Tyrosine Kinase Inhibitor Drugs Research Articles

A case of Ph+ acute lymphoblastic leukemia and EGFR mutant lung adenocarcinoma synchronous overlap: may one TKI drug solve two diseases?

BackgroundPhiladelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) refers to ALL patients with t(9;22) cytogenetic abnormalities, accounting for about 25% of ALL. Lung adenocarcinoma (LUAD) is the most common pathological type of non-small-cell lung cancer, which has a frequency of approximately 45% cases with mutations in EGFR. Both Ph+ ALL and EGFR mutant LUAD are involved in the pathogenesis of the abnormal activation of the tyrosine kinase pathway. Although the second primary hematological malignancy after the treatment of solid tumors is common in clinics, the synchronous multiple primary malignant tumors of hematological malignancy overlap solid tumors are uncommon, even both tumors involved in the pathogenesis of the abnormal activation of the tyrosine kinase pathway are extremely rare.Case presentationAn 84-year-old man with fatigue and dizziness was diagnosed with Ph+ ALL. Meanwhile, a chest CT indicated a space-occupying lesions, characterized by the presence of void, in the right lower lope with the enlargement of mediastinal lymph node and right pleural effusion. After a few weeks, the patient was diagnosed with LUAD with EGFR exon 19 mutation. Both tyrosine kinase inhibitors (TKI) (Flumatinib) and EGFR-TKI (Oxertinib) was used for the patients, and finally have controlled both diseases.ConclusionAs far as we know, we for the first time reported a case of Ph+ ALL and EGFR mutant LUAD synchronous overlap, of which pathogenesis is related to abnormal tyrosine kinase activation. This patient was successfully treated with two different TKIs without serious adverse events.

Differential Impact of Tumor Endothelial Angiopoietin-2 and Podoplanin in Lymphatic Endothelial Cells on HCC Outcomes with Tyrosine Kinase Inhibitor Treatment According to Sex.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Curative treatments are available to a minority of patients, as HCC is often diagnosed at an advanced stage. For patients with unresectable and multifocal HCC, tyrosine kinase inhibitor drugs (TKIs) are the only potential treatment option. Despite extensive research, predictors of response to these therapies remain elusive. This study aimed to analyze the biological and histopathological characteristics of HCC patients treated with TKIs, focusing on angiogenesis and lymphangiogenesis. Immunohistochemistry quantified the expression of angiopoietin-2 (Ang2), lymphatic endothelial cells (LEC) podoplanin, and C-type Lectin Domain Family 2 (CLEC-2), key factors in neoangiogenesis and lymphangiogenesis. An increased expression of endothelial Ang2 and LEC podoplanin predicted a lower risk of metastasis. Female patients had significantly longer overall survival and survival on TKIs, associated with higher tumor expression of endothelial Ang2 and LEC podoplanin. Moreover, LEC podoplanin expression and a longer time on TKIs were independently correlated with improved survival on TKI therapy at Cox regression analysis. These findings suggest that endothelial Ang2 and LEC podoplanin could be potential biomarkers for predicting treatment outcomes and guiding therapeutic strategies in HCC patients treated with TKIs.

Quantification and analyses of seven tyrosine kinase inhibitors targeting hepatocellular carcinoma in human plasma by QuEChERS and UPLC-MS/MS

Tyrosine kinase inhibitors (TKIs) are commonly used to treat various cancers. Literature suggests that the blood concentration of TKIs strongly correlates with their efficacy and adverse effects. Therefore, establishing a Therapeutic Drug Monitoring (TDM) methodology for TKI drugs is crucial to improving their clinical efficacy and minimizing the treatment-related adverse effects. However, quantifying their concentrations in the plasma using existing methods to avoid potential toxicity is challenging. Herein, seven TKIs, namely sorafenib tosylate, axitinib, erlotinib, cediranib, brivanib, linifanib, and golvatinib, were successfully analyzed in human plasma by following a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Briefly, biological samples were extracted using 1 mL of methanol, followed by the sequential addition of 250 mg of anhydrous magnesium sulfate and 25 mg of N-propylethylenediamine (PSA) for salinization and purification by adsorption, respectively. In this study, dovitinib was used as the internal standard. The seven TKIs were detected by the gradient elution method for 4 min in the positive ion electrospray mode. The mobile phase comprised methanol (phase A) and 0.1 % aqueous formic acid solution (phase B) on the Agilent Zorbax RRHD Stablebond Aq, (2.1 × 50 mm; 1.8 μm). Brivanib, linifanib, axitinib, sorafenib tosylate, and golvatinib exhibited good linearity in the range of 5–500 ng/mL, and erlotinib and cediranib exhibited good linearity in the range of 10–1000 ng/mL, with linear correlation coefficients (R2) ≥ 0.99. The limits of detection and quantification were 0.60–0.18 ng/mL and 5–10 ng/mL, respectively. The intraday and interday accuracy values ranged from −6.12 % to 7.31 %, with a precision (RSD) of ≤ 10.57 %. The method was rapid, accurate, specific, simple, reproducible, and suitable for the quantitative determination of the seven TKIs in human plasma.

Prognostic Role of Human Leukocyte Antigen Alleles and Cytokine Single-Nucleotide Polymorphisms in Patients with Chronic Myeloid Leukemia Treated with Tyrosine Kinase Inhibitor Drugs.

Tyrosine kinase inhibitor (TKI) drugs have significantly improved chronic myeloid leukemia (CML) outcomes. Neopeptides from CML cells may induce specific immune responses, which are crucial for deep molecular (DMR) and treatment-free remission (TFR). In this study of Ethiopian patients with CML (n = 162), the HLA alleles and single-nucleotide polymorphisms of five cytokines revealed significant associations with clinical outcomes. Clinically unfavorable outcomes correlated with HLA alleles A*03:01/02, A*23:17:01, B*57:01/02/03, and HLA-DRB4*01:01 (p-value = 0.0347, p-value = 0.0285, p-value = 0.037, and p-value = 0.0127, respectively), while HLA-DRB4*01:03:01 was associated with favorable outcomes (p-value = 0.0058). After assigning values for the 'low', 'intermediate', and 'high' gene expression of the SNPs' respective cytokine genes, Kaplan-Meier estimates for relapse-free survival, adjusted for age, treatment duration, and relapse risk among patients after the administration of TKIs, indicated that a gene expression ratio above the overall median of TNF-α, IL-6, and the combination of TGF-β1/IL-10, IFNγ, and IL-6/IL-10 TGF-β1 was correlated with a higher likelihood of treatment failure ((RR: 3.01; 95% CI: 1.1-8.3; p-value = 0.0261) and (RR: 2.4; 95% CI: 1.1-5.2; p-value = 0.022), respectively). Multi-SNPs, surpassing single-SNPs, and HLA allele polymorphisms showed promise in predicting outcomes of patients with CML during TKI treatment, prompting further exploration into their potential utility.

Abstract 1960: CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations

Abstract Bruton’s Tyrosine Kinase (BTK) inhibitor drugs are a mainstay in the treatment of CLL, WM and MCL. They are not curative because of the emergence of BTK inhibitor resistance mutations in as much as 67% of relapsed patients (in CLL) [1]. Frequently observed mutations include BTKC481S, which abrogates covalent binding of irreversible drugs such as acalabrutinib, ibrutinib and zanubrutinib, and BTKT474I and BTKV416L which both abrogate binding of the non-covalent inhibitor drug pirtobrutinib [1, 2]. To combat resistance, third generation BTK inhibitors are needed, which must 1. bind to unique regions in the tumor driving kinases that are not marred by resistance mutations, 2. have sufficient potency for sustained inhibition of BTK signaling, and 3. be sufficiently selective to afford efficacious doses and suitable for a combination regimen. We have developed a platform called Energetically Privileged Ligands (EPriLs), based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket. Their unique binding mode involves connecting the N-terminal and C-terminal lobes of the general kinase fold in a continuous β-sheet, in this way avoiding hotspots for kinase inhibitor resistance mutations. The potent binding of the macrocycle ensures a very long residence time. Using the EPriL platform, we have developed CFON-026, a next generation non-covalent BTK inhibitor with a covalent-like residence time as demonstrated by surface plasmon resonance (SPR) and cellular washout experiments. The biochemical binding KD to BTKWT, determined by SPR, is 0.14 nM. CFON-026 binds mutants with KDs of 0.4 nM (BTKC481S) and and 1.8 nM (BTKT474I). X-ray crystal structures of CFON-026 bound to BTKWT and BTKT474I confirm the unique binding mode and water network around the active site. CFON-026 inhibits the growth of the BTK dependent cell lines TMD8 and Rec-1 with IC50s of 0.45 nM and 1.7 nM, respectively. CFON-026 is very active on TMD8 variants containing endogenous BTK inhibitor resistance mutations (BTKC481S, BTKV416L, BTKT474I and BTKL528W, IC50 range 1-100 nM). CFON-026 has favorable metabolic stability, pharmacokinetic properties, and safety characteristics, and induces complete tumor regression in a TMD8 xenograft model. Since BTK resistance mutations can be detected diagnostically 12-18 months before clinical relapse, CFON-026 could be applied as combination partner to prolong progression free survival on first line BTK inhibitor therapy. Experiments with mixed populations of BTK mutant cell lines, to model a heterogenous tumor, show that CFON-026 can deepen the efficacy of ibrutinib, acalabrutinib and zanubrutinib. In conclusion, CFON-026 is a novel EPriL-based non-covalent BTK inhibitor that could enhance and prolong responses of existing covalent BTK inhibitor therapy. [1] Woyach et al., J Clin Oncol (2017). [2] Wang et al., N. Eng J Med (2022). Citation Format: Jos de Man, Michelle Muller, Joost C.M. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G.T.H. van Mil, Winfried R. Mulder, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1960.

Abstract 6110: Computational exploration of genomic differences in Black patients treated with chemoradiation for head and neck squamous cell carcinoma (HNSCC)

Abstract Background: Black patients treated with chemoradiation for head and neck squamous cell carcinoma (HNSCC) experience significantly worse survival outcomes than White patients treated with the same modality. Our group aims to explore potential sociodemographic and biological factors undermining this differing response to therapy. Here, we present our computational findings of genomic differences in Black compared with White HNSCC patients receiving chemoradiation. Methods: We obtained gene expression, phenotype, and clinical datasets for the Cancer Genome Atlas (TCGA) HNSCC cohort from the UCSC Xena web browser. Of the 528 HNSCC patients, 131 (Black=13, White=118) of those receiving chemoradiation had gene expression, phenotype, and clinical data available. Our analysis included anatomic sites involving the oral cavity, hypopharynx, and larynx, and excluded the oropharynx. We performed differential gene expression analysis in Black compared with White (reference population) patients via edgeR in R studio (-log2FC < -1 or log2FC >1; p value < 0.05; FDR < 0.05). The Database for Annotation, Visualization, and Integrated Discovery (DAVID) webtool was used to identify significantly enriched molecular processes and pathways. Results: 462 genes were found to be significantly differentially expressed between Black and White HNSCC patients receiving chemoradiation. Of these genes, 198 were found to be higher in Black patients, and the most significant included JPH3, PCSK1N, DCUN1D5, CRYGS, and CCL7 (p < 0.005, FDR < 0.005). 264 were found to be lower in Black patients, with the most significant being KBTBD12, CMYA5, NEFH, LDB3, PYGM (p < 0.005, FDR < 0.005). Genes found to be higher in Black patients showed enrichment for processes such as EGFR tyrosine kinase inhibitor resistance (p < 0.005), drug metabolism (p < 0.005), and chemical carcinogenesis (p < 0.005). Genes found to be lower in Black patients showed enrichment for muscle-related processes such as myogenesis (p < 0.005), actin-binding (p < 0.005), muscle protein (p < 0.005), and hypertrophic cardiomyopathy (p < 0.005). Conclusion: In conclusion, using TCGA, we identified differentially expressed genes between Black and White HNSCC patients receiving chemoradiation. Moreover, these findings suggest that biological processes may differ in Black compared with White patients receiving chemoradiation, and this may dictate differing response to therapy. Additional studies will involve further computational analysis using other datasets for validation. Citation Format: Chayil C. Lattimore, Caretia J. Washington, Dejana Braithwaite, Shama D. Karanth, Kristianna M. Fredenburg. Computational exploration of genomic differences in Black patients treated with chemoradiation for head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6110.

Tyrosine kinase inhibitor-induced hypothyroidism: mechanism and clinical implications.

Since the first experimentally proven tyrosine kinase inhibitor (TKI) imatinib was introduced in the clinical setting, TKIs have attracted widespread attention because of their remarkable therapeutic effects and improvement of survival rates. TKIs are small-molecule, multi-target, anti-cancer agents that target different tyrosine kinases and block downstream signaling. However, with in-depth research on TKI drugs, the adverse reactions-for example, thyroid dysfunction-have become a concern and thus have attracted the attention of numerous researchers. Thyroid dysfunction, especially hypothyroidism, that occurs in high incidence during TKI therapy has a close relationship with treatment efficacy, but the mechanism of TKI-induced thyroid dysfunction is obscure. This review discusses the epidemiology, possible mechanisms, and clinical significance of hypothyroidism in cancer patients treated with TKI.

Facilitated Transport of EGFR Inhibitors Plays an Important Role in Their Cellular Uptake.

Epidermal growth factor receptor (EGFR) is a transmembrane protein commonly targeted by tyrosine kinase inhibitors (TKIs) as a front-line therapy for patients with many cancers including nonsmall cell lung cancer (NSCLC). Effective treatment requires efficient intracellular drug uptake and target binding. However, despite the recent success in the development of new TKI drugs, the mechanisms of uptake for many TKIs are still poorly understood due to the difficulty in imaging and measuring nonfluorescent drug molecules at a subcellular resolution. It has previously been shown that weakly basic TKI drugs are sequestered in lysosomes. Leveraging this property, we apply hyperspectral stimulated Raman scattering imaging to directly visualize and quantify two Food and Drug Administration-approved EGFR inhibitor drugs (lapatinib and afatinib) inside living cells and the changes in their cellular uptake upon the addition of organic cation transporter inhibitors. These single-cell quantitative measurements provide new insight into the role of membrane transporters in the uptake of TKI drugs in living cells.

Skin reactions to tyrosine kinase inhibitors in pediatric patients with chronic myeloid leukemia.

Cutaneous adverse events are commonly reported in adult patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitors (TKIs); however, little is known about the cutaneous reactions in children receiving TKIs for CML. As pediatric patients may require lifelong TKI therapy, it is essential to understand the wide range of potential cutaneous toxicities. We examined all case studies, cohort studies, and clinical trials in PubMed/MEDLINE and Embase that reported cutaneous reactions to first-, second-, and third-generation TKIs in children 18 years or younger with CML. This review article focuses on the TKI drug types and doses, patient demographic characteristics, features of skin reactions, and clinical outcomes.

Prospective Assessment of Co-Morbidities and Framingham Risk Score in Newly Diagnosed Chronic Myeloid Leukemia (CML) Patients and Its Impact on Clinical Outcomes Following Frontline TKI Therapy: Toronto CML Genomic Alliance in Greater Toronto Area & Ontario (TCGA-GTA)

Introduction Front-line (1L) tyrosine kinase inhibitor (TKI) drug selection is a pearl of clinical practice in CML. It requires consideration of multiple aspects including the goal of therapy, potency of the drug as well as comorbidity of the patient (pt). Contemporary CML practice emphasizes the importance of comorbidity for TKI selection. However, such data is scarce particularly the prevalence of comorbidity and its impact on 1L drug selection. We are developing a shared care model with community hematologists in Greater Toronto Area (TCGA-GTA). Newly diagnosed CML pts in the community will be referred to the study core center for prospective enrolment into the CML registry, informed consenting for genetic testing/future research, and evaluation of baseline comorbidity. Pt will return to referring hematologists for management of CML, including 1L TKI treatment as per standard of care, and is scheduled to visit the study center at 6 and 12 months, then annually for 10 years. It gives us a unique opportunity to evaluate the impact of comorbidity on 1L TKI drug selection. Patients and method Adult pts with newly diagnosed CML of any phase within 2 months of diagnosis are eligible for the study unless they are not able to visit study core center. Comorbidities and Framingham risk score (FRS) were assessed at the time of initial evaluation. Mutation profile testing is done by a bar-coded error-corrected sequencing platform for 40 genes developed at the Ontario Institute for Cancer Research. Cumulative incidence of molecular response with 2 log (MR2), 3 log (MR3), 4 log reduction or deeper (MR4), and treatment failure (TF) were calculated using cumulative incidence method considering competing risk. Event-free survival (EFS) was defined as time from start of TKI until TKI stop/switch, TF or death. Failure-free survival (FFS) was defined as time from start of TKI to TF [primary resistance, loss of complete cytogenetic response, new additional cytogenetic abnormalities (ACAs), and progression to accelerated or blasts phase (AP/BP)] or death. Results From November 2020 until June 31, 2023 (data cutoff), a total of 77 pts were enrolled in the study. The median follow-up time was 12 months. The frequency of 1L TKI selection was as follows: IMATINIB (IM) (n=23, 30%), DASATINIB (n=25, 32%), NILOTINIB (n=22, 29%), BOSUTINIB (n=1, 1%), ASCIMINIB (n=6, 8%). Majority of the pts were male (n=51, 66%). Ninety-three percent of pts were in chronic phase at diagnosis, while 4% and 3% were in AP and BP, respectively. Presence of ACAs was detected in 18 pts (23%), 10 of which were high risk, while 6% (n=5) were not evaluable. The baseline clinical and disease characteristics of pts on 1L IM vs other TKIs (2G-TKI) are summarised in Table 1. Of note, pts on IM were significantly older with higher FRS and comorbidities, including coronary artery disease, diabetes mellitus, and hypertension. The OS of all pts at 1 year was 98.5% (95% confidence interval 90%-99.8%). At 1 year, pts with intermediate to high risk FRS had lower FFS (73.2% vs 86.5%, p=0.037) while history of hyperlipidemia was associated with lower EFS (59.3% vs 81.5%, p=0.023) and 6 times more likely to stop or switch therapy due to intolerance or resistance (HR 5.9, p=0.002). Baseline comorbidities did not demonstrate signifincant impact on MR2 and MR3 at 1 year but pts with low FRS were more likely to achieve MR4 within 1 year (22.3% vs 0%, p=0.023). Age did not affect any of the clinical and molecular endpoints. Compared to 2G-TKI, IM had worse EFS (HR 3.93, p=0.003), FFS (HR 5.66, p=0.011), and MR2 (47.3% vs 84.4%, p=0.003) at 1 year. Further, IM pts experienced higher rate of any side effects compared to those on 2G-TKI (69.6% vs 31.5%, p=0.003). Conclusion The TCGA-GTA study provides valuable insights into the impact of comorbidity on selection of 1L TKI and its outcome in a real-world setting. Pts who are older and with higher FRS and comorbidities tend to receive 1L IM. However, pts on 1L 2G TKIs are more likely to reach treatment goals faster with lower probability of TF. The present study did not reach a clear conclusion whether the worse outcomes in the pts receiving IM are from IM treatment or concurrent risk factors such as age and comorbidities, thus further study with an expanded cohort and longer follow-up is required. Elderly pts or those with comorbidities would need alternative treatment with better efficacy and tolerability, such as Asciminib, that can overcome comorbidity issues in the future.

The Efficacy and Safety of Olverembatinib Combined with Monoclonal Antibodies As Salvage Therapy for RR B ALL with ABL1 Fusion Gene Positive

Background: B cell acute Lymphoblastic Leukemia （B ALL） with ABL1 fusion gene(FG) positive（ABL1+B ALL） is classified as Ph or Ph like ALL and has a poor prognosis. With the therapy of tyrosine kinase inhibitors(TKI)CD19- or CD22-specific chimeric antigen receptor (CAR) T-cell therapy,more patients(pts)can achieve complete remission(CR) even relapsed after hematopoietic stem cell transplantation(HSCT). However, the pts relapsed/recurrence（RR）after HSCT /CART/ TKIs even ponatinib or cannot receive CART due to any reason have the worst prognosis. Recently, such pts have new hope with the wide application of a novel third-generation TKI Olverembatinib developed in China and Blinatumomab（CD19 antibody,BITE）and INOTUZUMAB OZOGAMICIN(CD22 antibody,Ino.). Aims: Observe the efficacy and safety of Olverembatinib combined with BITE or INO. therapy in RR ABL1+B ALL pts after chemotherapy combined with TKI or CART or even after transplantation. Methods: Four RR ABL1+B ALL pts received Olverembatinib with BITE or INO from June 2022 to July 2023. During treatment, the dose of Olverembatinib was adjusted and chemotherapy or veneclax may be given at the same time,and give appropriate treatment depending on the patient's condition.After treatment, efficacy was assessed by complete remission (CR) rate, minimal residual disease(MRD)neg (<0.01%) rate by MFC and complete molecular remission(CMR )(FG transcript<10-5) rate by real-time quantitative polymerase chain reaction (RT-qPCR). Results:Four pts were male, the median age was 45 (range13-72) years, 3 pts were BCR-ABL1 FG positive, 1 pts was AML1-ABL1 FG positive. Before treatment,3 pts presented with 5.5%-35.5% leukemic blasts in bone marrow（BM） and only 1 pts was MRD positive by MFC.And all pts's leukemic cell showed CD19 and CD22 expression confirmed by MFC.2 pts did not undergo HSCT: both them have T315I mutation occurred during disease progression. Pts 1 was 72 years and relapsed after chemotherapy combined with TKI( include ponatinib);Pts 2 was 61 years and relapsed after chemotherapy, TKI( include ponatinib), venetoclax ,BITE and CD19-CART CD22-CART treatments. 2 pts were eventually treated with Olverembatinib combined with INO. and chemotherapy and venetoclax. The other two pts relapsed after HSCT: neither of them had T315I mutation.Pts 3 was 29 years and had recurrence after chemotherapyTKIvenetoclax HSCT and three times CART. Pts 4 was 13 years and had reccurence after chemotherapyHSCTthree times CART and TKI( include ponatinib)venetoclax DLI. 2 pts were eventually treated with Olverembatinib combined with BITE and venetoclax.All pts survived safely and achieved CR:3 pts CMR,1 pts MRDneg by MFC and BCR::ABL1:0.0023%. Pts.2 bridged HSCT;pts.3 and 4 continued oral Olverembatinib combined with venetoclax. The longest follow-up time for 3 pts was one year nearly until today. Unfortunately, pts 1 died due to a secondary fungal infection post-COVID-19 and recurrence of ALL. Conclusions:For heavily treated RR ABL1+B ALL pts,including those after treatment with ponatinib and other TKICART and HSCT, Olverembatinib combined with monoclonal antibodies is effective and safe. In my cases, it is particularly noteworthy that one pts ,Ph like ALL with ABL1 FG, received Olverembatinib combination BITE and achieved a surprising response, so Olverembatinib may also be effective and safe for such pts. In addition, venetoclax may be used as a sensitizer for TKI or other antitumor drugs. Of course, the above needs more clinical practice to verify.

Final Report: Somatic Mutations and HMGCLL1 Haplotype Are Not Associated with Molecular Relapse-Free Survival in Patients with Chronic Myeloid Leukemia Who Attempt Treatment-Free Remission

Background Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder resulting from the BCR::ABL1 rearrangement. Patients with CML have a comparable life expectancy to that of age-matched individuals in the general population when they achieve an optimal response with tyrosine kinase inhibitor (TKI) therapy. With the aim of improving the quality of life of patients who are significantly affected by drug-related adverse events and financial burden, the practical goal of CML treatment has shifted to treatment-free remission (TFR), defined as the successful discontinuation (DISC) of TKI therapy without losing leukemia control. TKI treatment DISC is a safe procedure but requires appropriate candidate selection and frequent molecular monitoring. Several biomarkers were investigated, but no definite biomarker has yet been successfully identified and validated. We have investigated somatic mutations before TKI discontinuation and HMGCLL1 haplotype as a potential biomarker for TFR, given that they were previously described as predictors of CML outcomes. Patients and methods A total of 155 patients who attempted TKI discontinuation for TFR from 5 countries were included: Canada (n=71), Brazil (n=30), Argentina (n=28), Italy (n=23), and the Czech Republic (n=3). We applied our in-house single molecule-molecular inversion probe (smMIP) panel which is a barcoded error-corrected DNA-based sequencing with a limit of detection up to 0.2%. The panel encompasses 40 genes with 332 amplicon probes: epigenetic modifiers (n=7), activation signaling (n=12), myeloid transcription factor (TF; n=5), spliceosome (n=3), tumor suppressor (n=3), cohesion (n=4), miscellaneous (n=3) and 8 HMGCLL1 single nucleotide polymorphisms (SNPs). Two groups were constructed based on these eight SNPs: group 1: CTCAGGCA (C- haplotype) or group 2: ACGTAATG (A-haplotype). Samples were collected prior to TKI DISC when patients met the criteria for TFR attempt. Molecular relapse-free survival (mRFS) was calculated from the date of TKI DISC to the date of confirmed loss of deep molecular response (DMR). Loss of DMR includes single-time loss of major molecular response (MMR) or two consecutive episodes of loss of molecular response 4 log or deeper response (MR4). Kaplan-Meier survival estimate was used to evaluate mRFS and Cox's proportional hazard regression model was applied. Results The median MR4 duration prior to TKI DISC was 7.2 years (range 4.7-9.3), while overall TKI treatment duration was 8.5 years (range 6.0-11.6). With a median follow-up of 831 days (range: 460-1,415), out of the 155 patients, 68 (43.9%) lost response, among whom 6 lost MMR only, 12 lost MR4 only, and 50 lost MMR and MR4, both. Median time to MMR loss was 95 days (range 83.5-137). A total of 71 mutations were detected in 51 patients (32.9%). TET2 mutation was the most frequent mutation, detected in 17 cases (24%) followed by ASXL1 (n=14; 20%) and DNMT3A (n=12; 17%). When grouped by relevant biological pathways involved, mutations in epigenetic genes (n=42, 61%) were the most frequently involved. mRFS rate was 64% (95% CI [55.8-71.1%]) and 56% [48.2-64.0%] at 6 and 12 months, respectively. During the univariate analysis, no significant difference was observed in mRFS according to the presence, number, or type of somatic mutations, or the HMGCLL1 haplotype. Additionally, there were no significant differences in mRFS based on age, sex, Sokal risk group, line of TKI therapy, or TKI drug type prior to TKI DISC. However, MR4 duration prior to TKI DISC was associated with mRFS rate: patients with≥7.3 years (n=71) showed higher mRFS: 74.09% [62.05-82.82%] compared to those <7.3 years (n=74) with mRFS rate of 44.36% [32.54 -55.51] (p <0.01, HR 0.36 [0.21-0.63]). In addition, a longer duration of TKI treatment before TKI DISC was associated with higher mRFS: patients with ≥ 8.3 years of treatment (n=85) showed 71.8% mRFS [60.4-80.4], compared to 42.5% mRFS [30.5-54.0%] in those with <8.3 years (n=71) (p<0.01, HR 0.48 [0.31-0.74]). Multivariate analyses for mRFS confirmed that MR4 duration and the total duration of TKI treatment before TKI DISC were associated with TFR outcomes, but not the presence of epigenetic mutations, any mutation, or HMGCLL1 haplotype (Table1). Conclusion In CML, somatic mutations before attempting TFR are frequently observed. However, our current study does not support their prognostic significance as TFR-predictive biomarkers.

MicroRNA based combinatorial therapy against TKIs resistant CML by inactivating the PI3K/Akt/mTOR pathway: a review.

Chronic myeloid leukemia (CML) is characterized by presence of Philadelphia chromosome, which harbors BCR-ABL oncogene responsible for encoding BCR-ABL oncoprotein. This oncoprotein interferes with cellular signaling pathways, resulting in tumor progression. Among these pathways, PI3K/Akt/mTOR pathway is significantly upregulated in CML. Tyrosine kinase inhibitors (TKIs) are current standard therapy for CML, and they have shown remarkable efficacy. However, emergence of TKIs drug resistance has necessitated investigation of novel therapeutic approaches. Components of PI3K/Akt/mTOR pathway have emerged as attractive targets in this context, as this pathway is known to be activated in TKIs-resistant CML cells/patients. Inhibiting this pathway may provide a complementary approach to improving TKIs' efficacy and treatment outcomes. Given previous research indicating that miRNAs play an inhibitory role in cancer, current study used computational tools to identify miRNAs that specifically target pathway's core components. A comprehensive analysis was performed, resulting in identification of 111 miRNAs that potentially target PI3K/Akt/mTOR pathway. From this extensive list, 7 miRNAs was selected for further investigation based on their consistent downregulation across leukemia subtypes. Except for hsa-miR-199a-3p, remaining six miRNAs have been extensively studied in acute myeloid leukemia (AML). Given high similarity between AML and CML, it is believed that six miRNAs which are not studied in context of CML may also be advantageous for curing chemoresistance in CML. Building upon this knowledge, it is reasonable to speculate that a combination therapy approach involving use of miRNAs alongside TKIs may offer improved therapy for TKIs-resistant CML compared to TKIs monotherapy alone.

Final report of TKI discontinuation trial with dasatinib for the second attempt of treatment-free remission after failing the first attempt with imatinib: Treatment-free Remission Accomplished by Dasatinib (TRAD) study.

Multiple studies have reported a significant treatment-free remission (TFR) rate of 50%-60% in patients with chronic myeloid leukaemia (CML) who discontinue tyrosine kinase inhibitor (TKI) therapy. However, the remaining half of these patients still require re-initiation of TKI therapy for leukaemia control. It remains unclear if TKI drugs should be switched for re-therapy in patients who failed the first TFR (TFR1) attempt. Our study attempted to determine whether dasatinib therapy after TFR1 failure post-imatinib discontinuation could improve the likelihood of TFR2. Of 59 patients who lost molecular response after imatinib discontinuation for TFR1, 55 patients (93.2%) were treated with dasatinib, of whom 49 (89.1%) regained MR4.5 or deeper response, with a median time of 1.85 months to achieve MR4.5. Dasatinib was discontinued in 35 patients for TFR2 attempt, of whom 26 patients (74.28%) lost MMR and 6 (17.14%) MR4. Risk factor analysis for the TFR2 after dasatinib discontinuation suggested three significant factors: (1) doubling time of BCR::ABL1 transcript following TFR1 attempt, (2) rapid regaining of molecular response following dasatinib therapy and (3) undetectable BCR::ABL1 transcript prior to TFR2 attempt. The present study showed that dasatinib does not increase the TFR2 rate in general, but a selected group of patients could benefit from this approach.

Development of Novel Micellar-Enhanced High-Throughput Microwell Spectrofluorimetric Method for Quantification of Lorlatinib: Application to In Vitro Drug Release and Analysis of Urine Samples.

Lorlatinib (LOR) is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor drug. The Food and Drug Administration (FDA) has granted an approval for the use of LOR as a first therapeutic intervention for individuals diagnosed with ALK-positive metastatic and advanced non-small-cell lung cancer (NSCLC). The present study outlines, for the first time, the development and validation of an innovative microwell-based spectrofluorimetric (MW-SFL) method for the quantification of LOR. The proposed method involved the enhancement of the weak native fluorescence of LOR by its micellization into the sodium lauryl sulfate (SLS) micelles. The procedures of the method were conducted in white opaque plates with 96 microwells, and the enhanced fluorescence signals were measured by a fluorescence plate reader at 405 nm after excitation at 310 nm. The measured relative fluorescence intensity (RFI) had a linear relationship with LOR concentrations in the range of 60-1600 ng mL-1. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 19 and 56 ng mL-1, respectively. The method's accuracy and precision were assessed using a recovery study; the recovery values ranged from 99.98% to 101.40%, accompanied by relative standard deviation (RSD) values of 0.42% to 1.59%. The proposed MW-SFL method combined the advantages of the intrinsically high sensitivity of the spectrofluorimetric measurement and the excellent throughput of the microwell-based approach. The results proved the method is effective in the determination of LOR in its pharmaceutical tablets, tablet dissolution testing, as well as in spiked urine with a high degree of precision and accuracy. The MW-SFL method is notable for its simple procedures and utilization of water as a solvent, as well as minimal quantities of sample solutions. These features align with its ecofriendly approach to green chemistry principles. These advantages gave the proposed MW-SFL method a high potential value for the determination of LOR in clinical and quality control laboratories.

Hematological Adverse Events with Tyrosine Kinase Inhibitors for Chronic Myeloid Leukemia: A Systematic Review with Meta-Analysis.

Chronic myeloid leukemia (CML) is treated with tyrosine kinase inhibitors (TKI) that target the pathological BCR-ABL1 fusion oncogene. The objective of this statistical meta-analysis was to assess the prevalence of other hematological adverse events (AEs) that occur during or after predominantly first-line treatment with TKIs. Data from seventy peer-reviewed, published studies were included in the analysis. Hematological AEs were assessed as a function of TKI drug type (dasatinib, imatinib, bosutinib, nilotinib) and CML phase (chronic, accelerated, blast). AE prevalence aggregated across all severities and phases was significantly different between each TKI (p < 0.05) for anemia-dasatinib (54.5%), bosutinib (44.0%), imatinib (32.8%), nilotinib (11.2%); neutropenia-dasatinib (51.2%), imatinib (29.8%), bosutinib (14.1%), nilotinib (14.1%); thrombocytopenia-dasatinib (62.2%), imatinib (30.4%), bosutinib (35.3%), nilotinib (22.3%). AE prevalence aggregated across all severities and TKIs was significantly (p < 0.05) different between CML phases for anemia-chronic (28.4%), accelerated (66.9%), blast (55.8%); neutropenia-chronic (26.7%), accelerated (63.8%), blast (36.4%); thrombocytopenia-chronic (33.3%), accelerated (65.6%), blast (37.9%). An odds ratio (OR) with 95% confidence interval was used to compare hematological AE prevalence of each TKI compared to the most common first-line TKI therapy, imatinib. For anemia, dasatinib OR = 1.65, [1.51, 1.83]; bosutinib OR = 1.34, [1.16, 1.54]; nilotinib OR = 0.34, [0.30, 0.39]. For neutropenia, dasatinib OR = 1.72, [1.53, 1.92]; bosutinib OR = 0.47, [0.38, 0.58]; nilotinib OR = 0.47, [0.42, 0.54]. For thrombocytopenia, dasatinib OR = 2.04, [1.82, 2.30]; bosutinib OR = 1.16, [0.97, 1.39]; nilotinib OR = 0.73, [0.65, 0.82]. Nilotinib had the greatest fraction of severe (grade 3/4) hematological AEs (30%). In conclusion, the overall prevalence of hematological AEs by TKI type was: dasatinib > bosutinib > imatinib > nilotinib. Study limitations include inability to normalize for dosage and treatment duration.

Long-circulating thiolated chitosan nanoparticles of nintedanib with N-acetyl cysteine for treating idiopathic pulmonary fibrosis: In vitro assessment of cytotoxicity, antioxidant, and antifibrotic potential

Nintedanib (NIN) is one of the FDA-approved tyrosine kinase inhibitor drugs used to treat idiopathic pulmonary fibrosis (IPF). This study aimed to formulate a long-circulating injection of Nintedanib to treat bedridden patients with IPF. Nintedanib was incorporated into chitosan nanoparticles (NIN-NP) via the ionic gelation method, and N-acetyl cysteine (NAC), a known antioxidant and mucolytic agent, was added to the NIN-NP (NAC-NIN-NP). The lyophilized formulation had a particle size of 174 nm, a polydispersity index of 0.511, and a zeta potential of 18.6 mV. The spherical nanoparticles were observed in transmission electron microscopy, whereas field emission scanning electron microscopy showed irregular clusters of NP. The thiolation of the chitosan in NAC-NIN-NP was confirmed by ATR-FTIR and NMR, which improved drug release profiles showing >90 % drug release that was 2.42-folds greater than NIN-NP lasting for five days. The DPPH assay showed that adding NAC increased the % inhibition of oxidation in blank-NP (from 54.59 % to 87.17 %) and NIN-NP (58.65 %–89.19 %). The MTT assay on A549 cells showed 67.57 % cell viability by NAC-NIN-NP with an IC50 value of 28 μg/mL. The NAC formulation reduced hydroxyproline content (56.77 μg/mL) compared to NIN-NP (69.48 μg/mL) in WI-38 cell lines. Meanwhile, the healthy cells count with NAC-NIN-NP was higher (5.104 × 103) than with NIN-NP (4.878 × 103). In Hoechst staining, no significant damage to DNA was observed by the drug or formulation. Therefore, NAC-NIN-NP could be a promising treatment option for IPF patients and can be studied further clinically.

A Single-center, Real-world Experience of Chronic GVHD Treatment Using Ibrutinib, Imatinib, and Ruxolitinib and its Treatment Outcomes.

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Tyrosine kinase inhibitors (TKIs), including ruxolitinib, imatinib, and ibrutinib, have shown promising efficacy in cGVHD treatment. A total of 43 patients who developed cGVHD and received at least one line of TKI therapy for cGVHD treatment were evaluated retrospectively. The overall response, clinical benefit (CB), corticosteroid dose reduction, failure-free survival (FFS), and overall survival (OS) were assessed. A total of 62 lines of TKI therapy were evaluated, including ruxolitinib (n = 18), ibrutinib (n = 13), and imatinib (n = 31). With a 12-month median follow-up duration, 19/58 (32.8%), 20/41 (48.7%), and 17/29 (58.6%) responded to TKI therapy at 3, 6, and 12 months, respectively. The CB was observed in 80% of patients over time, allowing prednisone dose reduction in all 3 TKIs. The FFS rate at 12 months was higher in the imatinib (71%) and ruxolitinib groups (67%) than in the ibrutinib group (46%), while the OS rate at 12 months was similar among the three groups at 96%-100% in patients. In the sclerotic GVHD patient subgroup (n = 39), the overall response rate gradually increased over time. Ruxolitinib appeared to be as effective as imatinib and gradually improved the photographic range of motion score in sclerotic GVHD patients. TKI drugs ruxolitinib, imatinib, and Ibrutinib are effective and feasible for cGVHD treatment. Ruxolitinib is as effective as imatinib for sclerotic GVHD.

Synthesis, Docking Study, and Cytotoxicity Evaluation of New Hydroxy benzoic Acid Derivatives

Hydroxy benzoic acid derivatives are an important scaffold in medicinal chemistry. A review of the literature revealed that they have a wide range of biological activities, including anti-inflammatory, antibacterial, and anti-tumor properties. The current study aimed to synthesize three new hydroxy benzoic acid derivatives (compounds 8, 9, and 10) and test them as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in silico and in vitro. Traditional organic synthesis methods were applied to produce these compounds. Docking studies revealed that compound 8 had nearly equal binding energy (DG) to erlotinib, the standard EGFR tyrosine kinase inhibitor. -8.56 and -8.75 kcal/mol, respectively. Using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) viability assay, the cytotoxicity of the new hydroxy benzoic acid derivatives was tested against DLD-1 colorectal adenocarcinoma cells, HeLa cervical cancer cells, and MCF-7 breast cancer cells. Compound 8 showed cytotoxicity against the three cancer cell lines tested (25.05, 23.88, and 48.36 µM, respectively), while compound 9 showed cytotoxicity against HeLa cells only (37.67 µM), and compound 10 was cytotoxic to DLD-1 and HeLa cells (27.26 and 19.19 µM, respectively. The half maximal inhibitory concentrations (IC50) of compound 8 were comparable to those of the standard tyrosine kinase inhibitor drug erlotinib (13.86, 36.41, and 87.34 µM, respectively). In conclusion, the findings of the present study indicate that compound 8, through its EGFR inhibitory activity, is a promising cytotoxic agent.

Phase Ib/IIa study assessing the safety and efficacy of AL8326 monotherapy in patients with ≥3rd line small cell lung cancer (SCLC) treatment.

8585 Background: AL8326 is a novel, orally administered, small molecule tyrosine kinase inhibitor (TKI). The primary objective of this Phase Ib/IIa study is to evaluate the safety and efficacy of AL8326 monotherapy in patients with ≥3rd line small cell lung cancer (SCLC) treatment. Methods: Patients with a diagnosis of small cell lung cancer either limited or extended stage requiring third or further line treatment were eligible for enrollment. The regimen was a 28-day cycle with oral AL8326 administered at 60 mg once per day via RECISIT1.1 evaluation until disease progression (PD), or intolerable toxicity or any other reason discontinuations. Results: At the cut-off date Nov 30, 2022, total n = 30 patients were enrolled. The objective response rate (ORR) was 20% (6/30) (4 confirmed). The disease control rate (DCR) was 56.7% (17/30). Median progression-free survival (PFS) was 3.65 months (95% CI: 1.9 – 5.5) and median overall survival (OS) was 10.42 months (95% CI: 5.4, NE). The median duration of response (DOR) was 5.6months. All 30 subjects had reported AE at least once. Common treatment emergent adverse events (TEAE) were similar to other TKI drug and included (incidence ≥20%): thyroid stimulating hormone (TSH) increase (46.67%), proteinuria (43.33%), weight loss (40.00%), hypertension (36.67%), thrombocytopenia (33.33%), diarrhea (33.33%), positive fecal occult blood (30.00%), hypercholesterolemia (26.67%), hypertriglyceridemia (26.67%), hand- foot syndrome (HFS) (20.00%), loss of appetite (20.00%). Most of these AEs were grade 1-2 (CTCAE V5.0). Conclusions: AL8326 has demonstrated acceptable tolerability and positive efficacy on small cell lung cancer treatment. A US Phase 2 (NCT05363280) study is ongoing and a phase 3 study is in preparation. Clinical trial information: NCT04890795 .