Genomic landscape of lymphatic malformations: a case series and response to the PI3Kα inhibitor alpelisib in an N-of-1 clinical trial
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Evaluation Summary:
This manuscript could be of interest to physicians and researchers in the field of vascular anomalies. The cohort of patients with lymphatic malformations is reasonably sized (n=30) and the claims made by the authors are supported by the data as well as by current knowledge in the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Lymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases.
Methods:
We examined the genomic landscape of a patient cohort of LMs ( n = 30 cases) that underwent comprehensive genomic profiling using a large-panel next-generation sequencing assay. Immunohistochemical analyses were completed in parallel.
Results:
These LMs had low mutational burden with hotspot PIK3CA mutations ( n = 20) and NRAS ( n = 5) mutations being most frequent, and mutually exclusive. All LM cases with Kaposi sarcoma-like (kaposiform) histology had NRAS mutations. One index patient presented with subacute abdominal pain and was diagnosed with a large retroperitoneal LM harboring a somatic PIK3CA gain-of-function mutation (H1047R). The patient achieved a rapid and durable radiologic complete response, as defined in RECIST1.1, to the PI3Kα inhibitor alpelisib within the context of a personalized N -of-1 clinical trial (NCT03941782). In translational correlative studies, canonical PI3Kα pathway activation was confirmed by immunohistochemistry and human LM-derived lymphatic endothelial cells carrying an allele with an activating mutation at the same locus were sensitive to alpelisib treatment in vitro, which was demonstrated by a concentration-dependent drop in measurable impedance, an assessment of cell status.
Conclusions:
Our findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations.
Funding:
R.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953.
Clinical trial number:
NCT03941782
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Author Response
Reviewer #1 (Public Review):
“The presentation of the clinical data in table 1 is very short and patchy and seems incomplete, also some of the classifications don't appear to be correct E.g. PTEN hamartoma tumor syndrome is a genetically distinct entity, that does not harbor somatic PIK3CA mutations but rather germline PTEN mutations. There are 5 patients with CLOVES and 1 patient with KTS, these patients often have mixed (e.g. lymphatic-venous) malformations, are the analyzed samples truly pure LMs? There are some more instances where I wonder if the presented data allows the reader to understand the cases.”
Thank you for this thoughtful comment. One of the limitations of our study is that the CGP cohort was a retrospective study of data available from an international reference laboratory. While the use of data …
Author Response
Reviewer #1 (Public Review):
“The presentation of the clinical data in table 1 is very short and patchy and seems incomplete, also some of the classifications don't appear to be correct E.g. PTEN hamartoma tumor syndrome is a genetically distinct entity, that does not harbor somatic PIK3CA mutations but rather germline PTEN mutations. There are 5 patients with CLOVES and 1 patient with KTS, these patients often have mixed (e.g. lymphatic-venous) malformations, are the analyzed samples truly pure LMs? There are some more instances where I wonder if the presented data allows the reader to understand the cases.”
Thank you for this thoughtful comment. One of the limitations of our study is that the CGP cohort was a retrospective study of data available from an international reference laboratory. While the use of data from a reference laboratory enables the study of relatively high numbers of rare diseases, it limits us to only clinical information provided by the ordering physicians at the time of testing. The table data under the heading “Clinical syndrome” was collected from review of test order forms and pathology reports submitted to the reference laboratory at the time of testing. The scope of our study did not enable outreach to ordering physicians and pathologists to determine if and how the genomic results refined the working clinical diagnosis and/or pathologic diagnoses.
To more accurately describe the source of data in the “clinical syndrome” column, we have now edited the column to read “Submitted clinical syndrome” and clarified this in the Results section and, further, have clarified the limitations of this study in more detail in the Discussion.
“If the histology is described as kaposiform, these cases likely represent kaposiform lymphangiomatosis, which is a very different disease than common LMs. KLA belongs to the group of complex lymphatic anomalies and usually is caused by NRAS mutations, which would be in line with the presented data. Case 24 (conventional histology, NRAS mutation) could also be a generalized lymphatic anomaly. This distinction of common LMs and complex lymphatic anomalies (including GLA and KLA) should be made and should include what is known about the genetics of these diseases. Taken together with the first point, the presentation of the cases might benefit from a more structured description and classification.”
We agree that our NRAS mutant tumors with kaposiform histology are compatible with the entity of kaposiform lymphangiomatosis (KLA), and have therefore added additional details about the clinical, histologic, and genomic features of KLA to the Introduction and Discussion.
“In the discussion and other parts of the manuscript, terms describing LMs and tumors are interchanged frequently. This mistake is also present in the study protocol (NCT03941782), in which "locally advanced or metastatic cancer" is listed as an inclusion criterion. Other examples include "tumor nuclei". Much of the cited literature also focuses on oncology rather than vascular malformations. And LMs are directly compared to "other low-grade pediatric tumors".
Also, clonality is a concept not too often used in vascular malformations, as an aberrant development of vascular structures during embryogenesis is seen as the cause of vascular malformations, as opposed to clonal expansion in tumors (but this might warrant further investigation in the field). Thus, the manuscript mixes tumors and malformations, however, it should be stressed, that LMs are not tumors but vascular malformations.”
Thank you again. We have considered the diagnosis and classification of vascular anomalies (vascular malformations and others) to be a holistic integration of clinical examination, imaging studies, pathology diagnosis, and/or genomic results. As addressed above, one of the limitations of our study is that the CGP cohort was a study of data available from an international reference laboratory. While the use of data from a reference laboratory enables the study of relatively high numbers of rare diseases, it limits us to only clinical information provided by the ordering physicians at the time of testing and only to one representative pathology specimen submitted by the pathology laboratory. The scope of our study did not enable outreach to ordering physicians and pathologists to determine if and how the genomic results refined the working clinical diagnosis and/or pathologic diagnoses.
Recognizing these limitations, we agreed to use the term lymphatic malformations to describe the lesions in our cohort. Lymphatic malformation is widely accepted to include a clinicopathologic continuum of benign tumors of lymphatic origin (https://rarediseases.org/rare-diseases/lymphatic-malformations/), including cystic lymphangioma, kaposiform lymphangiomatosis, macro/microcystic lymphatic malformation. While evidently an imperfect solution, we have also clarified their use of this term in the Introduction.
“The explanation for the reduced EF doesn't quite make sense, as there should be little blood flow into the LM. This is different from the Venot paper in Nature, where the reduced EF was due to the presence of an AV malformation.”
We agree the EF was an unusual finding and our case with reduced EF is somewhat bewildering. However, EF was measured both by ECHO and by cardiac MRI with the same results. The LM in this patient was of a giant size and could potentially produce hemodynamic changes, as proposed in the Results. That is the only coherent explanation we could identify as the EF corrected after we achieved a response.
“The data on the (back at the start of treatment of the patient novel) alpelisib is presented as a rather new finding. However, clinical data on its use in PROS diseases have already been published starting in 2018 (the paper from Venot is also mentioned in the manuscript). At the moment, an international clinical trial on alpelisib in PROS disease is recruiting, which could be mentioned.”
Our index case of a giant LM treated with alpesilib was within an early trial when the drug was experimental, and it represents the only case of non-syndromic LM that achieved complete remission and remained in sustained remission for years, but relapsed after discontinuing the medicine. Thus, it provides mechanistic insight into the potential efficacy and need for continuous administration in sporadic non-syndromic cases. Interestingly, the FDA approval of alpelisib came while this manuscript was under review. The Discussion was revised to better describe the current state of the field.
“Treatment: The rather high dose of 350 mg/d is not further discussed. Also, a patient like this would usually first receive sirolimus, especially back when alpelisib was started in this patient since it was much more experimental at that time point. This should also be explained.”
When our trial patient was deemed a challenging surgical oncology case, we felt targeting the driver mutation made more sense than using mTOR inhibitors, and indeed we achieved complete response with the phase I selected dose with no detectable toxicity. The patient continues on alpesilib as of now, more than 5 years later.
Reviewer #2 (Public Review):
“This is not the first demonstration of somatic activating NRAS mutation associated with Kaposiform lymphangiomatosis. A prior study demonstrated that 10/11 patients with Kaposiform lymphangiomatosis had NRAS mutation (PMID 30542204).”
Thank you for the suggestion to add clarity to the statement that our findings of NRAS in LM with kaposiform histology align with prior studies showing NRAS driver mutations in kaposiform lymphangiomatosis which is considered to be a distinct histologic and clinical entity. We have now refined the statement to highlight the relationship between the NRAS mutant cases in our cohort and kaposiform lymphangiomatosis.
“This is not the first demonstration of somatic activating PIK3CA patients exhibiting malformation shrinkage with alpelisib and they only had a single treated patient. A prior study showed similar results in 6/6 patients treated with alpelisib for 6 months (PMID 34613809).”
Correct. We have now added this reference to our Discussion, and we appreciate the replicability of recently emerging findings. Of note, our treated index case provides unique mechanistic insight in terms of speed and depth of response, durability, and need for continued therapy despite sustained complete remission in this setting, as discussed.
“The effects of alpelisib on cell growth in vitro were tested on lymphatic cells from one patient, and the results would have been strengthened if cells from 2 or more patients had been tested.”
We agree that additional cell lines would have been desirable; however, as this was a confirmatory study to more fully understand our index patient data, we believe that one cell line is sufficient to demonstrate the activity of alpelisib. Future studies will confirm the findings in other cell lines.
“For the RNA-seq experiments, the significance of the gene expression remains unclear, especially given the numerous cell types present in their tissues.”
We agree that these neoplastic lesions represent mixture of cell types and most are reactive and non-clonal. But we still appreciated an intriguing gene expression profile that would otherwise provide other targets such as JAK3, for this entity that appears druggable from different angles. New Figure 4 may enable the further interrogation of the relevant gene expression profiles.
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Evaluation Summary:
This manuscript could be of interest to physicians and researchers in the field of vascular anomalies. The cohort of patients with lymphatic malformations is reasonably sized (n=30) and the claims made by the authors are supported by the data as well as by current knowledge in the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Shaheen and Tse et al. investigate the mutational landscape of lymphatic malformations, find numerous PIK3CA mutations (as expected), and NRAS mutations (in what likely is a cohort of complex lymphatic anomalies), as well as a GOPC-ROS1 fusion. They present data on the treatment of one patient with an unresectable LM, show immunohistochemistry, and in vitro data on alpelisib's effect on LEC cultures.
Strengths:
- The authors present a sample size of 30, which is a good number of patients with these rather rare diseases.
- The broad approach analyzing the mutational landscape led to the identification of a GOPC-ROS1 fusion, which I thought was very interesting, as this has not been described in the setting of vascular malformations so far.
- The attempt to perform RNA-Seq on LM tissue is laudable. However, I …Reviewer #1 (Public Review):
Shaheen and Tse et al. investigate the mutational landscape of lymphatic malformations, find numerous PIK3CA mutations (as expected), and NRAS mutations (in what likely is a cohort of complex lymphatic anomalies), as well as a GOPC-ROS1 fusion. They present data on the treatment of one patient with an unresectable LM, show immunohistochemistry, and in vitro data on alpelisib's effect on LEC cultures.
Strengths:
- The authors present a sample size of 30, which is a good number of patients with these rather rare diseases.
- The broad approach analyzing the mutational landscape led to the identification of a GOPC-ROS1 fusion, which I thought was very interesting, as this has not been described in the setting of vascular malformations so far.
- The attempt to perform RNA-Seq on LM tissue is laudable. However, I wonder, if more discussion on the selected tissue (also keeping in mind the low variant allele frequency), the low number of patients analyzed with RNA-Seq (only one case), and the choice of control tissue would be warrantedWeaknesses:
- The presentation of the clinical data in table 1 is very short and patchy and seems incomplete, also some of the classifications don't appear to be correct E.g. PTEN hamartoma tumor syndrome is a genetically distinct entity, that does not harbor somatic PIK3CA mutations but rather germline PTEN mutations. There are 5 patients with CLOVES and 1 patient with KTS, these patients often have mixed (e.g. lymphatic-venous) malformations, are the analyzed samples truly pure LMs? There are some more instances where I wonder if the presented data allows the reader to understand the cases.
- If the histology is described as kaposiform, these cases likely represent kaposiform lymphangiomatosis, which is a very different disease than common LMs. KLA belongs to the group of complex lymphatic anomalies and usually is caused by NRAS mutations, which would be in line with the presented data. Case 24 (conventional histology, NRAS mutation) could also be a generalized lymphatic anomaly. This distinction of common LMs and complex lymphatic anomalies (including GLA and KLA) should be made and should include what is known about the genetics of these diseases.
Taken together with the first point, the presentation of the cases might benefit from a more structured description and classification.- In the discussion and other parts of the manuscript, terms describing LMs and tumors are interchanged frequently. This mistake is also present in the study protocol (NCT03941782), in which "locally advanced or metastatic cancer" is listed as an inclusion criterion. Other examples include "tumor nuclei". Much of the cited literature also focuses on oncology rather than vascular malformations. And LMs are directly compared to "other low-grade pediatric tumors".
Also, clonality is a concept not too often used in vascular malformations, as an aberrant development of vascular structures during embryogenesis is seen as the cause of vascular malformations, as opposed to clonal expansion in tumors (but this might warrant further investigation in the field).
Thus, the manuscript mixes tumors and malformations, however, it should be stressed, that LMs are not tumors but vascular malformations.- The explanation for the reduced EF doesn't quite make sense, as there should be little blood flow into the LM. This is different from the Venot paper in Nature, where the reduced EF was due to the presence of an AV malformation.
- The data on the (back at the start of treatment of the patient novel) alpelisib is presented as a rather new finding. However, clinical data on its use in PROS diseases have already been published starting in 2018 (the paper from Venot is also mentioned in the manuscript). At the moment, an international clinical trial on alpelisib in PROS disease is recruiting, which could be mentioned.
- Treatment: The rather high dose of 350 mg/d is not further discussed. Also, a patient like this would usually first receive sirolimus, especially back when alpelisib was started in this patient since it was much more experimental at that time point. This should also be explained.In summary: The manuscript shows data that is in line with the current state of knowledge in the field of vascular anomalies. The presented data, while not novel, support the conclusions of the manuscript. The manuscript harbors some inaccuracies in the nomenclature of vascular malformations, which should be corrected.
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Reviewer #2 (Public Review):
In the manuscript "Genomic Landscape of Lymphatic Malformations: A Case Series and Response to the PI3Kα Inhibitor Alpelisib in an N-of-One Clinical Trial" by Shaheen et al., the authors performed genomic profiling in 30 patients with lymphatic malformations (LM). They performed hybrid capture next-generation sequencing of exons from a panel of >300 cancer-related genes and introns from 36 genes on resected tissue from the LM. The LM had a low mutation burden, with PIK3CA (n = 20) and NRAS (n = 5) being the most frequent, and mutually exclusive. In four patients, the LM had kaposiform pathology and each of these patients had NRAS mutations, suggesting a correlation between phenotype and genotype. Finally, one patient with PIK3CA mutation was treated with the PI3Kalpha inhibitor alpelisib, resulting in a …
Reviewer #2 (Public Review):
In the manuscript "Genomic Landscape of Lymphatic Malformations: A Case Series and Response to the PI3Kα Inhibitor Alpelisib in an N-of-One Clinical Trial" by Shaheen et al., the authors performed genomic profiling in 30 patients with lymphatic malformations (LM). They performed hybrid capture next-generation sequencing of exons from a panel of >300 cancer-related genes and introns from 36 genes on resected tissue from the LM. The LM had a low mutation burden, with PIK3CA (n = 20) and NRAS (n = 5) being the most frequent, and mutually exclusive. In four patients, the LM had kaposiform pathology and each of these patients had NRAS mutations, suggesting a correlation between phenotype and genotype. Finally, one patient with PIK3CA mutation was treated with the PI3Kalpha inhibitor alpelisib, resulting in a radiological shrinking of the size of the LM at 6 weeks and 6 months. The in vitro growth of lymphatic endothelial cells carrying PIK3CA mutation could be inhibited with alpelisib.
Strengths:
1. This is a large cohort of patients for such a rare disease, and thus the identification of mutations in the majority of these lymphatic abnormalities is of significant clinical importance.
2. Kaposiform lymphangiomatosis is rare and this study shows it is present in 4 of the 5 patients with activating NRAS mutations, suggesting a phenotype-genotype correlation.
3. Their single patient treated with the PIK3CA inhibitor alpelisib had a radiologic response with shrinkage of the LM, consistent with a prior study showing similar results in 6/6 patients, and treatment of lymphatic endothelial cells carrying PIK3CA mutations with alpelisib showed reduced growth in vitro.Weaknesses:
1. This is not the first demonstration of somatic activating NRAS mutation associated with Kaposiform lymphangiomatosis. A prior study demonstrated that 10/11 patients with Kaposiform lymphangiomatosis had NRAS mutation (PMID 30542204).
2. This is not the first demonstration of somatic activating PIK3CA patients exhibiting malformation shrinkage with alpelisib and they only had a single treated patient. A prior study showed similar results in 6/6 patients treated with alpelisib for 6 months (PMID 34613809).
3. The effects of alpelisib on cell growth in vitro were tested on lymphatic cells from one patient, and the results would have been strengthened if cells from 2 or more patients had been tested.
4. For the RNA-seq experiments, the significance of the gene expression remains unclear, especially given the numerous cell types present in their tissues.Overall, this manuscript will have a clinical impact on the field of lymphatic abnormalities and the results support the conclusions.
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