Immunofluorescent staining of skin biopsy specimens from all 5 patients with JEB taken from intact skin sites before treatment (day 0) and from healed periwound sites at 1 month (day 30) after gentamicin treatment using antibodies that specifically target the 3 chains (α3, β3, or γ2) of laminin 332 or the β4 integrin. Note that within the selected wound sites, gentamicin induced continuous expression of all 3 chains of laminin 332 at the dermal-epidermal junction of all 5 patients. In addition, the β4 integrin is increased and polarized along the entire dermal-epidermal junction after treatment. All images were obtained using the same camera and identical exposure times. Scale bars are 50 μm. HDIV indicates high-dosage intravenous; LDIV, low-dosage intravenous; NHS, normal human skin.
Representative photographs of the open erosions before treatment (day 0) and at 1 month and 3 months after treatment with intravenous gentamicin. Patients 1 to 3 were treated with low-dosage intravenous gentamicin, and patients 4 and 5 were treated with high-dosage intravenous gentamicin. All monitored wound sites demonstrated improved wound closure with intravenous gentamicin. Scale bars are 1 cm.
eMethods 1. Time Points of Primary and Secondary Outcomes for LDIV and HDIV Trials
eMethods 2. Antibody Selection and Immunofluorescence Analysis
eTable 1. Gentamicin Trough Levels, Peak, Serum Creatinine, and Audiometry Screening for JEB Patients throughout the LDIV Trial
eTable 2. Gentamicin Trough Levels, Peak, Serum Creatinine, and Audiometry Screening for JEB Patients throughout the HDIV Trial
eFigure 1. CONSORT Flow Diagram
eFigure 2. Intravenous Gentamicin is Capable of Inducing Sustained Laminin 332 Expression in Skin
eFigure 3. Graphic Visualization of EBDASI Total Activity Scores for JEB Patients in LDIV and HDIV Trials
eFigure 4. Graphic Visualization of 5-D Pruritus and Wong-Baker Pain Scale for JEB Patients in LDIV and HDIV Trials
eFigure 5. Intravenous Gentamicin Did Not Induce Anti-laminin 332 Antibodies
Data Sharing Statement
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Mosallaei D, Hao M, Antaya RJ, et al. Molecular and Clinical Outcomes After Intravenous Gentamicin Treatment for Patients With Junctional Epidermolysis Bullosa Caused by Nonsense Variants. JAMA Dermatol. 2022;158(4):366–374. doi:10.1001/jamadermatol.2021.5992
Can nonsense-mediated readthrough therapy using intravenous gentamicin safely increase the expression of laminin 332 in the skin of patients with junctional epidermolysis bullosa (JEB) caused by nonsense variants?
In this open-label nonrandomized clinical trial of 5 pediatric patients with JEB, all participants exhibited increased expression of laminin 332 at the dermal-epidermal junction in their skin and no signs of ototoxic effects, nephrotoxic effects, or anti–laminin 332 autoantibody induction after 14 or 24 days of intravenous gentamicin.
Pending further investigation, intravenous gentamicin may be a safe short-term systemic therapy for patients with JEB caused by nonsense variants.
Junctional epidermolysis bullosa (JEB) is an incurable blistering skin disorder with high infant mortality often caused by nonsense variants in the genes that encode laminin 332.
To evaluate the safety and outcomes following intravenous gentamicin readthrough therapy and subsequent laminin 332 expression in patients with JEB.
Design, Setting, and Participants
This open-label, pilot nonrandomized clinical trial assessed 1 course of low- or high-dose intravenous gentamicin, including follow-up at 30 and 90 days after treatment. Five pediatric patients with JEB (2 with intermediate JEB and 3 with severe JEB) and confirmed nonsense variants in LAMA3 or LAMB3 in 1 or 2 alleles and decreased expression of laminin 332 at the dermal-epidermal junction of their skin participated in the study, which was performed at a single institution in collaboration with physicians and home infusion services near the patients from April 1, 2019, to February 28, 2021, with follow-up until May 31, 2021.
Three patients received gentamicin at 7.5 mg/kg daily for 14 days, and 2 patients received gentamicin at 10 mg/kg daily for 24 days.
Main Outcomes and Measures
Primary outcomes were change in expression of laminin 332 in patients’ skin and assessments for safety (ototoxic effects, nephrotoxic effects, and autoimmune response). Secondary outcomes included wound healing in monitored wounds and Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) score.
After gentamicin treatment, all 5 patients (age range, 3 months to 10 years, 4 [80%] female) exhibited increased laminin 332 in the dermal-epidermal junction. By 1 month, 7 of 9 wounds in patients receiving low-dose intravenous gentamicin and all wounds in patients receiving high-dose intravenous gentamicin exhibited at least 50% wound closure. By 3 months, 8 of 9 wounds in patients receiving low-dose gentamicin and all wounds in patients receiving high-dose intravenous gentamicin exhibited greater than 85% closure. All 3 patients who were evaluated with EBDASI showed a decrease in total activity scores that met minimal clinically important differences 1 month after treatment. All 5 patients completed the study, and no ototoxic effects, nephrotoxic effects, or anti–laminin 332 antibodies were detected.
Conclusions and Relevance
In this nonrandomized clinical trial, intravenous gentamicin therapy was associated with induced readthrough of nonsense variants in patients with JEB, restored functional laminin 332 in their skin, and wound closure during the 3-month study period. Although long-term safety and efficacy requires further evaluation, a single cycle of intravenous gentamicin may be a safe and readily available therapy in the short term for this population of patients with JEB.
ClinicalTrials.gov Identifiers: NCT03526159 and NCT04140786
Junctional epidermolysis bullosa (JEB) is an incurable, autosomal recessive disorder with 2 major subtypes: severe and intermediate. Affected patients have widespread blisters and erosions of the skin and mucosa, and those with the severe subtype are likely to die within the first year of life.1-5 Current treatment is supportive, despite various preclinical studies and experimental therapeutic trials.6-9 Wound bandaging, antimicrobial treatment, transfusions, pain control, and other supportive medical care are burdensome for patients and expensive for their families. Although there are exceptions, severe and intermediate JEB subtypes are mainly caused by variations in LAMA3 (OMIM 600805), LAMB3 (OMIM 150310), or LAMC2 (OMIM 150292), causing loss of functional laminin 332, the major component of anchoring filaments that mediate dermal-epidermal adherence.1,10,11 Variations in the LAMB3 gene account for 80% of severe JEB, and 95% of these variants are nonsense variants at the R635* hotspot, leading to a premature termination codon (PTC).12 Of interest, the aminoglycoside class of antibiotics, besides being antimicrobial agents, have another property: the ability to readthrough PTCs induced by nonsense variants and allow translation to proceed and generate a full-length protein. This approach has been evaluated in various disease models, including cystic fibrosis, Duchenne muscular dystrophy, Hailey-Hailey disease, xeroderma pigmentosa, Nagashima-type palmoplantar keratosis, and recessive dystrophic epidermolysis bullosa.13-18
A previous study19 demonstrated in vitro that the addition of gentamicin to laminin β3-null cultured keratinocytes from patients with JEB and LAMB3 nonsense variants produced PTC readthrough and generated full-length laminin β3. Another study20 of 3 patients with JEB who had nonsense variants with topical gentamicin demonstrated improved wound healing and the generation of full-length laminin 332 chains. In the current study, we sought to evaluate prospectively the therapeutic capability, durability, and feasibility of a single 14- or 24-day cycle of systemic gentamicin for patients with JEB caused by nonsense variants.
This was a single-arm, open-label nonrandomized clinical trial conducted from April 1, 2019, to February 28, 2021, with follow-up until May 31, 2021, to evaluate changes in laminin 332 expression, safety, and clinical outcomes following intravenously administered gentamicin in 5 patients with JEB caused by nonsense variants. This was a single-institution study conducted at University of Southern California with the collaboration of physicians and home infusion services near patients. Written patient consent was obtained from respective legal guardians, and all patient data were deidentified. This study was approved by the University of Southern California Institutional Review Board. The study followed the Transparent Reporting of Evaluations with Nonrandomized Designs (TREND) reporting guideline. The study protocol is available in Supplement 1.
The inclusion criteria for this study were diagnosis of JEB with at least 1 nonsense variant in the LAMA3 or LAMB3 alleles and an absence or decrease in laminin 332 expression at the dermal-epidermal junction (DEJ) when compared with normal human skin (NHS). The exclusion criteria for this study were preexisting kidney or auditory impairment, allergies to aminoglycosides or sulfate compounds, pregnancy, and exposure to intravenous gentamicin within the past 6 weeks.
Dosing was based on previous studies15,21 of patients with Duchenne muscular dystrophy and cystic fibrosis who received intravenous gentamicin to induce PTC readthrough. Initially, 3 patients received daily intravenous gentamicin sulfate at a dosage of 7.5 mg/kg daily for 14 days (low-dose intravenous [LDIV] treatment) via peripherally inserted central catheters or ports (Gentamicin for Junctional Epidermolysis Bullosa trial). After observing the safe, tolerable, and encouraging response from the patients who received LDIV treatment, we conducted a second clinical study for dose optimization and treated 2 new patients with the high dosage of 10 mg/kg daily for 24 days (high-dose intravenous [HDIV] treatment) (Optimizing Intravenous Gentamicin in JEB trial).
Patients were assessed for primary and secondary outcomes at several time points until completion of the trial (eMethods 1 in Supplement 2). The primary outcome measures were the expression of laminin 332 correctly localized to the DEJ of patients’ skin measured via immunofluorescence and safety assessment for the development of ototoxic effects, nephrotoxic effects, or autoantibodies against laminin 332. The secondary outcome measures included the assessment of wound healing of monitored wound sites via measurements of the wounds’ open surface area by planimetry and a clinical score using a validated scoring method, the Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI).22 Posttreatment follow-up visits occurred at 1 and 3 months after the final dose of gentamicin.
Before treatment, patients had 4- or 6-mm skin biopsy specimens taken to assess the expression of the 3 chains of laminin 332 and α6β4 integrin compared with NHS via quantitative immunofluorescence (eMethods 2 in Supplement 2). In addition, patients underwent baseline blood tests (complete blood cell count, blood urea nitrogen, serum creatinine, calculated creatinine clearance, electrolytes, and liver function tests), audiometry (pure-tone audiometry or otoacoustic emissions test), and clinical assessment using the EBDASI. These parameters were measured before treatment and again 1 and 3 months after treatment.
Each week, patients’ parents completed standardized clinical questionnaires, which included the Skindex-1623 (a quality-of-life measure for patients with skin diseases), the 5-D Pruritus Scale,24 and the Wong-Baker Faces Pain Rating Scale.25 The patients’ families also kept a wound healing diary and provided standardized photographs of selected wound sites that were also monitored weekly. The open wound areas were determined with an ImageJ software analyzer (National Institutes of Health).26 The percentage of wound closure was assessed at approximately 1 and 3 months after treatment by using marked, matched photographs. See the trial protocol in Supplement 1 for details.
Serum circulating anti–laminin 332 antibodies were assessed by enzyme-linked immunosorbent assay using recombinant laminin 332–coated plates, as previously described,27 and indirect immunofluorescence analysis on salt-split human skin. Evaluation for the presence of anti–laminin 332 antibody deposition in patient skin was assessed via direct immunofluorescence staining on patient skin biopsy samples using fluorescein isothiocyanate–conjugated goat antihuman IgG (Sigma-Aldrich), as previously described.28-30
We enrolled 5 patients with JEB (age range, 3 months to 10 years; 4 [80%] female and 1 [20%] male), 3 with severe JEB (patients 2, 3, and 5) and 2 with intermediate JEB (patients 1 and 4). Patients 1, 2, and 3 received LDIV infusions at a dosage of 7.5 mg/kg daily for 14 days, and patients 4 and 5 received HDIV infusions at 10 mg/kg daily for 24 days (eFigure 1 in Supplement 2). A summary of patient demographic characteristics, baseline weights, genotypes, and baseline laminin 332 expression is included in Table 1. Race and ethnicity data are omitted to protect the identities of patients. Both patients with LAMA3 variants (patients 2 and 4) had extensive mucocutaneous involvement. Patient 4 had a tracheal tube placed before involvement in the study, whereas patients 2 and 3 were receiving corticosteroids and supplemental oxygen to treat airway disease. Patient 4 used a wheelchair and required a gastrointestinal tube. Patient 1 also had oropharyngeal and corneal involvement, whereas patient 5 had oropharyngeal and anogenital involvement.
As shown in Figure 1, all 5 patients at baseline had minimal laminin 332 expression at the DEJ of their skin as determined by immunofluorescence. Figure 1A shows the expression of laminin 332 from the healed wounds of 3 patients (patients 1-3) treated with the LDIV regimen. At 1 month after intravenous gentamicin treatment, increased laminin 332 expression ranged from 30.0% to 58.3% (α3 chain), 46.7% to 77.0% (β3 chain), and 31.5% to 52.4% (γ2 chain) of that seen in NHS. Immunofluorescence staining of the skin biopsy specimens from intact skin also showed an increase in all 3 chains (eFigure 2A in Supplement 2).
Figure 1B shows laminin 332 expression in 2 patients treated with the HDIV regimen. Intravenous gentamicin increased laminin 332 expression to 100% (α3 chain), 87.9% (β3 chain), and 98.5% (γ2 chain) for patient 4 and 57.5% (α3 chain), 73.0% (β3 chain), and 56.7% (γ2 chain) of that seen in NHS for patient 5. The expression of laminin 332 was sustained for 3 months in all 5 patients (eFigure 2B in Supplement 2).
We also assessed the expression of the β4 integrin, an integrin that pairs with the α6 integrin to form the α6β4 integrin in the hemidesmosomes of basal keratinocytes, a location that depends on the presence of laminin 332.31 All 5 patients had reduced β4-integrin expression at the DEJ before treatment, and gentamicin treatment increased β4-integrin expression to levels similar to NHS.
Marked mechanical skin fragility is a salient feature of JEB, and new erosions and blisters result from trauma, such as friction from clothing or handling by caretakers. Patients were not restricted from their typical daily activities. Before treatment, each patient had 3 open wounds selected for monitoring and quantification of the surface area. None of these wounds showed any evidence of infection before or after treatment. Wound areas were quantified via computer-assisted planimetry of standardized photographs taken by the patients’ families or health care practitioners. We also selected 2 intact sites to monitor for the development of new blisters and erosions. The degree of wound closure for the selected wound areas at 1 month and 3 months compared with baseline is shown in Table 2. By 1 month, 7 of 9 wounds monitored for the LDIV trial and all wounds monitored for the HDIV trial exhibited at least 50% wound closure. By 3 months, 8 of 9 wounds in the LDIV trial and all wounds in the HDIV trial exhibited greater than 85% closure. Representative images of wound healing for both the LDIV and HDIV arms are shown in Figure 2. Intact sites remained closed without wound development during the entire study period.
The EBDASI is a validated tool that objectively grades the severity of disease in patients with epidermolysis bullosa.22 We evaluated and quantified epidermolysis bullosa–related activity and damage of the skin and mucosa before and after treatment (Table 3). Patients 1, 4, and 5 demonstrated a decrease in their EBDASI skin activity scores at 1 month after treatment, although scores generally rebounded closer to baseline by 3 months. Patient 2 exhibited an increase in skin activity scores by 1-month follow-up and was not assessed with the EBDASI at 3 months. In all patients in whom mucosa was assessed, mucosal activity scores were decreased at 1 month and 3 months after treatment. These patients also demonstrated a decrease in total disease activity from baseline at 1 month after treatment. By 3 months after treatment, patient 1 retained a 17-point decrease, and patient 5 returned to baseline for total activity scores (eFigure 3 in Supplement 2).
The Skindex-16 quality-of-life survey, Wong-Baker Pain Rating Scale, and 5-D Pruritus Scale were also used to assess subjective clinical and quality-of-life improvements noted by patients and their parents (Table 3).23-25 The Skindex-16 survey was able to be completed in patients 1 and 4 because of their older age and ability to respond verbally. Their symptom scores remained relatively stable, yet both exhibited downward trending scores in the emotions and functioning components of the Skindex-16 survey. Overall results from the 5-D Pruritus Scale and Wong-Baker Pain Rating Scale were mixed and bidirectional, with no trend toward consistent improvement over 3 months (eFigure 4 in Supplement 2).
Gentamicin trough and peak levels, complete blood cell counts, blood urea nitrogen levels, serum creatinine levels, electrolyte levels, liver function test results, and hearing function test results remained within normal limits throughout the trial (eTables 1-2 in Supplement 2). Notably, the audiometry and creatinine clearance results were unchanged.
New laminin 332 could possibly promote the production of anti–laminin 332 autoantibodies, leading to an autoimmune response. Anti–laminin 332 antibodies cause mucous membrane pemphigoid, an autoimmune bullous disease associated with mucosal erosions and scarring.32,33 We tested patient serum samples obtained at baseline, 1 month, and 3 months after treatment using an enzyme-linked immunosorbent assay,27 except for in patient 5, whose parents declined a blood draw. None of the tested samples had anti–laminin 332 antibodies above their baseline values (eFigure 5A in Supplement 2). Indirect immunofluorescence staining of all patients’ serum samples using salt-split NHS found that none of the serum bound to NHS substrate (eFigure 5B in Supplement 2). The results of direct immunofluorescence staining of biopsy specimens from all 5 patients were negative for anti–laminin 332 antibodies at all 3 time points (eFigure 5C in Supplement 2).
In this open-label nonrandomized clinical trial, intravenous gentamicin was associated with the generation of new laminin 332 in the DEJ of 5 patients with JEB caused by nonsense variants, and the newly generated laminin 332 persisted for 3 months, the longest time tested. The induction of newly generated laminin 332 was associated with closure of monitored wound sites. No adverse effects occurred in any of the 5 patients, and no patient generated anti–laminin 332 autoantibodies despite the induction of laminin 332.
It has been suggested that the amount of laminin 332 required at the DEJ to attenuate JEB symptoms may be a minimal percentage of that expressed in NHS.34 In the current study, a single cycle of intravenous gentamicin was followed by increased expression of laminin 332. The increased expression of laminin 332 observed in the patients’ intact skin sites suggests that it could prevent future blistering and wounding. Consistent with a previous study of topical application of gentamicin to the skin wounds of patients with JEB,20 we found that gentamicin-associated laminin 332 expression after intravenous infusions was durable for 3 months. This finding is consistent with a study in which the half-life of laminin α3 in lung epithelium was determined to be 30 to 60 days,35 and it is likely that laminin 332 would have a similar half-life in the skin. With data indicating a robust response to short-term gentamicin treatment and the marked stability of laminin 332, we envision that gentamicin could be delivered as a short-term pulse therapy every 2 to 3 months for patients with JEB caused by nonsense variants.
We treated 2 patients with R635* hotspot variants in LAMB3, a pathogenic variant found in most patients with severe JEB. However, 3 other patients in this study harbored different nonsense variants, including C325* in LAMB3 (PT1), Q625* in LAMA3 (PT2), and Y63* in LAMA3 (PT4), highlighting the applicability of systemic gentamicin for a range of nonsense variants.
The outcomes following systemic gentamicin therapy in these patients with JEB are apparent on a molecular level, and we attempted to assess relevant clinical changes as well. We demonstrate closure of wounds in patients with JEB in this study; however, in the absence of a control, we cannot determine whether this differed from the natural history of wounds in patients with JEB. All but 1 of the monitored wounds in the LDIV and HDIV treatment arms exhibited near-complete closure by final follow-up. In addition, several of these wound sites in the older patients with intermediate JEB (patients 1 and 4) were chronic wounds that had been open for more than 6 months to a year. We observed sustained healing of wounds in low- and high-friction anatomical sites. Although the 3 patients who were able to be fully evaluated using the EBDASI score exhibited decreases in their total activity scores that met minimal clinically important differences (3 points) by 1 month after treatment, significant gaps in patient data limit conclusions regarding comprehensive benefit.22
A noteworthy element of our study was the inclusion of 2 children with intermediate JEB (patient 1 [7 years of age] and patient 4 [10 years of age]). The relatively older age of these children allowed for an assessment of their disease-related emotions and functioning via the Skindex-16 survey. After treatment, both children experienced improvements in their emotions (worry, frustration, embarrassment, and depression) and functioning (interpersonal interactions, showing affection, and daily activities) that was sustained up to the end of the trial period, indicating potential psychosocial benefits of intravenous gentamicin for individuals with this subtype of JEB.
Extracutaneous involvement in patients with JEB is common, and intravenous administration of gentamicin has the advantage of penetrating skin and mucosal surfaces. All patients who had their mucosae evaluated (patients 1, 2, and 5) by EBDASI experienced a decrease in disease-related mucosal activity. Involvement of the mucosa in the upper respiratory tract is seen in up to 50% of patients with severe JEB.2 Of interest, the parents and physicians of patients 2 and 3 noted significant improvement of airway symptoms in these infants, including decreased coughing, stridor, and hoarseness after treatment.
The mainstay treatment for JEB is limited to supportive management and palliative care. Nevertheless, several experimental therapies have been attempted in patients with JEB, including bone marrow transplantation and gene-corrected keratinocyte autografts.6,36-38 Recently, Hammersen et al12 retrospectively reviewed the systemic (intravenous and intramuscular) administration of 7.5 mg/kg daily of gentamicin during 21 days for 5 infants with severe JEB attributable to R635* hotspot variants in LAMB3. After treatment, they reported improved patient quality of life per parental questionnaire in 4 of 5 children and no adverse effects. Our results support the findings of the study by Hammersen et al12 that systemic gentamicin may be a safe treatment, for at least a short-term period, that is inexpensive and commercially available.
The limitations of our trial include that it was an open-label study with no placebo control and included only 5 patients. We did not formally track weight or survival as parameters for this study. The assessments of mucosal improvement are limited to the EBDASI results and subjective reports regarding airway status. Furthermore, several of the clinical scoring instruments used in this study for pain, pruritus, and quality of life have not been validated in children and infants. Future trials should incorporate instruments better tailored to the patients’ ages. Although no adverse effects occurred in our patients during the 3-month postinfusion period, the safety risks from long-term gentamicin treatment, including the known toxic effect profile (ototoxic and nephrotoxic effects), autoantibody induction, and bacterial resistance, warrant further investigation. Gentamicin has also been described as an immunomodulatory agent, and future studies should incorporate evaluations of inflammatory markers to further elucidate their potential role in wound healing.39,40
This prospective nonrandomized clinical trial demonstrated the potential of intravenous gentamicin to induce readthrough of nonsense variants in patients with JEB, generate new laminin 332 in the patients’ skin, and possibly enhance wound healing during a 3-month study period. We found a single cycle of intravenous gentamicin to be safe and feasibly administered in an outpatient setting over a short-term period. Future trials studying dosage optimization and multiple cycles of treatment during a long period will provide data on feasibility, safety, and efficacy as an indefinite therapy. This trial further contributes to the groundwork of systemic readthrough therapy as a potential treatment of other diseases caused by nonsense variants.
Accepted for Publication: December 20, 2021.
Published Online: March 2, 2022. doi:10.1001/jamadermatol.2021.5992
Corresponding Author: Mei Chen, PhD (firstname.lastname@example.org), and David T. Woodley, MD (email@example.com), Department of Dermatology, University of Southern California, 1441 Eastlake Ave, Room 6322, Los Angeles, CA 90033.
Author Contributions: Dr Chen had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Mr Mosallaei and Dr Hao are co–first authors.
Concept and design: Mosallaei, Woodley, Chen.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Mosallaei, Hao, Kwong, Cogan, Tang, Woodley, Chen.
Critical revision of the manuscript for important intellectual content: Mosallaei, Hao, Antaya, Levian, Cogan, Hamilton, Schwieger-Briel, Tan, Woodley, Chen.
Statistical analysis: Hao, Kwong, Cogan.
Obtained funding: Woodley, Chen.
Administrative, technical, or material support: Mosallaei, Hao, Levian, Kwong, Schwieger-Briel, Tan, Tang, Chen.
Supervision: Cogan, Chen.
Conflict of Interest Disclosures: Dr Woodley and Dr Chen both reported receiving grants from Epidermolysis Bullosa Research Partnership and Epidermolysis Bullosa Medical Research Foundation during the conduct of the study and personal fees from Phoenix Tissue Repair outside the submitted work. No other disclosures were reported.
Funding/Support: This work was supported in part by grants from Epidermolysis Bullosa Research Partnership and Epidermolysis Bullosa Medical Research Foundation (Drs Chen and Woodley) and award W81XWH-1810558 from the Congressionally Directed Medical Research Program (Dr Chen).
Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Data Sharing Statement: See Supplement 3.
Additional Contributions: We thank the patients for granting permission to publish this information.