Comprehensive Laboratory Diagnostic Workup for Patients with Suspected Intraocular Lymphoma including Flow Cytometry, Molecular Genetics and Cytopathology
, Paolo Mazzeo 3,†, Martin S. Zinkernagel 4, Myriam Legros 1, Naomi Porret 1, Lorenz Romagna 5, Detlef Haase 3, Georg Lenz 2, Urban Novak 5, Yara Banz 6, Thomas Pabst 5 and Ulrike Bacher 1,*Round 1
Reviewer 1 Report
In their work, Shumilov et al addressed the important topic of diagnosis in intraocular lymphoma. This rare disease is difficult to diagnose, mostly due to very low cell numbers. The authors concentrated on PVRL and recommend a combined approach of imaging, cytopathology, molecular genetics and flow cytometry. Adding flow cytometry and ddPCR for MYD88, two further patients with negative results in cytopatholagy could be diagnosed. Even for a rare diasease, the cohort is small with 7 cases analyzed.
Some aspects should be reevaluated:
L 13: formatting
L 30: flow cytometry alone is not able to really exclude the diagnosis, especially in a setting where many approaches are needed for diagnosis
Line 40 ff: for the definition of IOL, the rare involement of the uvea (iris, the ciliary body and the choroid) should be added (e.g. Cunningham, Ocul Immunol Inflamm 2021) or the statement on analyzing PVRL only would be helpful.
L 106: LST is the 'lymphoid screening tube'
L110: after clonality search is performed by FC it should be stated clearly, if or how the following marker combination allows differentiation of lymphoma subtypes.
L 125: "a" is missing in assay
L 155: in patient 5 flow cytometry was not negative. It was an acellular punctate which could not be evaluated. In total, flow cytometry was negative in two patients
L 159: better: "in a single case" instead of "once"
L 160: in some cases not all flow cytometry analyses could be performed. To further distinguish the lymphoma subtype and/or the IOL form information on paraproteinemia would be helpful (espacially in MYD88 mutated cases)
L 179: case 5 has an acellular punctate. Did the authors check the suspicious lymphocytes in the CSF with flow cytometry for CD19 high CD20 low cells? The patient received rituximab previously.
L 243: to better distinguish different IOL forms, peripheral imaging is as well crusial to exclude secondary involvement of the eye. This of course was performed (table 1) but should be stated in the text. Analysis of the CSF was performed in one patient. Do the authors recommend this in the case of PVRL and for the overall diagnostic approach?
L 236: that was not a really neglected tool. Shi et al published 14 PVRL pts. in 2019 (Ocular Immunol Inflamm) analyzed for MYD88 mutations with ddPCR. The authors stated ddPCR for MYD88 mutations as promising tool for the diagnosis of PVRL.
Figure 1: arrows indicate on first view, that the methods are used one after the other. Arrows directing form the biopsy to the methods used would be more helpful, because the methods are run in parallel.
Author Response
Reviewer 1:
In their work, Shumilov et al addressed the important topic of diagnosis in intraocular lymphoma. This rare disease is difficult to diagnose, mostly due to very low cell numbers. The authors concentrated on PVRL and recommend a combined approach of imaging, cytopathology, molecular genetics and flow cytometry. Adding flow cytometry and ddPCR for MYD88, two further patients with negative results in cytopatholagy could be diagnosed. Even for a rare diasease, the cohort is small with 7 cases analyzed.
Some aspects should be reevaluated:
- L 13: formatting
Authors: We would like to thank the reviewer for his attention to the small details. We adjusted the text in line 13.
- L 30: flow cytometry alone is not able to really exclude the diagnosis, especially in a setting where many approaches are needed for diagnosis
Authors: We agree with the review that this statement should be rephrased. We changed that as following: “and FC confirmed additionally the lack of IOL in the remaining patients”.
- Line 40 ff: for the definition of IOL, the rare involement of the uvea (iris, the ciliary body and the choroid) should be added (e.g. Cunningham, Ocul Immunol Inflamm 2021) or the statement on analyzing PVRL only would be helpful.
Authors: This is an important point indeed. We commented on that as follows: “Commonly, IOL arise in the vitreous body or in the retina and refer accordingly to vitreoretinal lymphoma (VRL). Rarely, IOL may occur in the uveal tract (iris, ciliary body, and choroid) and refer to so called uveal lymphomas presented mostly by indolent extranodal marginal zone lymphoma.[8] Mostly, VRL comprises aggressive diffuse large B-cell lymphoma (DLBCL; ≥95%) although intraocular T-cell lymphomas have also rarely been reported”. We cited Cunningham et al [8] as proposed.
- L 106: LST is the 'lymphoid screening tube'.
Authors: We corrected that.
- L110: after clonality search is performed by FC it should be stated clearly, if or how the following marker combination allows differentiation of lymphoma subtypes.
Authors: Considering the low cell numbers that are evaluable in the vitreal body by flow cytometry, complete flow panels are rarely applicable. Thus, in many cases, only a lymphoma screening panel can be applied. Additionally, it should be mentioned that flow cytometry can detect antigen patterns that may be characteristic for certain lymphoma subtypes, but a clear delineation of a distinct lymphoma entity is possible only for few entities that play a minor or no role for intraocular lymphoma (e.g. hairy cell leukemia or chronic lymphatic leukemia, CLL). Therefore, flow cytometry will be rather helpful for identifying B-cell lymphoma in the vitreal body but for the delineation of the exact subtype cytopathology will be more important. These limitations were outlined more strongly in the discussion part.
- L 125: "a" is missing in assay
Authors: We corrected that.
- L 155: in patient 5, flow cytometry was not negative. It was an acellular punctate which could not be evaluated. In total, flow cytometry was negative in two patients.
Authors: We re-wrote that as follows: “Flow cytometry detected a monoclonal B-cell population in four patients (#1-4) and was negative in two patients (#6-7). In the remaining case (#5), acellular punctate was present”.
- L 159: better: "in a single case" instead of "once"
Authors: We changed that accordingly.
- L 160: in some cases not all flow cytometry analyses could be performed. To further distinguish the lymphoma subtype and/or the IOL form information on paraproteinemia would be helpful (especially in MYD88 mutated cases).
Authors: The reviewer mentions an important point. The here described analyses performed on vitreal body should of course be combined with other diagnostic techniques and methods that may be necessary in the clinical context. This was pointed out more clearly in the discussion. However, the detection of MYD88 mutation is not specific for lymphoplasmacytic lymphoma but may occur as well in other lymphoma subtypes. Nevertheless, we focused on the necessity of other diagnostic assessments that may be relevant for the complete picture.
- L 179: case 5 has an acellular punctate. Did the authors check the suspicious lymphocytes in the CSF with flow cytometry for CD19 high CD20 low cells? The patient received rituximab previously
Authors: We totally agree with the review that it would make sense to check the CSF with flow cytometry for the presence of CD19+/CD20+ cell population in this situation, but this analysis had unfortunately not been initiated by the respective physicians.
- L 243: to better distinguish different IOL forms, peripheral imaging is as well crucial to exclude secondary involvement of the eye. This of course was performed (table 1) but should be stated in the text. Analysis of the CSF was performed in one patient. Do the authors recommend this in the case of PVRL and for the overall diagnostic approach?
Authors: Again, the reviewer addressed the very important aspects of diagnosing of IOL. We agree with the reviewer that peripheral imaging as well as CSF analysis should be performed within a diagnostic workup of IOL. We addressed these as following: “In order to distinguish different forms of IOL (e.g. isolated PIOL, PCNSL, or SIOL), imaging, in particular MRI of the brain, plays a crucial role in the identification of additional IOL CNS manifestations. Moreover, peripheral imaging (e.g. CT or PET-CT) is as well crucial enabling to exclude secondary involvement of the eye. The results of both, in turn, has a direct impact on the choice of subsequent treatment strategies. For the overall diagnostic approach, cerebrospinal fluid (CSF) analysis can be recommended as well to exclude occult CNS manifestations within a PCNSL or SIOL helping to differentiate it from PIOL.”
- L 236: that was not a really neglected tool. Shi et al published 14 PVRL pts. in 2019 (Ocular Immunol Inflamm) analyzed for MYD88 mutations with ddPCR. The authors stated ddPCR for MYD88 mutations as promising tool for the diagnosis of PVRL.
Authors: Following the recommendation of the reviewer, we corrected this statement as follows: “Acknowledging technical limits of cytopathology and flow cytometry in vitreous samples, molecular genetics represents an additional tool in the diagnostic workup of IOL”. We also cited the article of Shi et al as follows: “Therefore, molecular genetics may significantly contribute to the diagnosis of PIOL since up to 80% of PVRL cases are positive for the MYD88 L265P mutation detected by either polymerase-chain reaction (PCR) or droplet digital PCR”.
- Figure 1: arrows indicate on first view, that the methods are used one after the other. Arrows directing form the biopsy to the methods used would be more helpful, because the methods are run in parallel.
Authors: We adjusted the figure 1 according to the recommendations of the reviewer. Indeed, that was a very good proposal for the figure.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This is a wonderful presentation of a rare but highly relevant situation in hematology. The authors are well known experts in their field. It is a pleausure to read this contribution. Between an extended day in the clinic and laboratory and an evening preparing a post ASH talk I was able to read and understand this manuscript within the blink of an eye. It is a key example of how the english language can be used to present information in a succinct, an expression which unfortunately has become extinct and stands for a language feature nowadays rarely relied to, manner.
I congratulate the authors. Please, regarding patient two, can you comment if the mutational load in the vitreal fluid differed from systemic mutational load? This might help to differ between intraocular lymphoma versus lymphomatoid infiltration through physiologic blood flow in the eye, perhaps enhanced by inflammation due to causes differing from lympoma infiltration. This might add to the manuscript but should not hinder publication as is.
Author Response
Reviewer 2:
This is a wonderful presentation of a rare but highly relevant situation in hematology. The authors are well known experts in their field. It is a pleasure to read this contribution. Between an extended day in the clinic and laboratory and an evening preparing a post ASH talk I was able to read and understand this manuscript within the blink of an eye. It is a key example of how the english language can be used to present information in a succinct, an expression which unfortunately has become extinct and stands for a language feature nowadays rarely relied to, manner.
I congratulate the authors. Please, regarding patient two, can you comment if the mutational load in the vitreal fluid differed from systemic mutational load? This might help to differ between intraocular lymphoma versus lymphomatoid infiltration through physiologic blood flow in the eye, perhaps enhanced by inflammation due to causes differing from lympoma infiltration. This might add to the manuscript but should not hinder publication as is.
Authors: We fell much honored to receive such a positive feedback to our manuscript and are very grateful to the reviewer for that. We agree with the reviewer that the everyday clinical routine requires many efforts and energy, so we are very pleased that our article is able to provide a short but clear message to readers.
The point about mutation load and the role of blood flow and inflammation in the eye is definitely very interesting and worth investigating in the future. Unfortunately, we did not measure the systemic mutational load for the MYD88 mutation in this particular case so we cannot contribute with this valuable point to the written text.
Author Response File: Author Response.docx
