Diffuse Large B Cell Lymphoma Arising in Patients with Preexisting Hodgkin Lymphoma

page 4 line 158 The phrase "...HRS cells destructive somatic mutations..." doesn't make sense. Is there a word missing perhaps?

the sentence has been better clarified: “In addition, in some cases of cHL, HRS cells lack Ig gene transcription ability due to functional defects in the Ig gene regulatory elements. It has therefore been proposed that HRS cells originate from pre-apoptotic GCB cells with an acquired mutation that in normal conditions would have led to apoptosis”

page 5 line 208 "...age, 208 gender and disease burden are linked at the time of the first diagnosis"- could you please clarify what you mean by this sentence?

the sentence has been better clarified: “In this study, it emerged that age, gender, and disease stage at the time of the first diagnosis affect the likelihood to have sNHL

page 5-line 212 change badly to poorly

Done

page 6 last 2 paragraphs occur after the conclusions and seem out of place. 

Done

1.       Authors claim Hans’s algorithm and cell of origin analysis were contradictory, delineated in Table 1. Please provide the cell of origin analysis performed with Gene expression profile (GEP) analysis along with assessment from NanoString Lymphoma Subtype Assay. This was mentioned in the abstract.

2.       Abstract line 31: “not classified” has been replaced with “unclassified”.

3.       Table 1: “undeterm” has been replaced with “unclassified”.

4.       New paragraph was added in the Patients and methods: line 85.

5.       “Recently, Scott et al described a robust model for COO assignment named Lymph2Cx. With nCounter platform of NanoString Technologies we applied Lymph2Cx on formalin-fixed, paraffin embedded RNA. The results obtained are reported as one of two molecular subtypes, Activeted-B-Cell (ABC). or Germinal Center-B-Cell (GC), moreover, the Lymph2Cx assay recognized a group of unclassified cases, where confident assignment cannot be made to either ABC or GCB subtype”.

6.       We included in the bibliography: Scott DW et al. Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue. Blood 2014 Feb 20;123(8):1214-7. (No 11)

2.       If data above is available, please provide the Gene Express Omnibus number or its equivalent.

We didn't have Gene Express Omnibus number or its equivalent. The COO assignment was processed using the National Cancer Institute's Lymphoma/Leukemia Molecular Profiling Project Lymph2Cx DLBCL COO Classifier (patented algorithm, available through National Cancer Institute website via Data Use Agreement) (Scott et al 2014). The assay produces a calculated score on a scale of 0.00 to 1.00 to classify each DLBCL sample based on the probability that the sample is ABC type. Samples with a ≥90%probability of being ABC-DLBCL type (a score of 0.90–1.00) are classified as ABC-DLBCL, samples that have a ≥90%probability of being GCB-DLBCL type (a score of 0.00–0.10) are classified as GCB-DLBCL, and samples with scores >0.10 and <0.90 are categorized as Unclassifiable (UNC-DLBCL).

3.       The crucial question of whether the DLBCL occurrence are independent or correlated events were not sufficiently addressed with primary data from the manuscript.

The results were better expressed, and two new paragraphs were added in the discussion: “In our case study the cumulative incidence of sNHL was 1,5 %, in part comparable with the incidences found in the other studies. From the analysis of the clinical characteristics of our series, it is not possible to draw statistically significant conclusions due to the small number and the low incidence of onset; DLBCL occurred in 3 males and 1 female.  Conversely neither older age nor advanced stage at cHL diagnosis were correlated with DLBCL incidence. In fact, median age at cHL diagnosis was 28 years; The three males were young (less than 40 years old) while the woman was 78 years old. The stage of cHL was 2 in 3 cases and 4 in 1 case. However, the exiguity of our cases doesn’t allow any conclusion.”.… “In our case series 3 patients had a low chemotherapy load, having been treated only with ABVD, while one had undergone 2 lines of chemotherapy, radiotherapy and high-dose therapy followed by ASCT. No patient had undergone splenectomy”

4.       From the clinical perspective, how to maintain vigilance for patients with cHL owing to the possible transformation to DLBCL?

A new paragraph was inserted in the conclusions specifying the clinical perspectives: “Clinically, it is It is important to maintain vigilance for patients with cHL even if in complete remission due to the possible development of sNHL.”

1.       Age for the 4 patients in table 1 is not specified, only mean age of 35 (with age range 21-79) was mentioned. Age is crucial factor in determining survival outcomes.

Age was entered in table 1

2.       Please provide the histological comparison between cHL and DLBCL for patient 2 and 4. Only data for patient 1 and 3 was presented in figure 1 and 2 respectively

Done. Figure 3 was added. “Histological comparison between cHL (A-C, hematoxylin-eosin, original magnification 200x) and DLBCL (B-D hematoxylin-eosin, original magnification 200x and 100x), in the patient respectively 2 (A-B) and 4 (C-D)”.

3.       Please explain why adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) was chosen as a 1^st line therapy instead of epirubicin, bleomycin, vinblastine, and methotrexate (EBVM) plus extended field radiation therapy for the 4 patients.

The guidelines used for the treatment have been specified in the text and included in the bibliography (No. 10)

1 Only 4 patients were observed have this dual incense of neoplasm, and two of them died. This is insufficient to show any statistical significance or make any conclusions.

This criticality was included in the discussion: “Due to the low incidence (only 4 patients out of 269 -1.5%) it is not possible to draw statistically significant conclusions on the factors determining the occurrence of a DLBCL in patients with cHL.”

2- These patients have been continually treated with medications (as they should be) but making it difficult to understand how these courses of treatment affect this dual disease incidence.

All diagnoses of DLBCL were made when patients were free from cHL disease, this was better specified in the text: “Patients n. 1, 2 and 4 performed, as first line for cHL, 6 cycles of ABVD obtaining complete remission. At a median time of 33 months these showed suspected signs of disease recurrence, but a DLBCL was diagnosed on histological examination. Patient number 3, being a stage 2A, was treated with 4 cycles of ABVD followed by "involved field" radiotherapy, subsequently having histologically documented recurrence of cHL, he underwent salvage therapy according to IGEV and ASCT schemes”

3- Has there been an indication over the course of treatment that would alert to a new development. These incidences of DLBCL cannot just happen overnight.

All 4 patients were cHL free at the time of DLBCL diagnosis (specified in the text)

4- Has there been a change in the genetic make-up of patients who have dual cancers. Has that been investigated.

Sorry, the data is not available.

There are so many factors that are developing, which make it difficult to understand this anomaly, in addition to the low incidence.

Ihis criticality was included in the discussion: “Furthermore, as highlighted, there are numerous factors that can manifest themselves in the becoming of this second diagnosis of lymphoma which, in addition to the low incidence, makes it difficult to understand this anomaly”

In their Table 1, the authors analyzed 4 patients with both cHL and DLBCL but did not include patients with only cHL for comparison. 

Sorry, it was not the goal of the study.

Finally, even the authors themselves conclude that the incidence of DLBCL in cHL patients "is controversial". Based on scientific evidence, the authors did not show any relevant or specific explanation for the development of DLBCL in patients with cHL.

The crucial question of whether DLBCL occurrences are independent or related events has been best addressed by improving the results, inserting new paragraphs into discussion. The criticality regarding the numerous factors that can occur in the becoming of this anomaly has been inserted