Mutation analysis of BCR-ABL1 kinase domain in chronic myeloid leukemia patients with tyrosine kinase inhibitors resistance: a Malaysian cohort study.

OBJECTIVE: Mutational analysis of BCR::ABL1 kinase domain (KD) is a crucial component of clinical decision algorithms for chronic myeloid leukemia (CML) patients with failure or warning responses to tyrosine kinase inhibitor (TKI) therapy. This study aimed to detect BCR::ABL1 KD mutations in CML patients with treatment resistance and assess the concordance between NGS (next generation sequencing) and Sanger sequencing (SS) in detecting these mutations. RESULTS: In total, 12 different BCR::ABL1 KD mutations were identified by SS in 22.6% (19/84) of patients who were resistant to TKI treatment. Interestingly, NGS analysis of the same patient group revealed an additional four different BCR::ABL1 KD mutations in 27.4% (23/84) of patients. These mutations are M244V, A344V, E355A, and E459K with variant read frequency below 15%. No mutation was detected in 18 patients with optimal response to TKI therapy. Resistance to TKIs is associated with the acquisition of additional mutations in BCR::ABL1 KD after treatment with TKIs. Additionally, the use of NGS is advised for accurately determining the mutation status of BCR::ABL1 KD, particularly in cases where the allele frequency is low, and for identifying mutations across multiple exons simultaneously. Therefore, the utilization of NGS as a diagnostic platform for this test is very promising to guide therapeutic decision-making.

Comprehensive analysis of mutations and clonal evolution patterns in a cohort of patients with cytogenetically normal acute myeloid leukemia.

BACKGROUND: Relapsed acute myeloid leukemia (AML) is associated with the acquisition of additional somatic mutations which are thought to drive phenotypic adaptability, clonal selection and evolution of leukemic clones during treatment. We performed high throughput exome sequencing of matched presentation and relapsed samples from 6 cytogenetically normal AML (CN-AML) patients treated with standard remission induction chemotherapy in order to contribute with the investigation of the mutational landscape of CN-AML and clonal evolution during AML treatment. RESULT: A total of 24 and 32 somatic variants were identified in presentation and relapse samples respectively with an average of 4.0 variants per patient at presentation and 5.3 variants per patient at relapse, with SNVs being more frequent than indels at both disease stages. All patients have somatic variants in at least one gene that is frequently mutated in AML at both disease presentation and relapse, with most of these variants are classic AML and recurrent hotspot mutations including NPM1 p.W288fs, FLT3-ITD, NRAS p.G12D and IDH2 p.R140Q. In addition, we found two distinct clonal evolution patterns of relapse: (1) a leukemic clone at disease presentation acquires additional mutations and evolves into the relapse clone after the chemotherapy; (2) a leukemic clone at disease presentation persists at relapse without the addition of novel somatic mutations. CONCLUSIONS: The findings of this study suggest that the relapse-initiating clones may pre-exist prior to therapy, which harbor or acquire mutations that confer selective advantage during chemotherapy, resulting in clonal expansion and eventually leading to relapse.

Identification of FLT3 and NPM1 Mutations in Patients with Acute Myeloid Leukaemia.

Objective: The most frequent acquired molecular abnormalities and important prognostic indicators in patients with Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1) mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method for detection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patients with cytogenetically normal (CN) AML in our population. Methods: A total of 199 samples from AML patients (95 women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction (PCR) and gene sequencing. Result: Sixty-eight patients were positive for the mutations. FLT3-ITD mutations were detected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations of NPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencing and showed 100% concordance with in house method. Conclusion: The optimized in-house PCR assays for the detection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and cost effective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification of these mutations are important for prognostication and optimization of patient care.

Prevalence of BCR-ABL T315I Mutation in Malaysian Patients with Imatinib-Resistant Chronic Myeloid Leukemia

Objective: Chronic Myeloid Leukemia (CML) is caused by a reciprocal translocation between chromosomes 9 and 22, t(9;22) (q34;q11) which encodes for the BCR-ABL fusion protein. Discovery of Imatinib Mesylate (IM) as first line therapy has brought tremendous improvement in the management of CML. However, emergence of point mutations within the BCR-ABL gene particularly T315I mutation, affects a common BCR-ABL kinase contact residue which impairs drug binding thus contribute to treatment resistance. This study aims to investigate the BCR-ABL T315I mutation in Malaysian patients with CML. Methods: A total of 285 patients diagnosed with CML were included in this study. Mutation detection was performed using qualitative real-time PCR (qPCR). Results: Fifteen out of 285 samples (5.26%) were positive for T315I mutations after amplification with real-time PCR assay. From the total number of positive samples, six patients were in accelerated phase (AP), four in chronic phase (CP) and five in blast crisis (BC). Conclusion: Mutation testing is recommended for choosing various tyrosine kinase inhibitors (TKIs) to optimize outcomes for both cases of treatment failure or suboptimal response to imatinib. Therefore, detection of T315I mutation in CML patients are clinically useful in the selection of appropriate treatment strategies to prevent disease progression.

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis.

JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

Preclinical studies with JAA-F11 anti-Thomsen-Friedenreich monoclonal antibody for human breast cancer.

AIM: The Thomsen-Friedenreich antigen (TF-Ag) is a disaccharide hidden on normal cells, but selectively exposed on the surface of breast, colon, prostate and bladder cancer cells. JAA-F11, a highly specific monoclonal antibody to TF-Ag, reduces metastasis and prolongs survival in a mouse model. In addition,(124)I-JAA-F11 localizes 4T1 tumors in mice. These studies continue translation of JAA-F11 to human breast cancer. MATERIALS & METHODS & RESULTS: Of the 41 human breast cancer cell lines tested, 78% were positive for reactivity with JAA-F11 by whole-cell enzyme immunoassay and positivity occurred unrelated to estrogen, progesterone or HER2 receptor status. JAA-F11 inhibited the growth rate of the human cancer cell lines tested. At 1 h, approximately 80% of JAA-F11 internalized in the three cell lines tested. (124)I-JAA-F11 specifically imaged human triple-negative tumors in mice by microPET. CONCLUSION: The results highlight the potential that humanized JAA-F11 may have for immunotherapy and drug conjugate therapy in breast cancer patients.

Anti-Thomsen-Friedenreich-Ag (anti-TF-Ag) potential for cancer therapy.

Thomsen-Friedenreich antigen (TF-Ag) is the disaccharide (Gal beta1-3 GalNAc alpha), which is also known as the core 1 structure. The presence of this disaccharide on the surface of approximately 90 percent of carcinomas is due to altered glycosylation in these tumors. TF-Ag plays a role in the adhesive properties of tumor cells involved in metastasis. Treatment of mice with JAA-F11, a monoclonal antibody to TF-Ag alpha inhibited lung metastasis and improved prognosis in a mouse breast cancer model. The presence of naturally occurring antibodies to TF-Ag in cancer patients is related to improved prognosis. The pancarcinoma expression of TF-Ag, combined with the evidence of a mechanistic role for TF-Ag in cancer spread, show that this target would have clinical utility. The presence of naturally occurring antibody to TF-Ag indicates that increasing the anti-TF-Ag antibody would be safe for the cancer patient and indicates that tolerance would not have to be broken to create this immune response. Finally, the prognostic improvements seen clinically and in animal models indicate that this is an important vaccine target.

Identification and characterization of genes required for cell-to-cell fusion in Neurospora crassa.

A screening procedure was used to identify cell fusion (hyphal anastomosis) mutants in the Neurospora crassa single gene deletion library. Mutants with alterations in 24 cell fusion genes required for cell fusion between conidial anastomosis tubes (CATs) were identified and characterized. The cell fusion genes identified included 14 genes that are likely to function in signal transduction pathways needed for cell fusion to occur (mik-1, mek-1, mak-1, nrc-1, mek-2, mak-2, rac-1, pp2A, so/ham-1, ham-2, ham-3, ham-5, ham-9, and mob3). The screening experiments also identified four transcription factors that are required for cell fusion (adv-1, ada-3, rco-1, and snf5). Three genes encoding proteins likely to be involved in the process of vesicular trafficking were also identified as needed for cell fusion during the screening (amph-1, ham-10, pkr1). Three of the genes identified by the screening procedure, ham-6, ham-7, and ham-8, encode proteins that might function in mediating the plasma membrane fusion event. Three of the putative signal transduction proteins, three of the transcription factors, the three putative vesicular trafficking proteins, and the three proteins that might function in mediating cell fusion had not been identified previously as required for cell fusion.