This study capitalizes on one of the largest clinical trials for childhood ALL reported to date [22] to comprehensively dissect the prognostic implications of CD9. We unveiled CD9’s lineage-specific expression pattern and its survival impact on childhood ALL. This protein was expressed in only 27.2% of T-ALL but significantly in 88.5% of B-ALL cases, likely due to its inherent disparities within the developmental hierarchy of blood lineages [27]. An intriguing observation was that CD9 status affected B-ALL outcome while sparing T-ALL, indicating its context-dependent implications for cancer progression. This could be potentially driven by the hematopoietic lineage-specific difference in the composition of CD9 binding partners within the tetraspanin web, a well-recognized phenomenon in solid cancers to determine its oncogenic versus tumor suppressive role that deserves further biological studies in leukemias [12, 13].

In B-ALL, CD9+ patients experienced inferior EFS primarily due to increased relapse rates, findings consistently replicated in this extensive patient cohort. The less favorable prognosis of CD9+ patients seems linked to the interplay of pre-treatment risk factors. Specifically, CD9+ cases had higher WBC at presentation, a recognized unfavorable factor. CD9 was also associated with cytogenetics. For the two most common anomalies, hyperdiploidy occurred mostly in CD9+ patients while ETV6::RUNX1 was predominant in CD9– cases. Notably, when focusing solely on these two genotypes, the CD9+ group accounted for only 36.4% patients whereas CD9– patients constituted 50.4%, emphasizing the favorable cytogenetic profile of the latter. Additionally, CD9 positivity was notably linked to intermediate-risk TCF::PBX1. This distribution of prognostic parameters already contributed to more CD9– patients falling into the low-risk category. Yet, upon deeper analysis within risk groups, the prognostic potency of CD9 was moderate for low-risk patients but strikingly for the intermediate/high-risk group. Here, CD9+ patients faced twice the risk of relapse, underscoring CD9’s potency to pinpoint patients with genuinely high-risk diseases.

With this large cohort of patients, we were able to conduct a comprehensive analysis to discern the distinct impact of CD9 on various cytogenetic subtypes. Notably, among CD9– patients with the BCR::ABL1 subtype, a remarkable outcome was observed, with a 100% EFS rate. In contrast, CD9+ patients had a substantially lower EFS of only 56%. Accordingly, BCR::ABL1+ patients with a CD9– phenotype could be effectively managed by the current chemotherapy regimen and a tyrosine kinase inhibitor [28]. On the contrary, innovative therapies are warranted for CD9+ patients within this subgroup. In future protocols, CD9 positivity with concurrent BCR::ABL1 may be considered as an indication for allogeneic hematopoietic stem cell transplantation (HSCT) or upfront immunotherapies. Similarly, among patients with a normal karyotype, CD9– patients had a favorable outcome with CIR of only 6.8%, whereas CD9+ subjects had a notably higher CIR of 15%, underscoring the necessity for more intensive treatments. While our investigations, alongside those of others, have highlighted CD9’s influence on leukemia stem cell renewal, leukemia-stroma interaction, and leukemia dissemination [17,18,19,20,21], potentially explaining its oncogenic role in B-ALL in general, the differential impact of CD9 on cytogenetic subtypes identified by this study has provided new insights into its underlying biology. It is conceivable that cooperative pathways might be requisite for CD9 to fully manifest its function. For example, our prior work showed that CD9 activates the PI3K/Akt pathway to drive leukemia progression and chemoresistance in BCR::ABL1+ cells [29]. A systematic dissection of its underlying mechanisms in future studies, possibly by transcriptomic analyses comparing CD9+ and CD9– cases in each cytogenetic background, holds the potential for revealing important findings with therapeutic implications.

Importantly, when combined with MRD status, CD9 emerges as a potent prognostic determinant. Notably, our previous single-center study, conducted prior to the introduction of MRD monitoring, failed to capture this informative synergy. Specifically, on day 19 after initiation of induction therapy, CD9+ patients who had low or high MRD had a 1.9- and 3.1-fold augmented risk of relapse, culminating in EFS rates of 77.8% and 65.2%, respectively. Consequently, these subjects should be managed early with treatment intensification or innovative therapies to reduce the risk of relapse. However, by day 46, CD9 did not impart an additional value to predict relapse for patients who had already tested positive for MRD. Thus, CD9 should be embraced as an early marker, complementing the specific prognostic parameters identified in this study to inform proper patient management, where HSCT or immunotherapies could be timely introduced for patients with high risk of relapse. Although CD9 appeared to be highly associated with known risk factors, multivariate analyses underscore its autonomy as an independent predictive factor for adverse events and relapse.

This is the largest study to evaluate the significance of CD9 in childhood ALL. A prominent strength is the multicenter design, where all patients underwent treatment following a standardized protocol. This approach generated a wealth of reliable and unbiased data, solidifying the genuine prognostic impact of CD9. Leveraging this substantial cohort, we were able to perform subgroup analyses with other well-established risk factors, meticulously documenting its precise prognostic attributes. However, this study has some limitations. One notable limitation is the absence of standardized flow cytometry protocols across participating centers, specifically in regard to the fluorochromes employed for CD9 detection. Addressing this concern in future trials, guided by Euroflow’s recommendations [30], is essential to enhance consistency and vigor. Another shortcoming pertains to our cohort composition, therein approximately 50% of patients exhibited either a normal karyotype or an ALL categorization marked as “not otherwise specified.” Given the rapidly expanding molecular taxonomy of ALL in the era of genomic medicine [31], it becomes imperative for upcoming studies to incorporate such molecular information. This inclusion would enable a deeper exploration of genetic correlations with CD9 and its subtype-specific prognostic implications.

In sum, we validated the results of our previous single-center study with the findings from this nationwide multicenter study. CD9 positivity unequivocally correlates with a heightened probability of relapse, particularly among patients with intermediate/high-risk diseases, positive MRD status, or specific cytogenetic backgrounds. Notably, BCR::ABL1+ patients with a CD9– phenotype had excellent outcomes, potentially obviating the necessity for HSCT. Conversely, patients with MRD+ who also exhibit CD9 positivity had poor outcomes, underscoring the urgency of early interventions with innovative treatments to mitigate the risk of relapse. As major study groups have yet to mandate the measurement of CD9 [32, 33], we propose its integration into the diagnostic immunophenotyping panel as a ready-to-use prognostic marker to inform risk stratification and management of childhood ALL.