Effect of tyrosine kinase inhibitors on male fertility in patients with chronic phase chronic myeloid leukemia.

Advancements in the management of patients with chronic myeloid leukemia (CML) allowed them to achieve survival comparable with their healthy counterparts. Consequently, their care has widened with growing focus on quality of life, including parenting children. Although tyrosine kinase inhibitors (TKI) are contraindicated in pregnancy given their teratogenic effect, their effect on male fertility is less clear with contradictory results from animal studies and case reports/series. We compared the sperm analysis parameters, as the gold-standard assessment for male fertility, of 11 patients with CP- CML before and after TKI therapy. Median therapy duration was 5.1 years (range: 2.5-16.5). The sperm concentration, % progressive, and total motility before and after therapy were not significantly different (p = 0.376, 0.569, and 0.595, respectively). Our results suggest no impairment in fertility potential in male patients after TKI therapy. A larger sample size is crucial to support/refute our findings.

Treatment-free remission in CML patients with additional chromosome abnormalities in the Philadelphia-positive clone or variant Philadelphia translocations.

Probability of treatment-free remission (TFR) in CML patients with additional chromosomal abnormalities (ACA) in the Philadelphia-positive clone or variant Philadelphia translocations (ACA/Var-Ph group, blue panel), in those with no cytogenetic abnormality other than the classical Philadelphia translocation (c-Ph group, green panel) and in the subgroups of CML patients with high-risk ACA (HR-ACA, yellow panel) and Var-Ph (red panel).

Long-term outcomes after upfront second-generation tyrosine kinase inhibitors for chronic myeloid leukemia: managing intolerance and resistance.

Second-generation tyrosine kinase inhibitors (2GTKI) are more effective in inducing rapid molecular responses than imatinib when used first-line in patients with chronic myeloid leukemia in chronic phase (CML-CP). However, failure of first line-2GTKI (1L-2GTKI) still occurs and there is no consensus regarding subsequent management. We retrospectively analyzed the outcome of 106 CML-CP patients treated with 1L-2GTKI and with a median follow-up of 91 months. 45 patients (42.4%) switched to an alternative TKI, 28 for intolerance (26.4%) and 17 (16%) for resistance. Most patients who remained on 1L-2GTKI achieved deep molecular responses (DMR) and 15 (14.1%) are in treatment-free remission (TFR). Intolerant patients also obtained DMR, although most required multiple TKI changes and were slower to respond, particularly if treated with 2L-imatinib. Inferior outcomes were observed in resistant patients, who failed alternative 2L-2GTKI and required 3/4GTKI and/or allogeneic hematopoietic stem cell transplant (alloSCT). 7yr-OS was significantly lower for these individuals (66.1%) than for intolerant patients and those who remained on 1L-2GTKI (100% and 97.9%, respectively; p = 0.001). It is apparent that failure of 1L-2GTKI is a challenging problem in modern CML therapy. Intolerance can be effectively managed by switching to an alternative 2GTKI, but resistance requires early consideration of 3/4GTKI.

FISH-negative BCR::ABL1-positive e19a2 chronic myeloid leukaemia: the most cryptic of insertions.

BACKGROUND: Chronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 ('Philadelphia' or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these 'Philadelphia chromosome negative' patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR. CASE PRESENTATION: A 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient's BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR. CONCLUSIONS: We present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.

Third primary SARS-CoV-2 mRNA vaccines enhance antibody responses in most patients with haematological malignancies.

SARS-CoV-2 infection, and resulting disease, COVID-19, has a high mortality amongst patients with haematological malignancies. Global vaccine rollouts have reduced hospitalisations and deaths, but vaccine efficacy in patients with haematological malignancies is known to be reduced. The UK-strategy offered a third, mRNA-based, vaccine as an extension to the primary course in these patients. The MARCH database is a retrospective observational study of serological responses in patients with blood disorders. Here we present data on 381 patients with haematological malignancies. By comparison with healthy controls, we report suboptimal responses following two primary vaccines, with significantly enhanced responses following the third primary dose. These responses however are heterogeneous and determined by haematological malignancy sub-type and therapy. We identify a group of patients with continued suboptimal vaccine responses who may benefit from additional doses, prophylactic extended half-life neutralising monoclonal therapies (nMAB) or prompt nMAB treatment in the event of SARS-CoV-2 infection.

A retrospective observational research study to describe the real-world use of bosutinib in patients with chronic myeloid leukemia in the United Kingdom and the Netherlands.

OBJECTIVES: To describe the real-world effectiveness and safety of bosutinib in patients with chronic myeloid leukemia (CML). METHODS: This was a multi-center, retrospective, non-interventional chart review study conducted in 10 hospitals in the United Kingdom and the Netherlands. RESULTS: Eighty-seven patients were included. Bosutinib was the third-line tyrosine kinase inhibitor (TKI) in 33 (38%) and fourth-line in 44 (51%) patients. Median treatment duration was 15.6 months. Among 84 patients in chronic phase (CP) at baseline, 26 (31%) switched to bosutinib due to resistance and 57 (68%) due to intolerance to prior TKIs. Cumulative complete cytogenetic and major molecular response rates in CP patients were 67% and 55%, respectively. After a median follow-up of 21.5 months, nine (11%) patients in CP died; estimated overall survival rates at 1 and 2 years postbosutinib initiation were 95% and 91%, respectively. Overall, 33/87 (38%) patients discontinued bosutinib due to either lack of efficacy/disease progression (17%), adverse events (14%), death (2%), or other reasons (5%). Eighty-two (94%) patients experienced ≥1 adverse event possibly related to bosutinib, most commonly diarrhea (52%). CONCLUSIONS: Bosutinib used in routine clinical practice in heavily pretreated patients with CML is an effective treatment for patients in CP and is generally tolerable.

Assessment of quantitative polymerase chain reaction for BCR-ABL1 transcripts in chronic myeloid leukaemia: Are improved outcomes in patients with e14a2 transcripts an artefact of technology?

The clinical outcome of chronic myeloid leukaemia patients has vastly improved since the introduction of tyrosine kinase inhibitor treatment, with a significant proportion of patients able to achieve treatment-free remission. However, studies have shown that patients with the e13a2 transcript were less likely to achieve major molecular response compared to those with e14a2 transcripts. Most quantitative polymerase chain reaction (PCR) assays for detection of the BCR-ABL1 fusion gene do not differentiate between the two transcripts and we therefore hypothesised that technical bias linked to the qPCR assay could partially explain the discrepancy in outcomes. We designed an e14a2-specific assay and identified no difference in results compared to an e13a2 standard assay. We then demonstrated that the commercial e14a2 standards were causing a significant overestimation of the e13a2 transcripts. Finally, we reviewed patient management after the qPCR values were corrected, using our new evaluation. We concluded that despite statistically significant differences in qPCR results, there was no impact on patient management or outcome. We conclude that, at least in our institution, it would be inappropriate to perform separate assays for patients with e13a2 or e14a2.

Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis.

As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

Identification of genetic targets in acute myeloid leukaemia for designing targeted therapy.

Few effective therapies exist for acute myeloid leukaemia (AML), in part due to the molecular heterogeneity of this disease. We sought to identify genes crucial to deregulated AML signal transduction pathways which, if inhibited, could effectively eradicate leukaemia stem cells. Due to difficulties in screening primary cells, most previous studies have performed next-generation sequencing (NGS) library knockdown screens in cell lines. Using carefully considered methods including evaluation at multiple timepoints to ensure equitable gene knockdown, we employed a large NGS short hairpin RNA (shRNA) knockdown screen of nearly 5 000 genes in primary AML cells from six patients to identify genes that are crucial for leukaemic survival. Across various levels of stringency, genome-wide bioinformatic analysis identified a gene in the NOX family, NOX1, to have the most consistent knockdown effectiveness in primary cells (P = 5∙39 × 10-5 , Bonferroni-adjusted), impacting leukaemia cell survival as the top-ranked gene for two of the six AML patients and also showing high effectiveness in three of the other four patients. Further investigation of this pathway highlighted NOX2 as the member of the NOX family with clear knockdown efficacy. We conclude that genes in the NOX family are enticing candidates for therapeutic development in AML.

TKI dose reduction can effectively maintain major molecular remission in patients with chronic myeloid leukaemia.

Targeted therapy for chronic myeloid leukaemia (CML) has allowed for a near-normal patient life-expectancy; however, quality of life and aggravation of existing co-morbidities have posed new treatment challenges. In clinical practice, TKI dose reduction occurs frequently, often on multiple occasions, because of intolerance. We conducted a retrospective 'real-world practice' review of 246 patients receiving lower than standard dose (LD) TKI after the achievement of major molecular response (MR3), because of intolerable adverse events. In 274 of 298 cases of dose reduction (91·9%), MR3 was maintained at median follow-up of 27·3 months. One patient progressed to blast crisis while on LD TKI. Two patients developed two new ABL kinase domain mutations (T315I and V299L), of whom one had achieved deep molecular response on an alternative LD TKI at last follow-up. Seventy-six patients eventually discontinued LD TKI and the two-year treatment-free remission (TFR) rate in these patients was 74·1%. The majority of patients with CML in at least MR3 appear to be safely managed with LD TKI, although three of 246 patients had new events (progression and new mutation), indicating that this approach requires vigilance. TKI LD does not prevent the achievement of TFR in this patient population.

Somatic variants in epigenetic modifiers can predict failure of response to imatinib but not to second-generation tyrosine kinase inhibitors.

There are no validated molecular biomarkers to identify newly-diagnosed individuals with chronic-phase chronic myeloid leukemia likely to respond poorly to imatinib and who might benefit from first-line treatment with a more potent second-generation tyrosine kinase inhibitor. Our inability to predict these 'high-risk' individuals reflects the poorly understood heterogeneity of the disease. To investigate the potential of genetic variants in epigenetic modifiers as biomarkers at diagnosis, we used Ion Torrent next-generation sequencing of 71 candidate genes for predicting response to tyrosine kinase inhibitors and probability of disease progression. A total of 124 subjects with newly-diagnosed chronic-phase chronic myeloid leukemia began with imatinib (n=62) or second-generation tyrosine kinase inhibitors (n=62) and were classified as responders or non-responders based on the BCRABL1 transcript levels within the first year and the European LeukemiaNet criteria for failure. Somatic variants affecting 21 genes (e.g. ASXL1, IKZF1, DNMT3A, CREBBP) were detected in 30% of subjects, most of whom were non-responders (41% non-responders, 18% responders to imatinib, 38% non-responders, 25% responders to second-generation tyrosine kinase inhibitors). The presence of variants predicted the rate of achieving a major molecular response, event-free survival, progression-free survival and chronic myeloid leukemia-related survival in the imatinib but not the second-generation tyrosine kinase inhibitors cohort. Rare germline variants had no prognostic significance irrespective of treatment while some pre-leukemia variants suggest a multi-step development of chronic myeloid leukemia. Our data suggest that identification of somatic variants at diagnosis facilitates stratification into imatinib responders/non-responders, thereby allowing earlier use of second-generation tyrosine kinase inhibitors, which, in turn, may overcome the negative impact of such variants on disease progression.