Intention-to-treat outcomes utilising a stringent event definition in children and young people treated with tisagenlecleucel for r/r ALL through a national access scheme.

CAR T-cell therapy has transformed relapsed/refractory (r/r) B-cell precursor acute lymphoblastic leukaemia (B-ALL) management and outcomes, but following CAR T infusion, interventions are often needed. In a UK multicentre study, we retrospectively evaluated tisagenlecleucel outcomes in all eligible patients, analysing overall survival (OS) and event-free survival (EFS) with standard and stringent definitions, the latter including measurable residual disease (MRD) emergence and further anti-leukaemic therapy. Both intention-to-treat and infused cohorts were considered. We collected data on feasibility of delivery, manufacture, toxicity, cause of therapy failure and followed patients until death from any cause. Of 142 eligible patients, 125 received tisagenlecleucel, 115/125 (92%) achieved complete remission (CR/CRi). Severe cytokine release syndrome and neurotoxicity occurred in 16/123 (13%) and 10/123 (8.1%), procedural mortality was 3/126 (2.4%). The 2-year intent to treat OS and EFS were 65.2% (95%CI 57.2-74.2%) and 46.5% (95%CI 37.6-57.6%), 2-year intent to treat stringent EFS was 35.6% (95%CI 28.1-44.9%). Median OS was not reached. Sixty-two responding patients experienced CAR T failure by the stringent event definition. Post failure, 1-year OS and standard EFS were 61.2% (95%CI 49.3-75.8) and 55.3% (95%CI 43.6-70.2). Investigation of CAR T-cell therapy for B-ALL delivered on a country-wide basis, including following patients beyond therapy failure, provides clinicians with robust outcome measures. Previously, outcomes post CAR T-cell therapy failure were under-reported. Our data show that patients can be successfully salvaged in this context with good short-term survival.

Associations of granulocyte colony-stimulating factor with toxicities and efficacy of chimeric antigen receptor T-cell therapy in relapsed or refractory B-cell acute lymphoblastic leukemia.

Few studies have reported the associations of granulocyte colony-stimulating factor (G-CSF) with cytokine release syndrome (CRS), neurotoxic events (NEs) and efficacy after chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We present a retrospective study of 67 patients with R/R B-ALL who received anti-CD19 CAR T-cell therapy, 41 (61.2%) patients received G-CSF (G-CSF group), while 26 (38.8%) did not (non-G-CSF group). Patients had similar duration of grade 3-4 neutropenia between the two groups. The incidences of CRS and NEs were higher in G-CSF group, while no differences in severity were found. Further stratified analysis showed that the incidence and severity of CRS were not associated with G-CSF administration in patients with low bone marrow (BM) tumor burden. None of the patients with low BM tumor burden developed NEs. However, there was a significant increase in the incidence of CRS after G-CSF administration in patients with high BM tumor burden. The duration of CRS in patients who used G-CSF was longer. There were no significant differences in response rates at 1 and 3 months after CAR T-cell infusion, as well as overall survival (OS) between the two groups. In conclusion, our results showed that G-CSF administration was not associated with the incidence or severity of CRS in patients with low BM tumor burden, but the incidence of CRS was higher after G-CSF administration in patients with high BM tumor burden. The duration of CRS was prolonged in G-CSF group. G-CSF administration was not associated with the efficacy of CAR T-cell therapy.

Neuropsychological task outcomes among survivors of childhood acute lymphoblastic leukemia in Malaysia.

This study intended to explore the neuropsychological ramifications in childhood acute lymphoblastic leukemia (ALL) survivors in Malaysia and to examine treatment-related sequelae. A case-control study was conducted over a 2-year period. Seventy-one survivors of childhood ALL who had completed treatment for a minimum of 1 year and were in remission, and 71 healthy volunteers were enlisted. To assess alertness (processing speed) and essential executive functioning skills such as working memory capacity, inhibition, cognitive flexibility, and sustained attention, seven measures from the Amsterdam Neuropsychological Tasks (ANT) program were chosen. Main outcome measures were speed, stability and accuracy of responses. Mean age at diagnosis was 4.50 years (SD ± 2.40) while mean age at study entry was 12.18 years (SD ± 3.14). Survivors of childhood ALL underperformed on 6 out of 7 ANT tasks, indicating poorer sustained attention, working memory capacity, executive visuomotor control, and cognitive flexibility. Duration of treatment, age at diagnosis, gender, and cumulative doses of chemotherapy were not found to correlate with any of the neuropsychological outcome measures. Childhood ALL survivors in our center demonstrated significantly poorer neuropsychological status compared to healthy controls.

Infant Acute Lymphoblastic Leukemia-New Therapeutic Opportunities.

Infant acute lymphoblastic leukemia (Infant ALL) is a kind of pediatric ALL, diagnosed in children under 1 year of age and accounts for less than 5% of pediatric ALL. In the infant ALL group, two subtypes can be distinguished: KMT2A-rearranged ALL, known as a more difficult to cure form and KMT2A- non-rearranged ALL with better survival outcomes. As infants with ALL have lesser treatment outcomes compared to older children, it is pivotal to provide novel treatment approaches. Progress in the development of molecularly targeted therapies and immunotherapy presents exciting opportunities for potential improvement. This comprehensive review synthesizes the current literature on the epidemiology, clinical presentation, molecular genetics, and therapeutic approaches specific to ALL in the infant population.

Different Levels of Therapeutic Strategies to Recover the Microbiome to Prevent/Delay Acute Lymphoblastic Leukemia (ALL) or Arrest Its Progression in Children.

Changes in the components, variety, metabolism, and products of microbiomes, particularly of the gut microbiome (GM), have been revealed to be closely associated with the onset and progression of numerous human illnesses, including hematological neoplasms. Among the latter pathologies, there is acute lymphoblastic leukemia (ALL), the most widespread malignant neoplasm in pediatric subjects. Accordingly, ALL cases present a typical dysfunctional GM during all its clinical stages and resulting inflammation, which contributes to its progression, altered response to therapy, and possible relapses. Children with ALL have GM with characteristic variations in composition, variety, and functions, and such alterations may influence and predict the complications and prognosis of ALL after chemotherapy treatment or stem cell hematopoietic transplants. In addition, growing evidence also reports the ability of GM to influence the formation, growth, and roles of the newborn's hematopoietic system through the process of developmental programming during fetal life as well as its susceptibility to the onset of onco-hematological pathologies, namely ALL. Here, we suggest some therapeutic strategies that can be applied at two levels of intervention to recover the microbiome and consequently prevent/delay ALL or arrest its progression.

Shared genetic architecture between autoimmune disorders and B-cell acute lymphoblastic leukemia: insights from large-scale genome-wide cross-trait analysis.

BACKGROUND: To study the shared genetic structure between autoimmune diseases and B-cell acute lymphoblastic leukemia (B-ALL) and identify the shared risk loci and genes and genetic mechanisms involved. METHODS: Based on large-scale genome-wide association study (GWAS) summary-level data sets, we observed genetic overlaps between autoimmune diseases and B-ALL, and cross-trait pleiotropic analysis was performed to detect shared pleiotropic loci and genes. A series of functional annotation and tissue-specific analysis were performed to determine the influence of pleiotropic genes. The heritability enrichment analysis was used to detect crucial immune cells and tissues. Finally, bidirectional Mendelian randomization (MR) methods were utilized to investigate the casual associations. RESULTS: Our research highlighted shared genetic mechanisms between seven autoimmune disorders and B-ALL. A total of 73 pleiotropic loci were identified at the genome-wide significance level (P < 5 × 10-8), 16 of which had strong evidence of colocalization. We demonstrated that several loci have been previously reported (e.g., 17q21) and discovered some novel loci (e.g., 10p12, 5p13). Further gene-level identified 194 unique pleiotropic genes, for example IKZF1, GATA3, IKZF3, GSDMB, and ORMDL3. Pathway analysis determined the key role of cellular response to cytokine stimulus, B cell activation, and JAK-STAT signaling pathways. SNP-level and gene-level tissue enrichment suggested that crucial role pleiotropic mechanisms involved in the spleen, whole blood, and EBV-transformed lymphocytes. Also, hyprcoloc and stratified LD score regression analyses revealed that B cells at different developmental stages may be involved in mechanisms shared between two different diseases. Finally, two-sample MR analysis determined causal effects of asthma and rheumatoid arthritis on B-ALL. CONCLUSIONS: Our research proved shared genetic architecture between autoimmune disorders and B-ALL and shed light on the potential mechanism that might involve in.

EP300-ZNF384 transactivates IL3RA to promote the progression of B-cell acute lymphoblastic leukemia.

The EP300-ZNF384 fusion gene is an oncogenic driver in B-cell acute lymphoblastic leukemia (B-ALL). In the present study, we demonstrated that EP300-ZNF384 substantially induces the transcription of IL3RA and the expression of IL3Rα (CD123) on B-ALL cell membranes. Interleukin 3 (IL-3) supplementation promotes the proliferation of EP300-ZNF348-positive B-ALL cells by activating STAT5. Conditional knockdown of IL3RA in EP300-ZF384-positive cells inhibited the proliferation in vitro, and induced a significant increase in overall survival of mice, which is attributed to impaired propagation ability of leukemia cells. Mechanistically, the EP300-ZNF384 fusion protein transactivates the promoter activity of IL3RA by binding to an A-rich sequence localized at -222/-234 of IL3RA. Furthermore, forced EP300-ZNF384 expression induces the expression of IL3Rα on cell membranes and the secretion of IL-3 in CD19-positive B precursor cells derived from healthy individuals. Doxorubicin displayed a selective killing of EP300-ZNF384-positive B-ALL cells in vitro and in vivo. Collectively, we identify IL3RA as a direct downstream target of EP300-ZNF384, suggesting CD123 is a potent biomarker for EP300-ZNF384-driven B-ALL. Targeting CD123 may be a novel therapeutic approach to EP300-ZNF384-positive patients, alternative or, more likely, complementary to standard chemotherapy regimen in clinical setting.

Aggressive precursor B cell ALL of cervix with obstructive uropathy.

Involvement of the cervix with acute lymphoblastic leukaemia (ALL) is extremely rare. In this case report, we discuss an unmarried woman in her early 20s, who presented in the emergency with lower abdominal pain and irregular vaginal bleeding for 1 month. Clinical examination and imaging revealed a large cervical mass probably neoplastic with obstructive uropathy. On evaluation, she was diagnosed incidentally with CALLA-positive precursor B cell ALL in peripheral blood flow cytometry. Involvement of B cell ALL in cervical mass was confirmed by histopathological examination of cervical biopsy and immunohistochemistry markers. Her history was not suggestive of signs and symptoms pertaining to leukaemia. Literature is sparse with only a few cases reporting cervical leukaemic infiltration. The present case report is a rarest case where the primary/initial presentation of precursor B cell ALL was seen with cervical involvement and obstructive uropathy mimicking characteristics of advanced cervical malignancy.

Terminal deoxynucleotidyl transferase expression in different subtypes of childhood B-cell acute lymphoblastic leukemia.

The lack of expression of terminal deoxynucleotidyl transferase (TdT) is frequently associated with KMT2A-rearranged subtype of pediatric acute lymphoblastic leukemia (ALL). However, this association has not been investigated extensively in the Asian population. A retrospective analysis of TdT expression in pediatric B-cell ALL (B-ALL) was performed in patients treated using the Taiwan Pediatric Oncology Group (TPOG) ALL 2002 and 2013 protocols. Among the 331 patients with B-ALL, 12 patients showed TdT negativity at initial diagnosis. Among these, eight patients showed KMT2A rearrangement (66.7%). Other patients showing negative TdT expression had ETV6::RUNX1, MEF2D-rearranged, and other B-ALL subtypes. However, in the context of KMT2A-rearranged B-ALL (n = 20), only eight patients showed TdT negativity. The 5-year event-free survival and overall survival of patients with and without TdT expression were 83.8% versus 46.8% (P <0.001) and 86.3% versus 55.4% (P = 0.004), respectively. Moreover, several aberrant markers, such as CD2, CD56, CD7, and CD117, were rarely expressed in the B-ALL samples, and if expressed, they were enriched in specific genetic subtypes. The results of this study indicate that immunophenotypic features are correlated with specific genetic subtypes of childhood B-ALL.

Novel PIKfyve/Tubulin Dual-target Inhibitor as a Promising Therapeutic Strategy for B-cell Acute Lymphoblastic Leukemia.

OBJECTIVE: In B-cell acute lymphoblastic leukemia (B-ALL), current intensive chemotherapies for adult patients fail to achieve durable responses in more than 50% of cases, underscoring the urgent need for new therapeutic regimens for this patient population. The present study aimed to determine whether HZX-02-059, a novel dual-target inhibitor targeting both phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) and tubulin, is lethal to B-ALL cells and is a potential therapeutic for B-ALL patients. METHODS: Cell proliferation, vacuolization, apoptosis, cell cycle, and in-vivo tumor growth were evaluated. In addition, Genome-wide RNA-sequencing studies were conducted to elucidate the mechanisms of action underlying the anti-leukemia activity of HZX-02-059 in B-ALL. RESULTS: HZX-02-059 was found to inhibit cell proliferation, induce vacuolization, promote apoptosis, block the cell cycle, and reduce in-vivo tumor growth. Downregulation of the p53 pathway and suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway and the downstream transcription factors c-Myc and NF-κB were responsible for these observations. CONCLUSION: Overall, these findings suggest that HZX-02-059 is a promising agent for the treatment of B-ALL patients resistant to conventional therapies.

The CNS microenvironment promotes leukemia cell survival by disrupting tumor suppression and cell cycle regulation in pediatric T-cell acute lymphoblastic leukemia.

A major obstacle in improving survival in pediatric T-cell acute lymphoblastic leukemia is understanding how to predict and treat leukemia relapse in the CNS. Leukemia cells are capable of infiltrating and residing within the CNS, primarily the leptomeninges, where they interact with the microenvironment and remain sheltered from systemic treatment. These cells can survive in the CNS, by hijacking the microenvironment and disrupting normal functions, thus promoting malignant transformation. While the protective effects of the bone marrow niche have been widely studied, the mechanisms behind leukemia infiltration into the CNS and the role of the CNS niche in leukemia cell survival remain unknown. We identified a dysregulated gene expression profile in CNS infiltrated T-ALL and CNS relapse, promoting cell survival, chemoresistance, and disease progression. Furthermore, we discovered that interactions between leukemia cells and human meningeal cells induced epigenetic alterations, such as changes in histone modifications, including H3K36me3 levels. These findings are a step towards understanding the molecular mechanisms promoting leukemia cell survival in the CNS microenvironment. Our results highlight genetic and epigenetic alterations induced by interactions between leukemia cells and the CNS niche, which could potentially be utilized as biomarkers to predict CNS infiltration and CNS relapse.

Chemo-free treatment of adult patients with Ph-positive acute lymphoblastic leukemia: latest updates from the 2023 ASH annual meeting.

The chemo-free concept represents a new direction for managing adult patients with Ph-positive acute lymphoblastic leukemia (Ph + ALL). The tyrosine kinase inhibitors (TKIs), blinatumomab and venetoclax serve as the backbone of chemo-free regimens; several prospective studies involving these drugs have demonstrated high remission rates and promising, albeit short, survival outcomes. This review summarizes the latest updates on chemo-free regimens in the treatment of adult patients with Ph + ALL, presented at the 2023 ASH annual meeting.

A review of immunotargeted therapy for Philadelphia chromosome positive acute lymphoblastic leukaemia: making progress in chemotherapy-free regimens.

Philadelphia chromosome-positive acute lymphoblastic leukemia (PH + ALL) is the most common cytogenetic abnormality of B-ALL in adults and is associated with poor prognosis. Previously, the only curative treatment option in PH + ALL was allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Since 2000, targeted therapy combined with chemotherapy, represented by the tyrosine kinase inhibitor Imatinib, has become the first-line treatment for PH + ALL. Currently, the remission rate and survival rate of Imatinib are superior to those of simple chemotherapy, and it can also improve the efficacy of transplantation. More recently, some innovative immune-targeted therapy greatly improved the prognosis of PH + ALL, such as Blinatumomab and Inotuzumab Ozogamicin. For patients with ABL1 mutations and those who have relapsed or are refractory to other treatments, targeted oral small molecule drugs, monoclonal antibodies, Bispecific T cell Engagers (BiTE), and chimeric antigen receptor (CAR) T cells immunotherapy are emerging as potential treatment options. These new therapeutic interventions are changing the treatment landscape for PH + ALL. In summary, this review discusses the current advancements in targeted therapeutic agents shift in the treatment strategy of PH + ALL towards using more tolerable chemotherapy-free induction and consolidation regimens confers better disease outcomes and might obviate the need for HSCT.

The RAS-signaling-pathway-mutation-related prognosis in B-cell acute lymphoblastic leukemia: A report from South China children's leukemia group.

The next-generation sequencing technologies application discovers novel genetic alterations frequently in pediatric acute lymphoblastic leukemia (ALL). RAS signaling pathway mutations at the time of relapse ALL frequently appear as small subclones at the time of onset, which are considered as the drivers in ALL relapse. Whether subclones alterations in the RAS signaling pathway should be considered for risk group stratification of ALL treatment is not decided yet. In this work, we investigate the RAS signaling pathway mutation spectrum and the related prognosis in pediatric ALL. We employed an NGS panel comprising 220 genes. NGS results were collected from 202 pediatric ALL patients. 155 patients (76.7%) harbored at least one mutation. The incidences of RAS signaling pathway mutations are different significantly between T-ALL and B-ALL. In B-ALL, the RAS pathway is mostly involved, and NRAS (17.6%), KRAS (22.7%), and PTPN11 (7.7%) were the three most frequently mutated genes. Co-occurring mutations of CREBBP and NRAS, FLT3, or PTPN11 (p = 0.002, p = 0.009, and p = 0.003, respectively) were found in this cohort. The 3-year RFS rates for the RAS signaling pathway mutation-positive and negative cases was 76.5 % versus 89.7 % (p = 0.012). Four cases relapsed in the lately 3 years were RAS signaling pathway mutation-positive. RAS signaling pathway mutation is an important biomarker for poorer relapse-free survival in pediatric B-ALL patients despite good early MRD levels.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

BACKGROUND: Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin ß1 (CD29) or ß4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. AIMS: Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. METHODS AND RESULTS: Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. CONCLUSION: BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Epidemiological characteristics and influencing factors of acute leukemia in children and adolescents and adults: a large population-based study.

OBJECTIVE: To assess the epidemiological characteristics and prognostic factors of acute leukemia (AL) in children and adolescents, and make comparisons between pediatric and adult patients. METHODS: This retrospective cohort study enrolled AL patients from the Surveillance, Epidemiology, and End Results (SEER) 1975-2016. OS in children and adolescents and adults with AL was compared and analyzed separately by age and AL subtype. RESULTS: Totally 61,694 AL patients were identified, with 45,411 (73.6%) adults and 16,283 (26.4%) children and adolescents. From 2000 to 2016, the incidence rates of AL [annual percent changes (APC) = 1.2, 95%CI = 0.9-1.6, P < 0.05] and acute lymphoblastic leukemia (ALL) (APC = 1.5, 95%CI = 1.1-1.8, P < 0.05) in children and adolescents were significantly increasing. For adults, AL (APC = 0.9, 95%CI = 0.3-1.5, P < 0.05), ALL (APC = 2.5, 95%CI = 2.0-3.1, P < 0.05) and acute myeloid leukemia (AML) (APC = 0.9, 95%CI = 0.4-1.5, P < 0.05) had significantly elevated incidence rates. Overall survival (OS) in children and adolescents with AL was significantly higher than that in adults with AL (log-rank P < 0.0001). OS in children and adolescents and adults with ALL, AML and AUL decreased with age (all log-rank P < 0.0001). Older age, male sex and black race were risk factors for the survival of children and adolescents and adults with ALL, AML and AUL. CONCLUSION: From 2000 to 2016, the incidence rates of AL in children and adolescents and adults were increasing. Children and adolescents with AL had significantly better OS than adults with AL, and OS declined with age in both children and adolescents and adults with ALL, AML and AUL.

[Efficacy and safety of chimeric antigen receptor T-cell therapy followed by allogeneic hematopoietic stem cell transplantation in 21 patients with Ph-like acute lymphoblastic leukemia].

Objective: To evaluate the efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with Ph-like acute lymphoblastic leukemia (Ph-ALL) . Methods: Patients with Ph-ALL who underwent CAR-T therapy followed by allo-HSCT from March 2018 to August 2023 at the First Affiliated Hospital of Soochow University were included, and their clinical data were retrospectively analyzed. Results: Of the 21 patients, 14 were male and 7 were female. The median age at the time of CAR-T therapy was 22 (6-50) years. Seven patients had ABL1-like rearrangements, and 14 had JAK-STAT rearrangements. Prior to CAR-T therapy, 12 patients experienced hematologic relapse; 7 were multiparameter flow cytometry minimal residual disease (MFC-MRD) -positive and 2 were MFC-MRD-negative. CAR-T cells were derived from patients' autologous lymphocytes. Nine patients were treated with CD19 CAR-T cells, and 12 were treated with CD19/CD22 CAR-T cells. After assessment on day 28 after CAR-T therapy, 95.2% of the patients achieved complete remission, with an MRD-negative remission rate of 75%. Nineteen patients developed grade 0-2 cytokine release syndrome (CRS) and 2 patients suffered grade 3 CRS, all cases of which resolved after treatment. All patients underwent allo-HSCT after CAR-T therapy. The median time from CAR-T therapy to allo-HSCT was 63 (38-114) days. Five patients experienced relapse after CAR-T therapy, including four with hematologic relapse and one with molecular relapse. The 3-year overall survival (OS) rates in the ABL1 and JAK-STAT groups were (83.3±15.2) % and (66.6±17.2) %, respectively (P=0.68) . The 3-year relapse-free survival (RFS) rates were (50.0±20.4) % and (55.6±15.4) % in the ABL1 and JAK-STAT groups, respectively. There was no significant difference in 3-year OS or RFS between the two groups. Conclusions: CAR-T therapy followed by allo-HSCT leads to rapid remission in most patients with Ph-ALL and prolongs leukemia-free survival.

[Use of the ETV6/RUNX1 probe to verify the performance of the fluorescence in situ hybridization probe before clinical detection].

Objective: To explore the standardized performance of a FISH probe before clinical detection. Methods: The probe sensitivity and specificity of ETV6/RUNX1 were analyzed via interphase and metaphase FISH in 20 discarded healthy bone marrow samples. The threshold system of the probe was established using an inverse beta distribution, and an interpretation standard was established. Finally, a parallel-controlled polymerase chain reaction detection study was conducted on 286 bone marrow samples from patients at our hospital. The clinical sensitivity, specificity, and diagnostic coincidence rate of ETV6/RUNX1 FISH detection were analyzed, and the diagnostic consistency of the two methods was analyzed by the kappa test. Results: The probe sensitivity and specificity of the ETV6/RUNX1 probe were 98.47% and 100%, respectively. When 50, 100, and 200 cells were counted, the typical positive signal pattern cutoffs were 5.81%, 2.95%, and 1.49%, respectively, and the atypical positive signal pattern cutoffs were 13.98%, 9.75%, and 6.26%, respectively. The clinical sensitivity of FISH was 96.1%, clinical specificity was 99.6%, diagnostic coincidence rate was 99.00%, diagnostic consistency test kappa value was 0.964, and P value was <0.001. Conclusion: For FISH probes without a national medical device registration certificate, standardized performance verification and methodology performance verification can be performed using laboratory developed test verification standards to ensure a reliable and accurate reference basis for clinical diagnosis and treatment.