Learning from Patients: The Interplay between Clinical and Laboratory Research in AL Amyloidosis

Round 1

Reviewer 1 Report

The manuscript by Moshe E Gatt and Marjorie Pick is an exhaustive review about AL amyloidosis with an interesting emphasis on the role of the interplay between clinic and basic research to better understand the disease. However, the manuscript suffers from several approximations and hypothetical points of view and is confusing in its organization.

• Thorough the manuscript, the authors consider AL amyloidosis as a distinct entity compared to MGUS, SMM and MM. This remains controversial. AL amyloidosis is the consequence of the abnormal accumulation of a monoclonal LC in organs leading to their dysfunction. As for other amyloidoses, it is a conformational disease due to the physicochemical and structural properties of some monoclonal LCs. But the underlying PC clone (or B cell clone…) can be a MGUS, an SMM or a MM. As the authors state, most patients with AL amyloidosis have small PC clone, likely because the toxicity of the LC lead to organ damages before the evolution to SMM/MM, but about 30% have >10% PC in the bone marrow and meet the criteria for MM together with AL. Consequently, if AL amyloidosis PCs are found at the crossroad between MGUS and MM (refs 12 and 13 for ex), it’s likely because some AL patients have an MGUS-like PC clone and others an MM-like PC clone.

Overall, the statement that PC clones from AL patients are unique and different from other PCDs should be tone down thorough the manuscript and/or the intrinsic role of the pathogenic LC to explain the specificity of AL PCs should be more emphasize.

• Lines 61… and 199: cytogenetic feature t(11;14) is also associated with a frequent loss of the rearranged IgH gene leading to the production of a free LC only. Since AL amyloidosis is due to the aggregation of a free LC most of the time by a small clone, the loss of HC expression increases the production of free LC, accelerating the LC fibrilization and deposition. In that sense, the authors should take into account the LC only vs HC+LC PCs clones to evaluate their correlation between % BM PCs and levels of FLC in fig.1. It would be also of interest to discuss the high proportion of AL patients presenting with LC only PC clones (~50%). Also line 269, “FLC levels in AL may be 10 logs lower than MM”: Where the authors found this information. I did not find it in the cited papers but Nowrousian et al (Clin cancer res, 2004) or Mead et al (BJH, 2005) showed FLC levels not so different from AL (~100 to 1000 mg/L, similar to Fig.1).
• Line 175: whether ref 39 indeed describes a highest sensitivity to PI of AL cells , ref 38 did not show any higher apoptosis rates of AL PCs. They found less apoptosis rate than in MGUS. Also, the authors should discuss more the direct role of the LC in the PC stress as described in ref 39. Once again, it is more likely the LC (because of its abnormal folding and/or aggregation in the cells) that induces this sensitivity to PI than a proper feature of the PCs.
• Paragraph 4 is confusing. On one hand, the authors state that a deep hematologic response, i.e. a deep decrease in circulating pathogenic LC, is the only factor to correlate with survival and organ responses but on the other hand, they state that the LC still cause damages in the absence of increased amount of amyloid load. They cite review papers (67-69) but the clinical evidence are still scarce. The remaining toxicity in treated patients can also be due to the long persistence of amyloid deposits in organs because of their high resistance to proteolysis.
• Line 294: the fact that LC sequences (and so, structural properties) influence their propensity to aggregate and deposit is highly documented, at least in vitro (numerous papers from Marina Ramirez-Alvarado, Jonathan Wall, Johannes Buchner…). Consequently, it is not surprising to find no correlation between the PC burden / FLC levels and the organ damages.
• Line 327: several recent papers give information on the transcriptomic and proteomic profile of amyloid infiltrated tissues (Jordan et al, leukemia 2020; Kourelis et al, JACC Cardiooncol 2020) and should be cited and discussed.
• Line 333: sentence “dermal fibroblast… “ is confusing.
• Line 346: GWAS data: increased amount of monocytes: is it a cause or a consequence of amyloidosis?
• Many new therapeutic strategies in AL amyloidosis aim at removing actively amyloid fibrils from the organs. Could you consider adding a paragraph on these encouraging approaches?

Minor points:

• Line 315: “some are very toxic to these AL patients”. Is it specifically toxic to AL patients but not MM? Is there any explanation? Refs?
• Line 320: “mechanisms involved in … death caused by the amyloid proteins are poorly understood” Are you sure? The cause of death during AL amyloidosis are well known.
• Line 371: “That could sensitizers AL PCs”. Confusing
• Line 378: ref 92: JJN-3 do not express an amyloid kappa LC
• Please reread the manuscript to correct confusing sentences that miss verbs (see for instance the last sentence of the abstract) or are too long and have to be rephrased.

Author Response

We thank you for considering our manuscript: Learning from patients: the interplay between clinical and laboratory research in AL amyloidosis for publication and we thank the reviewers for their thorough remarks.

Here are our point by point corrections to the remarks and manuscript

Reviewer 1:

The manuscript by Moshe E Gatt and Marjorie Pick is an exhaustive review about AL amyloidosis with an interesting emphasis on the role of the interplay between clinic and basic research to better understand the disease. However, the manuscript suffers from several approximations and hypothetical points of view and is confusing in its organization.

• Thorough the manuscript, the authors consider AL amyloidosis as a distinct entity compared to MGUS, SMM and MM. This remains controversial. AL amyloidosis is the consequence of the abnormal accumulation of a monoclonal LC in organs leading to their dysfunction. As for other amyloidoses, it is a conformational disease due to the physicochemical and structural properties of some monoclonal LCs. But the underlying PC clone (or B cell clone…) can be a MGUS, an SMM or a MM. As the authors state, most patients with AL amyloidosis have small PC clone, likely because the toxicity of the LC lead to organ damages before the evolution to SMM/MM, but about 30% have >10% PC in the bone marrow and meet the criteria for MM together with AL. Consequently, if AL amyloidosis PCs are found at the crossroad between MGUS and MM (refs 12 and 13 for ex), it’s likely because some AL patients have an MGUS-like PC clone and others an MM-like PC clone.

Overall, the statement that PC clones from AL patients are unique and different from other PCDs should be tone down thorough the manuscript and/or the intrinsic role of the pathogenic LC to explain the specificity of AL PCs should be more emphasize.

We thank the reviewer for the emphasis, and will make the corrections as outlined below. However, we would like to stress that in writing this review, we did take into consideration that the theme issue will already include in detail some of the covered topics in this review, and therefore went to less details while mentioning these.

In addition, we do believe that AL is different than MM and MGUS, and not an incidental structural-only entity simply occurring in the either. We therefore brought arguments to support this claim, but did not disregard those that do not, and reference these too.

.

• Lines 61… and 199: cytogenetic feature t(11;14) is also associated with a frequent loss of the rearranged IgH gene leading to the production of a free LC only. Since AL amyloidosis is due to the aggregation of a free LC most of the time by a small clone, the loss of HC expression increases the production of free LC, accelerating the LC fibrilization and deposition. In that sense, the authors should take into account the LC only vs HC+LC PCs clones to evaluate their correlation between % BM PCs and levels of FLC in fig.1. It would be also of interest to discuss the high proportion of AL patients presenting with LC only PC clones (~50%). Also line 269, “FLC levels in AL may be 10 logs lower than MM”: Where the authors found this information. I did not find it in the cited papers but Nowrousian et al (Clin cancer res, 2004) or Mead et al (BJH, 2005) showed FLC levels not so different from AL (~100 to 1000 mg/L, similar to Fig.1).

We have re checked the data shown in figure 1 with patients presenting with or without a LC only clone, and the data is the same, although the IG heavy chain levels to those presenting having a HC secreting clone do correlate (Pearson R 0.38 with p=0.007) the PC BM load.

In addition, the FLC levels as measured by the 'binding site' in www.wikilite.com and directly comparing study as Kumar et al. and Snozek et al are in line with these measurements.

 

• Line 175: whether ref 39 indeed describes a highest sensitivity to PI of AL cells , ref 38 did not show any higher apoptosis rates of AL PCs. They found less apoptosis rate than in MGUS. Also, the authors should discuss more the direct role of the LC in the PC stress as described in ref 39. Once again, it is more likely the LC (because of its abnormal folding and/or aggregation in the cells) that induces this sensitivity to PI than a proper feature of the PCs.

Ref 39 was specifically looking in-vitro at PC apoptosis and autophagy in relation to LC levels, whereas ref 38 is looking at patients' primary cell analysis. We have corrected the comparison of apoptotic rates from ref 38 and added data from ref 39. More details of Ref 39 study added to text.

 

• Paragraph 4 is confusing. On one hand, the authors state that a deep hematologic response, i.e. a deep decrease in circulating pathogenic LC, is the only factor to correlate with survival and organ responses but on the other hand, they state that the LC still cause damages in the absence of increased amount of amyloid load. They cite review papers (67-69) but the clinical evidence are still scarce. The remaining toxicity in treated patients can also be due to the long persistence of amyloid deposits in organs because of their high resistance to proteolysis.

We agree with the reviewer that toxicity is obviously because of organ-amyloid persistence, and have added this to the paragraph. However, the new data accumulating on MRD significance argues for the addition of very low level LC persistence and added persistent toxicity. This is a very good example of a data coming from patient and not laboratory studies.

 

• Line 294: the fact that LC sequences (and so, structural properties) influence their propensity to aggregate and deposit is highly documented, at least in vitro (numerous papers from Marina Ramirez-Alvarado, Jonathan Wall, Johannes Buchner…). Consequently, it is not surprising to find no correlation between the PC burden / FLC levels and the organ damages.

              We have cited and discussed the papers in the text

 

• Line 327: several recent papers give information on the transcriptomic and proteomic profile of amyloid infiltrated tissues (Jordan et al, leukemia 2020; Kourelis et al, JACC Cardiooncol 2020) and should be cited and discussed.

            We have cited and discussed these papers in the text.

 

 

• Line 333: sentence “dermal fibroblast… “ is confusing.

We have clarified the sentence and its meaning.

 

• Line 346: GWAS data: increased amount of monocytes: is it a cause or a consequence of amyloidosis?

We have clarified the sentence and its meaning

 

• Many new therapeutic strategies in AL amyloidosis aim at removing actively amyloid fibrils from the organs. Could you consider adding a paragraph on these encouraging approaches?

We thank the reviewer for this suggestion and have added a short paragraph as noted.

Minor points:

• Line 315: “some are very toxic to these AL patients”. Is it specifically toxic to AL patients but not MM? Is there any explanation? Refs?

Due to their frailty as compared with MM patients. We have added this to the text.

• Line 320: “mechanisms involved in … death caused by the amyloid proteins are poorly understood” Are you sure? The cause of death during AL amyloidosis are well known.

We refined the sentence to " organs dysfunction and cellular-death"

 

• Line 371: “That could sensitizers AL PCs”. Confusing

We have changed the sentence to: "This system will also allow the study of novel agents that may be used and screened as treatment and adjuvant treatments for AL PCs."

 

• Line 378: ref 92: JJN-3 do not express an amyloid kappa LC

We corrected the typo error

• Please reread the manuscript to correct confusing sentences that miss verbs (see for instance the last sentence of the abstract) or are too long and have to be rephrased.

We have modified the manuscript

 

 

Reviewer 2 Report

Moshe E Gatt and Marjorie Pick wrote a comprehensive review on learning from patients about the interplay between clinical and laboratory research in AL amyloidosis.

The review is well written, highlights the challenges in research on AL amyloidosis and provides deep insights into current knowledge about characteristics and pathophysiology of AL amyloidosis.

The manuscript might be further improved by addressing the following points:

General

• The authors use numerous abbreviations. It might be easier to read (especially for non-hematologists) if the number of abbreviations could be reduced and some abbreviations could be omitted and the words completely spelled instead.
• Please provide the complete terminus an abbreviaton stands for, the first time the abbreviaton is introduced (f.e. „BM“ in line 49 or OS in line 250)
• The manuscript might benefit from another orthographic revision. There are some typos as well as several additional spaces or punctuation marks (f.e. bracket after „AL“ in line 44)

 

Individual

• The sentence lines 80-83 seems to be mixed up. Please revise.
• Table 1: „large complexes“ might better be called „high voltage QRS“ in ECG
• Table 1: „unexplained peripheral edema“ and „unexplained peripheral polyneuropathy“ often are not unexplained but rather wrongly explained by concomitant diseases (diabetes) or well explained by heart failure (which, in turn, is caused by AL). I would recommend to remove the „unexplained“
• Lines 132-135: the presentation oft he odds ratios is rather unusual. It might be sufficient to present OR without decimales.
• There is the expression „light chains (LC)“ and „free light chains (FLC)“. Please briefly explain the difference between both expressions to the non-hematologist, once you introduce FLC.
• Line 156. Is there a difference between AL and LC AL? If so, please explain, if not, please remove the LC.

 

Author Response

We thank you for considering our manuscript: Learning from patients: the interplay between clinical and laboratory research in AL amyloidosis for publication and we thank the reviewers for their thorough remarks.

Here are our point by point corrections to the remarks and manuscript:

The manuscript might be further improved by addressing the following points:

We thank the reviewer for the remarks and have corrected all.

General

• The authors use numerous abbreviations. It might be easier to read (especially for non-hematologists) if the number of abbreviations could be reduced and some abbreviations could be omitted and the words completely spelled instead.
• Please provide the complete terminus an abbreviaton stands for, the first time the abbreviaton is introduced (f.e. „BM“ in line 49 or OS in line 250)
• The manuscript might benefit from another orthographic revision. There are some typos as well as several additional spaces or punctuation marks (f.e. bracket after „AL“ in line 44)

 

Individual

• The sentence lines 80-83 seems to be mixed up. Please revise.

The sentence has been modified.

• Table 1: „large complexes“ might better be called „high voltage QRS“ in ECG

Changed

• Table 1: „unexplained peripheral edema“ and „unexplained peripheral polyneuropathy“ often are not unexplained but rather wrongly explained by concomitant diseases (diabetes) or well explained by heart failure (which, in turn, is caused by AL). I would recommend to remove the „unexplained“

Removed and amended

• Lines 132-135: the presentation oft he odds ratios is rather unusual. It might be sufficient to present OR without decimales.

Changed

• There is the expression „light chains (LC)“ and „free light chains (FLC)“. Please briefly explain the difference between both expressions to the non-hematologist, once you introduce FLC.

Thank you for this remark. We have added a clarification

• Line 156. Is there a difference between AL and LC AL? If so, please explain, if not, please remove the LC.

removed

 

Round 2

Reviewer 1 Report

The manuscript has been clearly improved and, although some hypotheses and statements remain, in my opinion very questionable, the manuscript likely deserves to be published.

However, I still do not agree with the text claiming that FLC are log lower in AL than in MM. Even in Kumar et al and Snozek et al, the difference is not log (~double or triple) and many other studies show no difference. See for example, Kyrtsonis et al. 2007 Brit J Haematol who observed a median of 83mg/l and 213mg/l of FLCkappa and FLClambda respectively in MM and Bochtler et al, 2008 Haematologica who observed a median of 322 mg/l and 195mg/L of FLCkappa and FLClambda respectively in AL. In all of the MM studies (including Snozek), FLC only rarely reach 1000mg/L as stated in the text line 385 (of course there are exceptions but it's not the norm for MM).

Please correct typos especially in the added text.

 

Author Response

See Attached please

Author Response File: Author Response.pdf

Reviewer 2 Report

thank you for sending the revised version of the manuscript. It is much easier to read now and improved a lot. 

The review met all my suggestions.

Author Response

Thank you for your remarks