Successful Therapy over 12 Months of People with Cystic Fibrosis with Rare Non-phe508del Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutations with Elexacaftor/Tezacaftor/Ivacaftor (ETI)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe Authors describe the clinical parameters of six patients with cystic fibrosis before and after one year of treatment with ETI. Although the genotype of the six patients is rare, no conclusions can be drawn about the effect of ETI on any given genotype by considering only one case. The study should be conducted on at least triplicate of each genotype.
Furthermore, the choice of controls is completely unsuitable, i.e. the number of controls is too low, it is not matched for age and sex and, above all, 1/4 controls is homozygous for one of the most frequent mutations (phe508del).
Minor comments:
- acronyms should be reported in full when cited for the first time, e.g. ENT lines 29, 74, 131;
- line 130: how was lung function assessed? Please, describe it;
- lines 149-150: non-parametric paired comparison between more than two groups should be performed using the Friedman test.
Author Response
Comment 1: The Authors describe the clinical parameters of six patients with cystic fibrosis before and after one year of treatment with ETI. Although the genotype of the six patients is rare, no conclusions can be drawn about the effect of ETI on any given genotype by considering only one case. The study should be conducted on at least triplicate of each genotype.
Response 1: We agree with Reviewer 1 that our data alone cannot provide definitive conclusions on the effects of ETI for specific CFTR genotypes. We therefore have revised the abstract, discussion, and conclusion sections to reflect this limitation. Nevertheless, recent in vitro data (Bihler et al., 2024, now cited in lines 399-400 and 429) demonstrate responsiveness of CFTR mutation M1101K (present in five of our pwCF) and borderline responsiveness of mutation R347P (present in one pwCF) to ETI therapy. Consistent with these findings, our study revealed positive ETI effects on CFTR function, as indicated by reduced sweat chloride levels, along with clinical improvements in all six ETI-treated pwCF with rare mutations. These findings are noteworthy and should therefore be made available for colleagues in the field.
While we recognize the inherent limitations of this observational study with a small cohort, we took this into account by submitting our findings as brief report rather than a research paper. The cohort size reflects the rarity of the mutations studied: among approximately 300 pwCF treated at the two CF centers involved, the prevalence of such mutations is exceedingly low. Larger studies would be logistically unfeasible within a manageable timeframe. A search of the CFTR2 database (cftr2.org), which includes nearly 40.000 CF patients, underscores this challenge. For instance, only two patients share the mutation combination R347P;R1066C, none the combination Q220X;M1101K, and very few other combinations, such as 2789+5G->A;M1101K (three patients), G542X;M1101K (three patients), CFTRdele2,3;M1101K (two patients), and G551D;M1101K (six patients). Even in the French Compassionate Use Program, the 45 ETI responders had highly heterogeneous mutation combinations, limiting subgroup analyses to those with N1303K (Burgel et al., 2023). We have expanded the discussion of the manuscript to address this issue in lines 359–370.
Given the constraints mentioned above, forming treatment or control groups for clinical trials involving rare mutations is rather impractical. However, the pwCF in our cohort share two key features: they have CFTR mutations that unequivocally cause CF and result in a classical clinical CF phenotype, although at least one mutation permits production of low CFTR protein levels. Instead of comparing patients, our study focused on evaluating individual patient trajectories before and during ETI treatment over a 12-month observation period, using both clinical and objective parameters, such as sweat chloride levels. We have now addressed these points in the discussion section of our revised manuscript, lines 330-338.
Importantly, we limited our study to patients with mutations recently confirmed as responsive to ETI in vitro, as pointed out in the abstract, the introduction, the results and discussion.
To account for the small cohort size, we used only non-parametric statistical tests.
In summary, we strongly believe that data from patients with rare diseases should be published in journals, which are renown in the field, provided their limitations are transparently discussed as in our manuscript. In doing so, our study may fill an important gap in CF research by providing real-world data on a population underrepresented in other clinical trials.
Comment 2: Furthermore, the choice of controls is completely unsuitable, i.e. the number of controls is too low, it is not matched for age and sex and, above all, 1/4 controls is homozygous for one of the most frequent mutations (phe508del).
Response 2. We do not agree with this assessment. We are aware that the group of untreated pwCF included into our study is small. Therefore, the results can only serve as an indication, that the expected course of pwCF without ETI may also be observed in a clinical setting, where all pwCF have comparable access to common CF treatment, no matter what the underlying CFTR mutations are. For the same reason, the rarity of non-phe508del mutations, the inclusion of age and sex matched controls are impossible to achieve. Nevertheless, the results from the small study group suggests that our methods of evaluation were carried out correctly, e.g. that in the untreated group the objective parameter sweat chloride stayed high over the observation period. Given that there were only very few pwCF available without ETI treatment, we were forced to consider any pwCF with a classical phenotype as suitable to serve as a “control” patient, regardless of CFTR mutations. Moreover, to increase reliability and robustness of our clinical observations, our study continuously monitored the disease course over 12 months, instead of picking out a few time points only. Since most pwCF have at least one phe508del variant, the majority of these pwCF receive ETI therapy in industrial countries and are therefore no longer available as controls. Nevertheless, despite the low number of cases and heterogeneity of our cohort, we have attempted to carry out a comparable data collection and analysis. Even in the small series of cases of pwCF reported here, we observed clearly detectable positive effects of ETI therapy such as on the objective parameter sweat chloride.
Comment 3: Minor comments: - acronyms should be reported in full when cited for the first time, e.g. ENT lines 29, 74, 131;
Response 3: We thank the author for this comment and have adjusted the manuscript, now avoiding the acronyms, where possible such as in the abstract and reporting them in full at their first appearance, e.g. ENT now in line 31 and 77, BMI, now line 30, 67-68, FEV1 in line 67, CFQ-R, line 71.
Comment 4: - line 130: how was lung function assessed? Please, describe it;
Response 4: Lung function was performed using body plethysmography (Vyaire Master Screen Body with Vyaire software), according to Global Lung Function Initiative/European Respiratory Society guidelines. This information has now been included in the Methods section of our revised manuscript, now lines 133-135.
Comment 5: - lines 149-150: non-parametric paired comparison between more than two groups should be performed using the Friedman test.
Response 5: We thank reviewer 1 for this comment and have adapted our statistical analysis accordingly. Please see the corrected statistics, lines 155-158. Importantly, the modifications did not result in major changes regarding statistical significances.
Reviewer 2 Report
Comments and Suggestions for AuthorsThank you very much for this very interesting manuscript about ETI treatment in people with CF with rare non-phe508del CFTR mutations.
I have only some questions and suggestions
Line 199: "sweat chloride values 199 dropped >30 (<60) mmol/L in three pwCF"; I think you could write "between 30 and 60 mmol/L"
Why do you insert a patient with phe508del*/phe508del* in the control group? Why didn't he take ETI? Why didn't you evaluated another patient without treatable mutations instead of this treatable patients? Don't you think that this patient with F508del/F508del could affect the tests and evaluations of the control group? Please clarify.
Line 389: "Four of ETI- treated pwCF in our cohort have the CFTR mutation M1101K"; looking at Table 1 patients they are five; are you excluding the patient who developed a malignancy? was this patients teh patient number 1 (R347P/R1066C)? So why didn't you say that five patients had M1101K? Please clarify.
Please write clearly better if all the measurement of the intervention group included the patient who developed a malignancy 6 months after ETI initiation. Or for the comparison with the cnotrol group you evaluated only five treated patients vs 4 control patients. Many thanks
Many thanks
Author Response
Comment 1: Line 199: "sweat chloride values 199 dropped >30 (<60) mmol/L in three pwCF"; I think you could write "between 30 and 60 mmol/L"
Response 1: We thank reviewer 2 for pointing this misleading wording out to us. We intended to express that after 1-3 months of ETI therapy, sweat chloride levels decreased more than 30 mmol/l in three, and more than 60 mmol/l in two pwCF. This decrease resulted in a milder CF phenotype (30<X<60 mmol/l) and in two cases even in normal sweat chloride levels (<30 mmol/l). We have modified this sentence in the results section accordingly, now lines 207-209, and complemented the discussion in lines 338-339.
Comment 2: Why do you insert a patient with phe508del*/phe508del* in the control group? Why didn't he take ETI? Why didn't you evaluated another patient without treatable mutations instead of this treatable patients? Don't you think that this patient with F508del/F508del could affect the tests and evaluations of the control group? Please clarify.
Response 2: The one pwCF with the phe508del*/phe508del mutations so far has refused ETI because of a psychiatric disorder, which he would like to get under control before starting ETI. His phenotype resembles the phenotype of the other untreated pwCF, which is why he was included in the control group. In the untreated group the objective parameter sweat chloride stayed high over the observation period, which is what all pwCF with the classical phenotype have in common. Given the above-mentioned limitations, we consider any pwCF with a classical phenotype as suitable to serve as a “control” patient, regardless of CFTR mutations, which we describe in the discussion, lines 383- 394. Moreover, our study monitors a disease course of over 12 months instead of single time points to ensure that the observed effects hold over a longer observation period.
Comment 3: Line 389: "Four of ETI- treated pwCF in our cohort have the CFTR mutation M1101K"; looking at Table 1 patients they are five; are you excluding the patient who developed a malignancy? was this patients the patient number 1 (R347P/R1066C)? So why didn't you say that five patients had M1101K? Please clarify.
Response 3: We thank reviewer 2 for pointing this mistake out to us. It is correct that five pwCF have M1101K and the one pwCF with R347P/R1066C developed a (semi)malignant disease (MGRS from a longer persistent MGUS). So far, ETI by itself has not been described to cause malignancies, according to literature and case reports. This one pwCF initially improved regarding her CF symptoms under ETI, and therefore was able to tolerate MGUS treatment pretty well. Currently, she is in a stable condition and would like to continue ETI treatment. We have added the details in the discussion lines 348-354.
Comment 4: Please write clearly better if all the measurement of the intervention group included the patient who developed a malignancy 6 months after ETI initiation. Or for the comparison with the control group you evaluated only five treated patients vs 4 control patients. Many thanks
Response 4: The pwCF who developed a malignant disease (see above) during the observation period was included in the evaluation at time points 0, 3 and 6 months, but her data were excluded from time points 9 and 12 months, during which she received a proteasome inhibitor (in addition to ETI). We have adjusted the results section in lines 185-189 accordingly. In the two CF centers participating in this study, only 5 pwCF without ETI therapy met all inclusion criteria. We are fully aware that the small group of untreated pwCF can only serve as a hint, that the expected course of pwCF without ETI may also be observed in a clinical setting, where all pwCF have comparable access to common CF treatment, no matter what the underlying CFTR mutations are. On the other hand, the small group size suggests that our methods of evaluation were carried out correctly, since in the untreated group the objective parameter sweat chloride stayed high and no significant change of lung function or BMI occurred over the entire observation period. We consider any pwCF with a classical phenotype as suitable to serve as a “control” patient, regardless of CFTR mutations. The focus of this brief medical report was the course of disease. To ensure that our observations are reliable, we therefore chose an extended observation period of 12 months. The pwCF in our cohort have in common that they all have CFTR mutations which result in the classical clinical CF phenotype.
Reviewer 3 Report
Comments and Suggestions for AuthorsI read this paper with great interest. This study fills an important gap in CF research by providing real-world data on a population underrepresented in clinical trials. The use of robust methodology, including well-defined outcome measures and appropriate statistical analyses, strengthens the study.
However, I have several major concerns which authors must address before accepting this paper for publication.
- The cohort is limited to only six patients in the intervention group and four in the control group, reducing the statistical power of the study. This limitation is somewhat inherent given the rarity of non-phe508del mutations, but future research with larger cohorts is essential to confirm these results. Please better address this in discussion.
- The control group consists of only four patients, which further limits comparisons. Moreover, the absence of ETI in the control group leaves open questions about the relative effectiveness of ETI compared to other treatments or supportive care options. The study would benefit from a larger and more matched control group. Please comment on that.
- A portion of the study rationale is based on in vitro data from the pharmaceutical company. These findings are promising, but additional clinical evidence is necessary to strengthen the argument for expanding ETI approval to rare mutations in the European Union. Please address this point.
- I found several minor English errors throughout the manuscript. Please have a deep language revision.
Comments on the Quality of English Language
moderate revision required.
Author Response
Comment 1: - The cohort is limited to only six patients in the intervention group and four in the control group, reducing the statistical power of the study. This limitation is somewhat inherent given the rarity of non-phe508del mutations, but future research with larger cohorts is essential to confirm these results. Please better address this in discussion.
Response 1. We are grateful for this comment and fully agree that our small cohort greatly limits the statistical power of our study. As reviewer 3 points out, this limitation is inherent in face of the rarity of non-phe508del mutations. Even though it would be desirable to confirm these results in larger cohorts this is an unrealistic goal to achieve in a mid-term time range. Since we are aware of this limitation, we decided to submit this manuscript as a brief medical report, rather than a research paper. Even in the cited publication from the French Compassionate Use Program (FCUP, now addressed lines 339-347) CFTR mutation combinations among the 45 ETI responders were very heterogeneous. Groups of pwCF with the same mutations could not be analyzed, except for N1303K, since 6 pwCF were homozygous for that CFTR mutation (Burgel et al. 2023, citation No. 23 in our paper). Although the cohort in our study is much smaller than in the cited publication from the FCUP, data for our study were collected independently from a pharmaceutical sponsor and our observation period is significantly longer. We are convinced that it is in the best interest of pwCF with rare mutations to publish real world data, not only larger studies driven mainly by the interests of pharmaceutical companies. We have now addressed these points in the discussion section of our revised manuscript (lines 395-397).
Comment 2: - The control group consists of only four patients, which further limits comparisons. Moreover, the absence of ETI in the control group leaves open questions about the relative effectiveness of ETI compared to other treatments or supportive care options. The study would benefit from a larger and more matched control group. Please comment on that.
Response 2: We agree with reviewer 3 that a larger control group would be desirable. However, in the two CF centers participating in this study, only 5 pwCF without ETI, who did not meet exclusion criteria, were available. More than 80% of pwCF receive a CFTR modulator these days. We are aware that our small group of untreated pwCF can only serve as an indication, that the expected course of pwCF without ETI may also be observed in our clinical environment, where all pwCF have comparable access to common CF treatment, no matter what the underlying CFTR mutations are. Nevertheless, the results from the small study group suggest that our methods of evaluation were carried out correctly, e.g. that in the untreated group the objective parameters sweat chloride stayed high and the lung function or BMI remained unchanged over the observation period. We therefore consider any pwCF with a classical phenotype as suitable to serve as a “control” patient, regardless of CFTR mutations. Since the focus of this brief medical report was the course of disease, and to ensure that our observations were reliable, we chose a rather long period of 12 months. The pwCF in our cohort thus have in common that they exhibit CFTR mutations, which distinctly cause CF including the classical clinical CF phenotype. We have extended our discussion, please see lines 383-394.
Comment 3: - A portion of the study rationale is based on in vitro data from the pharmaceutical company. These findings are promising, but additional clinical evidence is necessary to strengthen the argument for expanding ETI approval to rare mutations in the European Union. Please address this point.
Response 3. In the long run it would be desirable that the EMA approves the use of certain drugs for rare diseases based on in vitro data with strict requirements for the monitoring of in vivo effects to decide whether such a treatment should be continued or discontinued. We agree that additional evidence is necessary to strengthen the argument for expanding ETI approval to rare CFTR mutations in the EU. However, due to the inherent rarity of these patients it appears unrealistic that larger studies can be conducted. Decisions on treatment may be supported in the future by personalized in vitro testing of patient-derived respiratory epithelia, although such tests are not yet part of routine diagnostics. These aspects have now been included into the discussion and conclusion of the revised manuscript, see lines 438-440 and 459. Combining such in vitro approaches with stringent clinical monitoring may justify treatment based on hospital exemptions. Since CF patients with rare CFTR mutations that were ETI-responsive in vitro cannot wait for an approval that has been delayed for years or may never be granted, we have made the effort in our two CF centers to conduct a strict and detailed monitoring of ETI effects over more than 12 months.
Comment 4: - I found several minor English errors throughout the manuscript. Please have a deep language. Comments on the Quality of English Language: moderate revision required.
Response 4: We thank reviewer 3 for this comment and have corrected several English errors while revising the entire manuscript. All changes, including those of the content, are highlighted in yellow.
Reviewer 4 Report
Comments and Suggestions for AuthorsThis study report evaluated the effectiveness of Elexacaftor/Tezacaftor/Ivacaftor (ETI) in people with cystic fibrosis (pwCF) carrying rare non-PHE508del CFTR mutations. It is a 12-month retrospective study assessing six pwCF patients of different ages (ranging from 6 to 66 years) and included four control individuals who did not receive ETI treatment. The study focused on the off-label use of ETI and explored its clinical outcomes.
However, the study raises the following concerns:
1. Only six patients received ETI treatment, and the control group included just four individuals. Such a limited sample size is insufficient to represent the broader population of pwCF with rare non-PHE508del CFTR mutations, limiting the strength of evidence for policy changes.
2. The study did not use randomization, blinding, or a controlled trial design, which could lead to observer bias and patient bias (e.g., patients may have high expectations for the drug's effectiveness).
3. There were discrepancies between the control group and the treatment group in baseline lung function, mutation types, and disease severity.
4. Only a few survey responses were collected at the 6- to 8-month mark, making the assessment of quality-of-life improvements incomplete.
5. The study heavily relied on subjective questionnaires, such as SNOT-22 and CFQ-R, to assess ENT symptoms and quality of life, with limited use of physiological indicators.
Author Response
Comment 1. Only six patients received ETI treatment, and the control group included just four individuals. Such a limited sample size is insufficient to represent the broader population of pwCF with rare non-PHE508del CFTR mutations, limiting the strength of evidence for policy changes.
Response 1: We are aware of the limitations of this observational study with a small cohort, which is why we submitted it as a brief report rather than a research paper. We do not claim that we have evidence for any definitive policy changes. However, our small study is providing a hint that patients with certain non-PHE508del CFTR mutations may benefit from ETI treatment. Although the cohort in our study is much smaller than for example in the cited publication from the French Compassionate Use Program (FCUP), data for our study were collected independently from a pharmaceutical sponsor and our observation period is significantly longer. We are convinced that it is in the best interest of pwCF with rare mutations to publish real world data, not only larger studies driven mainly by the interests of pharmaceutical companies. We have now addressed these points in the discussion section of our revised manuscript (also see lines 328-338, 372-397).
Comment 2. The study did not use randomization, blinding, or a controlled trial design, which could lead to observer bias and patient bias (e.g., patients may have high expectations for the drug's effectiveness).
Response 2: As outlined above, forming treatment or control groups for clinical trials in face of the rarity of non-phe508del mutations is widely considered impractical. Burgel and colleagues who report data from an observational study design involving a heterogeneous cohort of pwCF with non-phe508del mutations (citation No. 23) point out that it would be extremely difficult to perform a randomized-controlled trial in pwCF and rare CFTR variants, especially in patients with advanced lung disease who are usually excluded from clinical trials. Of note, our observation period of 12 months was by far longer compared to the 4-6 weeks reported by Burgel et al. According to the latter, all ETI-treated pwCF in our cohort could be defined as ETI responders because of a sweat chloride reduction of at least 20 mmol/l and/or FEV1 increase of at least 10 percent, with stabilization of lung function over 12 months. We cannot rule out observer bias and patient bias but tried to ameliorate this by an extended observation period. To transparently address these limitations, we have completely revised the abstract and discussion of our manuscript.
Comment 3. There were discrepancies between the control group and the treatment group in baseline lung function, mutation types, and disease severity.
Response 3: We agree with reviewer 4 that our small cohort which served as control group and our small ETI-treated group had discrepancies, but they had in common that all were pwCF with a classical phenotype with pathological sweat test levels before ETI treatment except for the one with G551D, which is explained by previous treatment with the CFTR modulator Ivacaftor. The original sweat chloride level of this individual was not available but can be presumed to have been much higher, as can be seen in the 6 pwCF reported in the cftr2.org database (average sweat chloride: 106 mmol/l). The reason for reporting the clinical course in the control group despite its great heterogenicity was to show that the expected course of pwCF without ETI could also be observed in our clinical environment, where all pwCF have comparable access to common CF treatment, no matter what the underlying CFTR mutations are. The results from the small study group suggests that our methods of evaluation were carried out correctly, e.g. that in the untreated group the objective parameters sweat chloride stayed high and the lung function or BMI remained unchanged over the observation period. We therefore consider any pwCF with a classical phenotype as suitable to serve as a “control” patient, regardless of CFTR mutations. Since the focus of this brief medical report was the course of disease, and to ensure that our observations were reliable, we chose a rather long period of 12 months. The pwCF in our cohort thus have in common that they exhibit CFTR mutations, which distinctly cause CF including the classical clinical CF phenotype. Since most pwCF have at least one phe508del variant, these pwCF receive ETI therapy in industrial countries in most cases and are therefore no longer available as controls. As outlined above, due of the rarity of non-phe508del mutations, controls with more similar baseline lung function or disease severity are unrealistic to achieve. Nevertheless, despite the low number of cases and heterogeneity of pwCF in our cohort, we have attempted to carry out a comparable data collection and analysis. Even in the small series of cases of pwCF reported here, we observed clearly detectable positive effects of ETI therapy such as on the objective parameter sweat chloride. Therefore, our study may fill an important gap in CF research by providing real-world data on a population underrepresented in other clinical trials.
Comment 4. Only a few survey responses were collected at the 6- to 8-month mark, making the assessment of quality-of-life improvements incomplete.
Response 4: We politely point out that this statement is not correct, please see Table S2. As far as quality-of-life questionnaires CFQ-R, the SNOT-22 Score and the activity questionnaires (AQ) are concerned, we have collected survey responses from all individuals, both from the ETI-treated as well as from the untreated individuals at month 0 and at 6-8 months. The only difference was that we additionally evaluated the ETI- treated group with the Treatment Satisfaction Questionnaire for Medication (TSQM), since this obviously applies to ETI-treated individuals only.
Comment 5. The study heavily relied on subjective questionnaires, such as SNOT-22 and CFQ-R, to assess ENT symptoms and quality of life, with limited use of physiological indicators.
Response 5: Our observational study used both, questionnaires, which are validated for clinical trials, and objective parameters such as sweat chloride reduction of more than 20 mmol/l (which was achieved in all treated individuals) as well as the clinical improvement of lung function over an extended observation period of 12 months.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe Authors significantly improved the manuscript following the reviewer's suggestions/comments. The choice of the type of publication also appears suitable.
Author Response
We thank Reviewer 1 for the time spent on the review and for accepting our manuscript.
Dorit Fabricius
Reviewer 3 Report
Comments and Suggestions for AuthorsAuthors fully replied to my comments. Therefore I believe that this paper can now be accepted.
Author Response
We thank Reviewer 2 for the time spent on the review and for accepting our manuscript.
Dorit Fabricius
Reviewer 4 Report
Comments and Suggestions for Authorsno
Author Response
We thank Reviewer 4 for the time spent on the review and hope that our latest improvements to the manuscript based on the academic editor's suggestions will be satisfactory.
Dorit Fabricius
