Long Non-Coding RNA Expression in B-Cell Precursor Acute Lymphoblastic Leukemia: Analysis of LINC-PINT, MEG3, BALR6, and ZEB1-AS1

Comments 1: The major drawback of the study is the lack of comparable healthy control group. The lncRNA analyses discussed here are on the patients who already have the disease irrespective of their MRD status. What we do not see is the level of expression of these lncRNAs in control vs leukemia patients. That will give us a basis to compare the alterations in the expression levels of various lncRNAs with regard to leukemia.

Response 1:

Thank you for this valuable observation. The primary objective of our study was not to compare the expression levels of the selected lncRNAs between healthy individuals and patients diagnosed with leukemia. Rather, our aim was to evaluate changes in lncRNA expression from the time of diagnosis to MRD assessment performed at the end of induction therapy.

To achieve this objective, the study was designed as a longitudinal paired analysis in which each patient served as his or her own control. Bone marrow samples collected at diagnosis were directly compared with samples obtained from the same patient during MRD evaluation following induction treatment. This approach allowed us to assess treatment-associated changes in lncRNA expression while minimizing the impact of interindividual biological variability.

Comments 2: Sample size analysed is very small. This makes it difficult to agree with the conclusions made with relation to lncRNA expression in MRD+ and MRD- groups.

Response 2: Thank you for this valuable comment. We acknowledge that the sample size represents an important limitation of the present study and that caution is required when interpreting the findings.

Patient recruitment and sample collection were conducted immediately after the COVID-19 pandemic period (2020-2022), which substantially affected the availability of eligible participants and access to healthcare services. Consequently, several patients initially enrolled in the study were unable to complete the planned clinical follow-up (MRD assessment) and these cases were excluded from the final analysis, further reducing the number of evaluable patients.

To address this concern, we have explicitly acknowledged the limited sample size as a study limitation and have revised the manuscript to present our findings as preliminary evidence that warrant validation in larger, independent cohorts. Accordingly, the conclusions have been moderated to avoid overinterpretation of the observed associations between lncRNA expression patterns and MRD status.

Although the cohort size is limited, the longitudinal design based on paired samples collected at diagnosis and during end-of-induction MRD evaluation allowed us to assess intra-patient changes in lncRNA expression, with each patient serving as his or her own control. This approach partially reduced the inter-individual variability commonly observed in leukemia studies and enabled the evaluation of treatment-associated changes in lncRNA expression over time.

The corresponding statement regarding the limited cohort size has been included in the Discussion section, and can be found on page 10, paragraph 3, lines 292-295.

Comments 3: After induction therapy there is a decrease in linc-PINT and increase in BALR6 expression in the MRD+ group. Does that mean both these non-coding RNAs counteract each other's action or confer similar attributes under different conditions? Is there any correlation in the activities of these two non-coding RNAs?

Respose 3: Thank you for this insightful observation. In this particular case, patients who failed to achieve remission appeared to exhibit a complementary expression pattern, characterized by increased BALR6 expression and a significant decrease in linc-PINT expression. However, considering the biological mechanisms previously described for these lncRNAs, this relationship may be interpreted as synergistic rather than antagonistic. Increased BALR6 expression has been associated with enhanced proliferative capacity and resistance to cell death, whereas reduced linc-PINT expression may impair the cellular response to DNA damage and cell cycle regulation. Together, these alterations could potentially contribute to leukemic cell persistence during therapy by simultaneously promoting cell survival and reducing the effectiveness of mechanisms involved in the response to treatment-induced DNA damage.

This aspect is already addressed in the manuscript on page 9, paragraphs 5 and 6, lines 262–280.

Comments 4: Figure 3 is the repetition of Fig. 2. What additional information does it provide?

Response 4: Thank you for this valuable observation. The original rationale for presenting Figures 2 and 3 separately was to facilitate the visualization of lncRNA expression from two different perspectives.

However, we agree that presenting the data in separate figures may create the impression of redundancy and does not substantially improve data interpretation. Therefore, following the reviewer's suggestion, we have condensed the results into a single. This modification improves the clarity of the presentation while avoiding unnecessary repetition.

The revised figure has been included in the manuscript accordingly on page 7.

Comments 5: In the section 3.3.2, the authors mention “MEG3 expression showed a statistically significant decrease in patients who achieved remission”. This suggests MEG3 expression levels remained unaffected after induction therapy in the MRD+ patients whereas it decreased in the MRD- patients. Do the authors have any explanation why is it like this?

Response 5: We propose that the relatively high levels of MEG3 expression observed at diagnosis (compared with post-induction levels) may contribute to disease remission by promoting apoptotic processes in leukemic cells. This effect is likely mediated, at least in part, through the well-established tumor suppressor functions of MEG3, including activation of p53-dependent pathways and the regulation of genes involved in cell-cycle arrest and apoptosis. However, emerging evidence suggests that MEG3 can also exert biological effects through p53-independent and non-canonical mechanisms, such as epigenetic regulation, interactions with chromatin-modifying complexes, modulation of signaling pathways, and competitive endogenous RNA activity (as described by Wang, A. et al (2022).

As leukemic clones undergo apoptosis and are eliminated during treatment, the overall expression of MEG3 may gradually return to baseline levels. Subsequently, the bone marrow is repopulated by newly generated hematopoietic cells, which may exhibit lower MEG3 expression than leukemic clones, thereby contributing to the observed decrease in the average MEG3 expression after induction therapy.

The corresponding changes can be found on page 9, paragraph 4, lines 258-267.

Comments 1: The rationale for selecting the specific lncRNAs should be described more systematically, including clearer criteria linking each molecule to particular oncogenic hallmarks.

Response 1: Thank you for this valuable comment. We agree that the rationale for selecting the investigated lncRNAs should be presented more systematically. Accordingly, we revised the Introduction to more clearly describe the evidence linking each lncRNA to specific cancer hallmarks and biological processes involved in leukemogenesis. These modifications provide a clearer justification for the inclusion of the selected lncRNAs in the study.

The corresponding changes can be found on page 2, paragraph 4, lines 81–94.

Comments 2: The discussion should be improved from including more explicit references to statistical strength, effect sizes, or cohort characteristics to better support the biological interpretations.

Response 2: Thank you for this valuable comment. We agree that the biological interpretation of our findings should be considered in the context of the statistical and cohort-related limitations of the study. Accordingly, we have revised the Discussion section to include a statement emphasizing that the results should be interpreted with caution due to the limited cohort size and the exploratory nature of the study.

The corresponding changes can be found on page 10, paragraph 3, lines 292-295.

In addition, we have clarified that detailed sociodemographic and clinical characteristics of the study population are available in the Supplementary Materials, allowing readers to better assess the context and representativeness of the cohort.

The corresponding changes can be found on page 4, paragraph 5, line 176.

Comments 3: Although numerous studies are cited, the discussion could better synthesize how the present findings align with or diverge from prior evidence, especially regarding differences between solid and hematologic malignancies.

Response 3: Thank you for this insightful comment. We have expanded the Discussion section to better integrate our findings with previously published evidence. Specifically, we now compare the expression patterns and reported biological functions of the analyzed lncRNAs in solid tumors and hematological malignancies, highlighting both concordant and divergent observations. We further discuss how differences in cellular context, disease biology, and treatment response may contribute to the distinct expression profiles observed in our cohort.

The corresponding changes can be found on page 10, paragraph 4, lines 297-305.

Comments 4: Terms such as “MRD-negative,” and “poor prognosis” should be defined to avoid ambiguity.

Response 4: Thank you for this comment. To improve clarity, we revised the manuscript to explicitly describe the cutoff criteria used for the classification of patients as MRD-positive or MRD-negative.

The corresponding changes can be found on page 4, paragraph 3, lines 157–161.

Furthermore, we revised page 9, paragraph 5, line 272 to provide a clearer definition of the term “poor prognosis” as used throughout the manuscript.

Comments 5: The clinical implications of implementing lncRNA panels in routine leukemia monitoring could be expanded, including potential advantages, limitations, and challenges for clinical application.

Response 5: Thank you for this valuable suggestion. We agree that the potential clinical implications of lncRNA-based biomarkers deserve further investigation.

Accordingly, we expanded the Conclusions sections to address the potential advantages associated with the implementation of lncRNA.

We also acknowledge the current limitations and challenges associated with the clinical application of lncRNA-based biomarkers as validation in larger multicenter cohorts before routine implementation can be considered.

These additions have been incorporated into the revised manuscript page 10, paragraph 3 and 4, lines 292-305, and paragraph 6, lines 319-328.

Comments 6: The idea of a final paragraph related to how lncRNA profiling could complement existing MRD methodologies and contribute to personalized leukemia management.

Response 6: Thank you for this valuable suggestion. We agree that the potential role of lncRNA profiling as a complementary tool to conventional MRD assessment deserves further emphasis. Therefore, we incorporated a concluding paragraph discussing how their evaluation may provide additional biological information regarding leukemic persistence beyond conventional MRD methodologies, potentially contributing to improved risk stratification and more personalized disease-monitoring and therapeutic decision-making strategies.

The corresponding changes have been incorporated into page 9, paragraph 6, line 319-328.