Keyword(s):

Bone Marrow ◽

Phase I ◽

Dose Level ◽

Research Funding ◽

Hematologic Malignancies ◽

Clinical Activity ◽

Bone Marrow Biopsies ◽

Infusion Reactions ◽

Equity Ownership ◽

Dose Levels

Abstract Background: EphA3 is a novel drug target involved in cell positioning in fetal development. In adults it is an oncofetal antigen, that is re-expressed in hematologic malignancies (blood and bone marrow, leukemic stem cells) and solid tumors. It is also upregulated in diseases characterized by abnormal proliferation and fibrosis, such as idiopathic pulmonary fibrosis and diabetic kidney disease. KB004 is a Humaneered® high affinity antibody (KD = 610 pM) targeting EphA3 with at least 3 possible mechanisms of action: direct apoptosis in tumor cells, activation of ADCC and disruption of tumor vasculature. Objectives: The primary objectives of the Phase I study component are to determine safety and MTD for KB004 in patients with hematologic malignancies, refractory to or unfit for chemotherapy. Secondary objectives are to characterize PK, immunogenicity, and preliminary clinical activity of KB004. Exploratory objectives include evaluation of EphA3 expression on tumor, stromal, and endothelial cells. Methods: Multicenter Phase I/II study. Key eligibility criteria: unsuitable for standard of care or relapsed or refractory hematologic malignancy, ECOG PS 0-1, adequate organ function, platelets ≥ 10,000/uL (untransfused for 7 days) and normal coagulation times. KB004 was administered as a 1-2 hr intravenous infusion on days 1, 8, and 15 of each 21-day cycle, at incremental doses of 20, 40, 70, 100, 140, 190, 250 and 330 mg. At 70 mg and above infusion reaction prophylaxis included H1 and H2 blockers, acetaminophen and IV steroids. Safety and activity by IWG response criteria were assessed. Peripheral blood and bone marrow biopsies for PK analysis and EphA3 expression were also collected. Results: A total of 50 patients (AML 39, MDS 7, DLBCL 1, MF 3) received KB004 in the phase I/dose finding component of the study, which has been completed. The most common toxicities were transient grade 1 and grade 2 transient infusion reactions (IRs) in 79% of patients. IRs were characterized by chills, elevated temperature, fever, rigors, back pain, nausea, vomiting, hypotension, hypertension and transient hypoxia (in 2 cases). No other significant KB004 related toxicity was observed. Two patients discontinued KB004 due to an IR. One of these (grade 3) defined a DLT at the 330mg dose level. A second patient at 330mg had grade 2 infusion reactions associated with multiple infusion delays. These observations prompted expansion of the next lowest dose cohort, 250mg. Six evaluable patients were treated at this dose level. No clinically significant IRs or DLTs were observed. This is therefore the recommended phase 2 dose (RP2D). At all dose levels observed Cmax for KB004 was approximately dose proportional. Sustained exposure above the predicted effective concentration (1ug/mL) to cover the 7-day interval between doses was achieved above 190mg. Responses according to IWG criteria were observed in patients with AML, MF and MDS at the 20 mg, 140g and 250mg dose levels, respectively. At 20mg, a 78 yr-old patient with relapsed AML achieved CRp. Remission was sustained for over 18 months and relapse was preceded by a rise in EphA3 expression. Serial bone marrow biopsies with KB004 treatment show decreased reticulin and collagen fibrosis. At 140mg, a 67 yr old patient with JAK2 V617F mutant previously untreated myelofibrosis whose predominant clinical problem at diagnosis was anemia achieved Clinical Improvement [CI]. Transfusion independency (both RBC and platelets) has been sustained for 8+ months with improvement in constitutional symptoms and improved splenomegaly. At 250 mg an 84 yr-old patient with MDS/MPN (intermediate risk) achieved a Hematologic Improvement [HI, erythroid]. A > 50% reduction in marrow blast percentage was seen in 8 patients. Bone marrow biopsies positive for EphA3 expression with a cut-off of 10% of nucleated cells were obtained in greater than 70% of AML patients. Of 20 patients for whom EphA3 expression data exists with time, 7 (35%) had at least a 50% reduction in expression with treatment. Conclusion: KB004 is a novel agent targeted against EphA3 that is well tolerated when given as a weekly 2 hour infusion. The promising clinical activity profile is postulated to be consistent with the antifibrotic mechanism. The Phase II component of the study is ongoing in which the activity of KB004 will be characterized in disease specific cohorts including AML, MDS and MF at the RP2D of 250mg. Disclosures Durrant: KaloBios: Research Funding. Advani:KaloBios: Research Funding. Greenberg:Celgene: Research Funding; Novartis: Research Funding; GSK: Research Funding; Onconova: Research Funding; KaloBios: Research Funding. Cortes:KaloBios: Research Funding. Yarranton:KaloBios: Employment; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; Stemline Therapeutics: Equity Ownership. Walling:KaloBios, Corcept Therapeutics, Prothena, NewGen Therapeutics, Valent Technologies, LBC Pharmaceuticals: Consultancy, Equity Ownership; Amgen, BioMarin: Equity Ownership; Crown BioScience: Membership on an entity's Board of Directors or advisory committees.

Download Full-text