Plasmablastic Transformation of CLL/SLL: The Role of Early NGS Diagnosis and Targeted Multimodal Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The introduction has to be widened 

In the "Case report" PET: please describe SUV of lymph nodes of the different areas, in particular the axillary and retroperitoneal ones

It lacks on description of peripheral blood, morphological and phenotypical analysis. Moreover you should widen even hematochemical markers.

At line 97, before "Tumor cells" you have to insert (Fig 1G-L)

Lines 259-260 Why didn't you consider ALLO TMO for this young patient?

Author Response

Comment 1. The introduction has to be widened.

Response: We thank the reviewer for this valuable suggestion. The Introduction has been expanded to provide a broader epidemiological and clinical background on chronic lymphocytic leukemia, Richter transformation, and the rarity of plasmablastic transformation, particularly in immunocompetent patients. The manuscript has been revised accordingly. The changes are highlighted in red (lines 49-55).

Comment 2.

In the "Case report" PET: please describe SUV of lymph nodes of the different areas, in particular the axillary and retroperitoneal ones.

Response:
We thank the reviewer for this important comment. SUVmax values of the retroperitoneal and pelvic lymph nodes have now been explicitly reported in the Case Report section. The rationale for selecting the axillary lymph node for biopsy based on accessibility has also been clarified. The manuscript has been revised accordingly. The changes are highlighted in red in color (lines 67-72).

Comment 3.
It lacks description of peripheral blood, morphological and phenotypical analysis. Moreover you should widen even hematochemical markers.

Response:
We thank the reviewer for highlighting this point. A detailed description of peripheral blood findings, including complete blood count, blood smear morphology, and flow cytometric immunophenotyping, has been added. Hematochemical parameters have also been clarified and expanded in the Case Report section. The changes are highlighted in red (lines 73-78). The manuscript has been revised accordingly.

Comment 4.
At line 97, before "Tumor cells" you have to insert (Fig 1G–L).

Response:
We thank the reviewer for this helpful observation. The suggested figure reference has been inserted at the appropriate position, and the text now correctly refers to Figure 1G–L (line 120).

Comment 5.
Lines 259–260: Why didn't you consider ALLO TMO for this young patient?

Response:
We thank the reviewer for this relevant clinical question. We thank the reviewer for this comment. We have clarified that AHSCT was performed as consolidation therapy, reflecting an individualized treatment decision in the absence of standardized therapeutic recommendations. This change is highlighted in red (Therapy, line 174).

Reviewer 2 Report

Comments and Suggestions for Authors

1. What is the prevelance of  CLL and DLBCL Globally.
2. Explain the molecular mechanism reposnsible for the development of PBL.
3. How you have selected the immnophenotype panels.

Author Response

Comment 1.
What is the prevalence of CLL and DLBCL globally?

Response:
We thank the reviewer for this important comment. Data on the global prevalence and incidence of CLL and DLBCL have been added to the Introduction. These changes are highlighted in red (lines 49-55).

Comment 2.
Explain the molecular mechanism responsible for the development of PBL.

Response:
We thank the reviewer for this insightful suggestion. A dedicated paragraph discussing the molecular mechanisms underlying plasmablastic lymphoma, including MYC deregulation and loss of B-cell lineage program, has been added to the Discussion. The modifications are highlighted in red (lines 267–276).

Comment 3.
How you have selected the immunophenotype panels.

Response:
We thank the reviewer for this constructive comment. The rationale for the selection of the immunohistochemical panels has been clarified in the Methods/Case Report section. These changes are highlighted in red (lines 80–86).

Reviewer 3 Report

Comments and Suggestions for Authors

Please include results of EBER staining on plasmablastic lymphoma component. Please also document PAX5 staining pattern, and if positive, please comment on overall B-cell lineage expression. PAX5 negativity is an essential criterion for plasmablastic lymphoma in WHO 5th edition, so this result needs to be described for your case. Referenced in line 218 in discussion.

Arrows in figure 3 should be uniformly applied to either indicate only positive finding (MYC breakapart; preferred approach) or to highlight representative result across all FISH tests, and significance of arrow(s) should be indicated in figure legend.

Please clarify language in section 3.2, which implies that NGS results are presented in figure 4.

Figure 4. clone peak size boxes need to be formatted to fit the entire number.

Table 1 has discrepancy versus text, describing presence of plasmablastic lymphoma in same axillary LN an CLL/SLL. Text states plasmablastic lymphoma was present in retroperitoneal mass biopsy.

Please clarify and uniformly describe features in tables 1 and 2, especially viral status, genetics, and localization. For viral status, the use of "others" is unclear. Either describe all 3 viruses mentioned in heading as positive, negative or not tested, or include only positive results. For genetics, what is the difference between N/A and "not documented"? For localization, please use uniform language (concurrent versus simultaneous; one case simply described as pleural fluid). Overall, please carefully review and edit tables for clarity, uniformity, and correctness.

Line 197: please separate "as we revised the cases" into its own sentence more clearly describing that the hypothesis that early/concurrent versus late/post-therapy cases may have different biology/mechanisms was your rationale for separating cases as such in tables 1 and 2.

Line 211 immunohisotchemistry already abbreviated as IHC in text above. same for next-generation sequencing (line 212).

Line 223: Burkitt lymphoma ruled out by morphology and phenotype, not lack of BCL2 and BCL6 rearrangements.

Author Response

Comment 1.
Please include results of EBER staining on plasmablastic lymphoma component and document PAX5 staining pattern.

Response:
We thank the reviewer for this important comment. Results of EBER in situ hybridization and PAX5 immunohistochemistry are now explicitly described in the Results section, with reference to Supplementary Figure S1 (line 146). The diagnostic relevance of PAX5 loss according to WHO 5th edition criteria is discussed in the Discussion. These changes are highlighted in red (Results: lines 121–124; Discussion: lines 260–263).

Comment 2.
Arrows in figure 3 should be uniformly applied.

Response:
We thank the reviewer for this careful observation. Figure 3 has been revised so that arrows uniformly indicate the MYC break-apart signal, and the figure legend has been updated accordingly. These changes are highlighted in red in the figure legend.

Comment 3.
Please clarify language in section 3.2, which implies that NGS results are presented in figure 4.

Response:
We thank the reviewer for this helpful comment. The wording in section 3.2 has been corrected to clearly state that Figure 4 illustrates PCR-based clonality analysis, while NGS results are described in the text. The modification is highlighted in red (lines 153–156).

Comment 4.
Figure 4. clone peak size boxes need to be formatted to fit the entire number.

Response:
We thank the reviewer for this technical suggestion. The formatting of the frames in Figure 4 was unintentionally altered during file conversion. The figure has now been re-exported and provided in TIFF format to ensure correct and stable layout. No changes were made to the data or analysis.

Comment 5.
Table 1 discrepancy regarding localization of plasmablastic lymphoma.

Response:
We thank the reviewer for pointing out this discrepancy. Tables and text have been corrected to uniformly indicate that plasmablastic lymphoma was identified in the retroperitoneal lymph node. These changes are highlighted in yellow in Table 1 and the corresponding text highlighted in red.

Comment 6.
Please clarify and uniformly describe features in Tables 1 and 2.

Response:
We thank the reviewer for this important comment. Tables 1 and 2 have been thoroughly revised for clarity and uniformity regarding viral status, genetic data, localization, and terminology. All revisions are highlighted in red.

Comment 7.
Line 197: please separate hypothesis into its own sentence.

Response:
We thank the reviewer for this helpful suggestion. The sentence has been separated and rewritten to clearly state the rationale for distinguishing synchronous and metachronous cases. This change is highlighted in yellow (lines 232-238).

Comment 8.
Line 211–212: abbreviations already defined.

Response:
We thank the reviewer for this careful observation. Redundant definitions of IHC and NGS have been removed. The correction is highlighted in red (line 253).

Comment 9.
Line 223: Burkitt lymphoma ruled out by morphology and phenotype.

Response:
We thank the reviewer for this important correction. The sentence has been revised to state that Burkitt lymphoma was excluded based on morphology and immunophenotype. This change is highlighted in red (lines 267-269).