A comprehensive, multidisciplinary, precision medicine approach to discover effective therapy for an undiagnosed, progressive, fibroinflammatory disease

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A comprehensive, multidisciplinary, precision medicine approach to discover effective therapy for an undiagnosed, progressive, fibroinflammatory disease BERNADETTE R. GOCHUICO, SHIRA G. ZIEGLER, NICHOLAS S. TEN, NICHOLAS J. BALANDA, CHRISTOPHER E. MASON, PAUL ZUMBO, COLLEEN A. EVANS, CARTER VAN WAES, WILLIAM A. GAHL, and MAY C.V. MALICDAN BETHESDA, AND BALTIMORE, MARYLAND; AND NEW YORK, NEW YORK

Precision medicine has generated diagnoses for many patients with challenging undiagnosed disorders. Some individuals remain without a diagnosis despite comprehensive testing, and this impedes their treatment. This report addresses the role of personalized medicine in identifying effective therapy for an undiagnosed disease. A 22-year-old woman presented with chronic severe recurrent trismus, facial pain, progressive multicentric inflammatory and fibrotic masses, and high C-reactive protein. Sites of disease included the pterygomaxillary region, masseter muscles, mandible, lung, pericardium, intrabdominal cavity, and retroperitoneum. A diagnosis was not established after an extensive assessment, including multiple biopsies. The patient was subsequently evaluated under the Undiagnosed Diseases Program at the National Institutes of Health. Large scale genotyping, proteomic studies, and in vitro and gene expression analyses of fibroblasts obtained from a major disease locus were performed. Germline genetic testing did not identify strong candidate genes; proteomic studies of the patient’s serum and bronchoalveolar lavage fluid and gene expression analyses of her cells were consistent with dysregulation of the tumor necrosis factor-alpha pathway. The patient’s cultured fibroblasts were incubated with selected drugs, and cell proliferation was inhibited by hydroxychloroquine. Treatment of the patient with hydroxychloroquine conferred prolonged beneficial clinical effects, including stabilization of trismus and reduction of corticosteroid dose, C-reactive protein, and size of masses. This case represents an example

Clinical Trial registration: Registrar: ClinicalTrials.gov Website: www.clinicaltrials.gov Registration Numbers: NCT00369421 and NCT00084305. From the Section of Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland; Department of Genetics and Pediatrics, Johns Hopkins University School of Medicine, Bloomberg Children’s Center, Baltimore, Maryland; NIH Undiagnosed Diseases Program, Common Fund, Office of the Director, National Institutes of Health, Bethesda, Maryland; Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York; The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, New York; The World Quant Initiative for Quantitative Prediction, Weill Cornell Medicine, New York, New York; The Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, New York; Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of HealthBethesda, Maryland. Submitted for Publication May 2, 2019; received submitted August 15, 2019; accepted for publication August 22, 2019. Reprint requests: Bernadette R. Gochuico, 10 Center Drive, MSC 1851, Bethesda, MD 20892-1851. e-mail: [email protected]. 1931-5244/$ - see front matter Published by Elsevier Inc. https://doi.org/10.1016/j.trsl.2019.08.008

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of precision medicine applied to discover effective treatments for individuals with enigmatic undiagnosed disorders. (Translational Research 2019; 000:1 10) Abbreviations: NIH = National Institutes of Health; UDN = undiagnosed diseases network; UDP = undiagnosed diseases program

AT A GLANCE COMMENTARY Gochuico BR, et al. Background

Precision medicine can guide the clinical approach to diagnosing and treating common and rare diseases; limited information is available about its role in discovering medical therapy for complex undiagnosed disorders.

multimodal cross-disciplinary strategies developed by the UDP and UDN to diagnose these highly perplexing medical conditions, many cases remained unsolved. Among these undiagnosed patients, nonetheless, several individuals benefited from enhanced symptomatic care. This report demonstrates the ability of comprehensive precision medicine to identify effective treatment for patients despite the absence of a definitive diagnosis.

MATERIAL AND METHODS Translational Significance

Comprehensive, multidisciplinary, and precision medicine strategies can enable the identification of effective treatment for patients without a definitive diagnosis. This personalized therapeutic approach could provide potential clinical benefit to some patients with undiagnosed diseases.

INTRODUCTION

Precision medicine now influences the clinical approach to diagnosing and treating several diseases, including common and rare disorders. Targeted therapies are approved for various malignancies, including colorectal cancer, breast carcinoma, lung cancer, and leukemia.1-4 Personalized medicine has also guided treatment for chronic diseases (eg, cystic fibrosis, X-linked hypophosphatemia) and rare disorders (eg, Erdheim-Chester disease, Fabry disease, transthyretintype familial amyloid polyneuropathy).5-12 The Undiagnosed Diseases Program (UDP) of the National Institutes of Health (NIH) was created in 2008 to address the unmet needs of patients with challenging and often multisystemic disorders of unknown etiologies.55 In its initial 32 months, medical records were reviewed from hundreds of individuals, and approximately 400 of the first 1400 applicants were accepted into the program.12,13 A diagnosis was established in 20% 25% of the initial 272 patients evaluated at the NIH Clinical Center, and new or rare diseases were identified. In 2015, the UDP was extended and became part of the national multicenter Undiagnosed Diseases Network, which was launched with prospects of fostering collaborations to enhance the identification of new disorders, accelerate the diagnosis of rare diseases, expand the phenotype of known disorders, and advance scientific discovery.14-17 Despite the successful

Patient consent. Written informed consent was obtained from the patient and her parents, who enrolled in protocol 76-HG-0238 (Clinical Trials NCT00369421, “Diagnosis and Treatment of Inborn Errors of Metabolism and Other Genetic Disorders”), and from healthy research volunteers, who enrolled in protocol 04-HG-0211 (Clinical Trials NCT00084305, “Procurement and Analysis of Specimens from Individuals with Pulmonary Fibrosis”). The protocols were approved by the institutional review board of the National Human Genome Research Institute. Clinical testing. Magnetic resonance imaging of the sinuses and brain as well as computed tomography scans of the orbits, neck, chest, and abdomen were performed at the NIH Clinical Center in Bethesda, Maryland. Pulmonary function tests were performed in accordance with guidelines from the American Thoracic Society/European Respiratory Society as described.18 Fiberoptic bronchoscopy with lavage was performed and bronchoalveolar lavage fluid was isolated as described.19 Proteomic, genetic, and RNA sequencing analyses.

Concentrations of 165 cytokines, chemokines, growth factors, and proteases in serum and bronchoalveolar lavage fluid were measured by multiplex ELISA (Aushon Biosystems, Inc., Billerica, MA). Serum values were compared with those from 4 normal volunteers; bronchoalveolar lavage fluid measurements from 3 of these 4 normal volunteers were used for comparisons. A single-nucleotide polymorphism array using the HumanOmniExpress DNA Analysis BeadChip (Illumina, San Diego, CA) and the GenomeStudio software (Illumina) was used to analyze genomic DNA isolated from peripheral blood as described.20 Exome and genome sequencing of the patient’s and her parents’ genomic DNA was performed by the NIH Intramural Sequencing Center using the HiSeq2000 (Illumina) and the Illumina Genome Analyzer Pipeline software

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(V.1.13.48.0) as described.20 Raw FASTQ files were processed using the Illumina DRAGEN Bio-IT Platform (Version D01.011.254.02.06.05.49892). The resulting joint genotyped VCF was annotated with GnomAD (2.0.2) and loaded into a Gemini SQLite database (Version 0.20.1). Variants were filtered by segregation with disease and population frequency. Genes in the tumor necrosis factor-alpha pathway were specifically interrogated for variants. A clinically-indicated biopsy of the patient’s right pterygomaxillary mass was performed for histopathology, and a portion was used to culture the patient’s cells. Primary fibroblasts cultured from the mass were maintained in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 1% penicillinstreptomycin-glutamine. Adherent cells were washed with PBS, and RNA was isolated using standard methods. Dermal fibroblasts cultured from a skin biopsy performed in an uninvolved region of the patient’s forearm were used for controls. Poly-A selected RNA-Seq libraries were constructed from 1 mg mRNA using the Illumina TruSeq RNA Sample Preparation Kit (Illumina) protocol from a total of 6 samples derived from the patient’s pterygomaxillary mass fibroblast and dermal fibroblast cell cultures in triplicate. Unique barcode adapters were applied to each library. Libraries were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and pooled in an equimolar ratio. The pooled libraries were paired-end sequenced (2 £ 51 nucleotides) on an Illumina Genome Analyzer IIx. The sequences of the paired-end 2 £ 51 base pair raw reads were returned in compressed FASTQ format consisting of sequences with corresponding Phred33 (Sanger) base call quality scores for each short read.20,21 Quality control on the initial raw FASTQ files was performed with FastQC v11.6 from Babraham Bioinformatics to screen for the presence of adapters and to identify low-quality reads for exclusion from the analysis.22 Trimmomatic v0.39 was used to filter any paired-end reads from the FASTQ files in which at least 1 read in a pair had a Phred33 base call quality score average of less than 20 across the entire length of the 51 base pair read.23 Quality-filtered FASTQ files were then aligned to the human reference genome build GRCh37/hg19 using STAR version 2.5.3a in manual 2-pass mapping mode.24 GENCODE v19 annotation was used for genome annotation while preparing STAR genome indices for alignment.25 Gene-level raw read counts were determined using featureCounts v1.5.2 from the Subread package with a RefSeq transcript annotation set consisting of 27,090 gene features.26-28 Only short reads overlapping RefSeq exon features of known mRNAs or ncRNAs and were counted (‘NM’ and ‘NR’ accession prefixes), and those

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that were only predicted mRNAs or ncRNAs (‘XM’ and ‘XR’ accession prefixes) were excluded. Primary fibroblast cell proliferation and cytotoxicity

Cells (1 £ 103 per well in 96-well plates) in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum and 1% penicillin-streptomycinglutamine were incubated for 24 hours at 37˚C with hydroxychloroquine, pentoxifylline, or sunitinib (Sigma, St. Louis, MO). Cell proliferation and cytotoxicity assays (Cell Counting Kit-8, Dojindo Molecular Technologies, Rockville, MD) were performed as directed in triplicate and repeated twice. Statistical analysis. Tabular data are shown as mean § standard error of the mean. Cell proliferation data are shown as mean § standard deviation of the mean (GraphPad Prism 5, GraphPad Software, San Diego, CA). For gene expression studies, statistical tests on raw and processed data were performed using the SARTools R package v1.5.0.29 The DESeq2 R v1.22.0 package from Bioconductor was used to process raw data by normalizing read count values to account for library size discrepancies and to predict differential expression using a model that employs the negative binomial distribution.30 Genes with Benjamini-Hochberg-adjusted FDRs > 0.05 were excluded from the rest of the analysis after the DESeq2 normalization procedures were applied to the raw count numbers, resulting in a final total of 6750 statistically significant differentially expressed genes between experimental sample and control groups with varying magnitudes of differential expression.31 Expression values for each gene are reported by DESeq2 as log2(fold change) values, which are log2 transformations of the fold change in normalized counts between experimental and control groups for each gene. A volcano plot was produced using the EnhancedVolcano package in R.32 Pathway analysis software platform Ingenuity Pathway Analysis from QIAGEN (Redwood City, CA) was used to visualize expression log2(fold change) values mapped onto the disease and molecular/cellular function pathways.33 Genes with a differential expression adjusted P value < 0.001 were included. Experimental gene expression values were mapped to the KEGG tumor necrosis factor signaling pathway using the Pathview R package.34-37 assays.

RESULTS Clinical presentation. The patient (UDP1033) is a 22-year old woman with multicentric inflammatory and fibrotic masses of unknown etiology. She initially presented at 16 years of age with jaw pain and swelling after an orthodontic procedure. Her symptoms progressed, and she experienced severe recurrent trismus and pain; her clinical course showed progression of disease with

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A

B

C

D

E

F

G

H

Fig 1. Radiographic imaging and histopathology of fibroinflammatory mass lesions in multiple organs. A, Computed tomography scan images show progressive enlargement of right pterygomaxillary fossa mass (circle). Left image was 6 years prior to admission; center image was 3 years prior to admission, and right image was at admission to the National Institutes of Health Undiagnosed Disease Program. B, Multiple nodules (arrows) are detected by computed tomography scan in bilateral lungs. C, Extensive disorganized intraabdominal and right retroperitoneal inflammatory soft tissue lesions (open arrows) and right hydronephrosis are found by computed tomography imaging. D and E, Histopathology of right medial teratoid tissue demonstrates dense fibrosis and chronic inflammation. F H, Lung histopathology shows dense fibrosis along the pulmonary septa and chronic inflammation composed of lymphocytes, histiocytes, and plasma cells. Large aggregates of plasma cells and prominent follicular lymphoid hyperplasia are found. (hematoxylin and eosin; D-100£, E-200£, F-100£, G-100£, and H-100£ magnification).

development of bilateral masseter muscle infiltration, mandibular and pterygomaxillary masses, mastoid and sinus disease, recurrent pericarditis, lung nodules, hepatomegaly, as well as intra-abdominal, pelvic, and retroperitoneal soft tissue involvement (Fig 1A C). Additional manifestations included fever, anemia, and greatly elevated C-reactive protein. Testing for infectious diseases, malignancy and rheumatologic disorders was negative. Bone marrow biopsy was nondiagnostic. Pericardiocentesis evacuated 40 mL of serosanguinous fluid. Open lung biopsy was notable for intrathoracic adhesions and postoperative hemorrhage. Biopsies from her cheek soft tissue, right medial teratoid, masseter, maxillary sinus, and lung revealed chronic inflammation and dense fibrosis

(Fig 1D H). Although 3 different relatives had a history of Crohn’s disease, multiple sclerosis, and juvenile diabetes mellitus, none had a history of fibrotic disorders. The patient was admitted to outside institutions on multiple occasions, underwent several surgical procedures to relieve recurrent severe trismus, and required endodontic therapy for severe dental caries. Treatment with nonsteroidal anti-inflammatory drugs, prednisone, and azathioprine improved the patient’s pericarditis, but not her recurrent trismus. Her severe facial pain was treated with narcotics. The patient was also treated with prednisone, azathioprine, rituximab and required a temporary nephrostomy tube for hydronephrosis. Although intra-abdominal and retroperitoneal disease

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Table I. Clinical testing Parameter Blood urea nitrogen (mg/dL) Creatinine (mg/dL) Alanine aminotransferase (U/L) Aspartate aminotransferase (U/L) Creatine kinase (U/L) Angiotensin converting enzyme (U/L) Rheumatoid factor (IU/mL) Total protein (g/dL) Immunoglobulin G4 (mg/dL) Human herpes virus-8 immunoglobulin G Erythrocyte sedimentation rate (mm/h) C-reactive protein (mg/L) White blood cells (K/uL) Hemoglobin (g/dL) Platelets (K/uL)

UDP1033

Normal range

18

8 22

0.4 21

0.70 1.30 6 41

13

9 34

8 31

38 252 16 52

<15 7.0 11.7

<15 6.4 8.2 2.4 121.0

Negative

Negative

26.0

0.0 42.0

123 16.1 12.4 372

<3.0 3.98 10.0 11.2 15.7 173 369

tomography imaging of the head demonstrated a large mass involving the right maxillary sinus with invasion of the sinus wall and extension into the pterygoid fossa and mandibular condyle (Fig 1A). Magnetic resonance imaging revealed a heterogeneously enhancing, infiltrating mass in the right maxillary sinus extending into the pterygoid fossa, infratemporal fossa, medial and lateral pterygoid muscles, and masseter. Partial opacification of the right maxillary, sphenoid and ethmoid sinuses, and bony erosions of the right pterygoid plates and right maxillary sinus posterior lateral wall were visualized. Computed tomography scans of the chest and abdomen showed numerous bilateral upper lobe nodules with an inflammatory component, normal liver, and normal kidneys (Fig 1B). Echocardiogram demonstrated normal wall motion, cardiac function, and pericardium; no pericardial effusion was found. Fiberoptic laryngoscopy revealed friable posterior nasal mucosa and no structural abnormalities, and fiberoptic bronchoscopy showed normal airways without endobronchial lesions. Multiple microbiological tests of bronchoalveolar lavage fluid were negative. Proteomic,

Bold values are abnormal (i.e., not within the normal range).

improved, her trismus, pain and masseter, mandibular, pterygomaxillary and pulmonary masses progressed. At admission to the UDP at the NIH Clinical Center, the patient was experiencing chronic severe facial pain and pronounced trismus. Physical examination was notable for facial asymmetry, interincisal opening of 13 mm, and temporomandibular joint tenderness. Laboratory testing showed markedly increased C-reactive protein and high peripheral white blood cell concentration with normal cell differential count (Table I). Total protein, serologies, angiotensin converting enzyme, serum and urine protein electrophoresis, immunoglobulin subtyping, and immunoglobulin G4 values were normal, and human Herpes virus-8 immunoglobulin G was negative. Computed

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genetic,

and

RNA

sequencing

gene

Given the multisystem manifestations including the lung, proteomic profiling of serum and bronchoalveolar lavage fluid was performed to identity potential molecular pathways contributing to disease and possible therapeutic targets. Concentrations of 11 analytes were higher in the patient’s serum and bronchoalveolar lavage fluid compared to normal volunteers (Table II). Overall, these data suggested that the tumor necrosis factor-alpha pathway may be associated with the patient’s fibroinflammatory disorder. Given her family history of autoimmune disorders, large scale germline genotyping using a single nucleotide polymorphism array, whole exome sequencing, and whole genome sequencing were performed. Potential candidate disease-associated genes were not identified. Analysis of genes in the tumor necrosis factor-alpha expression analysis.

Table II. Upregulated proteins in serum and bronchoalveolar lavage fluid Protein

UDP1033 serum

NV serum

UDP1033 BALF

NV BALF

C-reactive protein (ng/mL) Matrix metalloprotease-9 (ng/mL) Interleukin 17E (pg/mL) Prolactin* GM-CSF (pg/mL) Chorionic gonadotropin alpha (pg/mL) Angiopoietin-2 (pg/mL) TNF-R1 (pg/mL) VEGF-R2 (ng/mL) CCL23 (pg/mL) Interleukin 6R*

4570 1709 350.7 12.0 42.0 481.2 8.6 952.7 12.7 857.3 12.3

90.2 § 19.5 114.1 § 49.9 53.1 § 34.6 2.22 § 0.77 11.8 § 9.5 147.0 § 83.0 2.7 § 1.5 345.0 § 125.0 5.49 § 0.94 396.0 § 86.1 6.0 § 0.38

2.43 11.2 72.8 3.3 50.0 18.4 152.1 37.2 163.8 38.6 198.2

0.05 § 0.02 2.41 § 2.17 32.3 § 17.1 0.73 § 0.15 9.67 § 3.71 8.17 § 1.61 40.1 § 36.1 15.7 § 3.66 69.2 § 26.2 3.20 § 2.31 91.8 § 39.3

Abbreviations: BALF, bronchoalveolar lavage fluid; NV, normal volunteers. * Serum concentrations (ng/mL); BAL concentrations (pg/mL).

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Fig 2. Differential gene expression of pterygoid mass fibroblasts. A, Histopathology of pterygoid mass biopsy demonstrates dense fibrosis and mild chronic inflammation (hematoxylin and eosin; 400£ magnification). B, Volcano plot of RNA sequencing results demonstrates differential expression between the patient’s pterygoid mass and unaffected dermal fibroblast samples. C, Top 5 upstream regulators as reported by Ingenuity Pathway Analysis are displayed in tabular format. D, DESeq2 log2 (fold change) values for differentially expressed genes are mapped onto the KEGG tumor necrosis factor-alpha pathway signaling pathway. (red—upregulated genes; green—downregulated genes). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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pathway identified some variants of unknown significance (Supplementary Table I) that could explain the phenotype and the response to treatment observed. The patient also underwent a clinically-indicated biopsy of her progressively enlarging pterygomaxillary mass. Histopathology showed inflamed dense fibrous tissue, mild chronic inflammation without granulomas or eosinophilic infiltration, hemosiderin pigment, and rare plasma cells positive for kappa and lambda, and these findings were inconsistent with IgG4-related sclerosing disease or sarcoidosis (Fig 2A). Stains for infectious etiologies were negative. Fibroblasts cultured from her pterygomaxillary mass were studied and comparisons were made to her own dermal fibroblasts procured from uninvolved forearm tissue. A volcano plot of RNA sequencing results showed differential expression between pterygoid mass fibroblasts and control cells (Fig 2B). A list of the top 50 differentially expressed genes is included in Supplementary Table II. Consistent with the serum and bronchoalveolar lavage fluid proteomic analyses, RNA sequencing gene expression results showed upregulation of the tumor necrosis factor-alpha pathway, which was 1 of the top 5 upstream regulators (Fig 2C). The transforming growth factor beta1 and the tumor-suppressor TP53 protein pathways were also in the top 5 list and predicted to be activated. Differential gene expression of the pterygomaxillary mass fibroblasts mapped to the KEGG tumor necrosis factor

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signaling pathway showed several up- and down-regulated genes (Fig 2D)(Table III). Identification of candidate drug and therapeutic

To identify a candidate drug as treatment for this patient’s undiagnosed fibroinflammatory disease, we studied her pterygomaxillary mass fibroblasts in vitro. The patient’s cells were incubated with hydroxychloroquine, pentoxifylline, or sunitinib, which were selected for testing due to their inhibitory effects on the tumor necrosis factor-alpha pathway and fibrosis.38-43 Cell proliferation and cytotoxicity assays revealed that the inhibitory effects of hydroxychloroquine on these primary fibroblasts were dose-dependent and were greater than those of pentoxifylline or sunitinib (Fig 3A). Given the results of these in vitro studies and the patient’s progressive disease, treatment with hydroxychloroquine was initiated. Response to therapy was assessed longitudinally for 4 years, and clinical outcome measures included corticosteroid dosage, severity of trismus, serum C-reactive protein concentrations, and size of masses. Treatment of the patient with hydroxychloroquine 400 mg daily was associated with long-term stabilization of trismus and fewer therapeutic surgical release procedures under a lower prednisone dose. The patient’s interincisal opening was generally maintained at 19 mm. In addition, serum C-reactive protein concentrations stabilized at reduced levels, and sizes of the pterygomaxillary mass and lung nodules were smaller (Fig 3B D). response.

Table III. Differential expression of TNF-alpha pathway genes in pterygomaxillary mass Gene TNFR1 Pathway RIPK3 MAP2K1 CREB3 MAP3K5 CASP3 CASP10 TRADD FADD MLKL BAG4 TNFRSF1A TNFR2 Pathway TNFRSR1B TRAF1 AKT3 DAB2IP TNFR1 and TNFR2 NFKB1 JUN MAPK8 NFKBIA TRAF2

Direction Log2 (fold change) P value

+ + + + + + +

+ +

+ + + +

4.372 0.774 1.108 0.870 0.659 0.721 1.000 0.683 0.503 0.451 0.280

2.08E-68 1.42E-09 3.31E-08 8.79E-07 2.94E-06 8.97E-04 3.05E-03 8.63E-03 0.01085 0.01282 0.04614

1.025 0.786 0.710 0.529

1.58E-07 2.61E-04 1.08E-03 6.65E-03

0.965 0.836 0.835 0.504 0.537

9.13E-11 1.29E-09 1.16E-05 6.21E-03 0.03391

“+” indicates upregulation; “ ” indicates downregulation.

DISCUSSION

Precision medicine has been a strategy used to identify a molecular basis of disease for previously unknown disorders, to provide estimated risk of disease or prognoses for different malignancies, and to determine targeted therapy for several disorders. For example, patients enrolled in the NIH UDP have been diagnosed with several genetic disorders, including Arterial Calcification due to Deficiency of CD73 (ACDC) and COPA syndrome, and exome or genome sequencing established diagnoses for many patients evaluated by the Undiagnosed Diseases Network.17,44,45 Detection of BRCA1 or BRCA2 mutations is associated with high risk of developing breast, ovarian, and contralateral breast cancer and may affect the clinical management of individuals harboring these mutations.56 Identification of tumor markers are used to guide targeted therapy for various disorders, such as the EML4-ALK fusion oncogene in nonsmall cell lung cancer and BRAF mutations in Erdheim-Chester disease.7,8,46,47 Furthermore, drugs that potentiate or modulate the cystic fibrosis transmembrane conductance regulator are approved by the Food and Drug Administration as treatment for

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A

B

Absorbance

2

CRP

8

1

40

20

0

0

0

100

0

12

24

36

48

Hydroxychloroquine Treatment (mo)

500

Hydroxychloroquine (uM)

1.0

A b s or b a nc e

50

C

0.5

0.0

0

500 1000 1500 2000 2500

0 mo

24 mo

48 mo

0 mo

24 mo

48 mo

D

Pentoxifylline (uM)

1.0

A bso r b an c e

5000

0.5

0.0

0

5

10

15

20

25

50

Sunitinib (uM)

Fig 3. Effect of hydroxychloroquine in vitro and as treatment for an undiagnosed fibroinflammatory disorder. A, Cell proliferation of the patient’s pterygomaxillary mass fibroblasts decreased in vitro with increasing concentrations of hydroxychloroquine, pentoxifylline, and sunitinib. B, Treatment with hydroxychloroquine was associated with a decline and stabilization of C-reactive protein (CRP) levels. C and D, Radiographic images demonstrated reduction in sizes of the pterygomaxillary mass (circle) and lung nodules (arrows) from baseline to 24 and 48 months after starting treatment with hydroxychloroquine 400 mg daily.

patients with specific CFTR variants.48-52 In this work, we demonstrate that precision medicine can successfully guide individualized therapy even in the absence of an established diagnosis. In our patient with an undiagnosed progressive multiorgan fibroinflammatory disorder, we utilized a comprehensive multidisciplinary precision medicine approach to identify candidate therapy. Serum proteomic profiling suggested that the tumor necrosis factor-alpha pathway may be associated with the pathobiology of this patient’s disorder. These results prompted in vitro studies of the patient’s own fibroblasts from a predominant site of disease involvement, and large-scale gene expression data independently showed that the tumor necrosis factor-alpha pathway was one of the main upstream regulators in these cells. Tumor necrosis factor-alpha, a proinflammatory

cytokine capable of inducing fibrotic effects, is a plausible driver and therapeutic target in this case, but other mechanisms may also be contributing to her disease. Given the progressive growth of the patient’s fibroinflammatory masses, we hypothesized that proliferation of her affected fibroblasts is a clinically relevant phenotype. We compared the inhibitory responses of her cells to potential therapeutic agents. One candidate drug identified by these in vitro studies was hydroxychloroquine, which suppresses inflammation and cell signaling induced by the tumor necrosis factor-alpha pathway as well as fibroblast proliferation and activation.38-41 Hydroxychloroquine is a widely-prescribed drug approved as treatment for malaria, systemic lupus erythematosus, and rheumatoid arthritis; it is generally tolerated without significant adverse effects. However, patients using hydroxychloroquine are at risk of

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retinopathy.53,54 Treatment with hydroxychloroquine at a standard dose resulted in clinical improvement in the patient’s previously aggressive and progressive disorder and was not associated with ocular toxicity. We acknowledge that resources of the NIH UDP were utilized to perform comprehensive multidisciplinary personalized testing, and limited funding is a likely impediment to performing a similar level of precision medicine in other clinical settings at this time. Progress in precision medicine would be facilitated by improving the resources available to medical institutions and clinical facilities to conduct similar advanced molecular and cellular biology testing. The potential benefits of expanding medical care beyond common clinical testing to generate favorable outcomes of patients with challenging undiagnosed conditions are exemplified by the results in this case. Overall, this case expands the role of precision medicine by illustrating how personalized approaches can identify effective therapy for challenging cases in the absence of a diagnosis. Similar strategies could be considered for other patients with undiagnosed diseases to develop individualized therapeutic plans. ACKNOWLEDGMENTS

Conflict of Interest: C.E.M is a cofounder and board member for Biotia and Onegevity Health. All authors have read the journal’s authorship agreement and policy on disclosure of potential conflicts of interest. We thank our patients who participated in our studies. We also thank the Epigenomics Core Facility and SCU at Weill Cornell Medicine. This research was supported in part by the Intramural Research Programs of the National Human Genome Research Institute and National Institute on Deafness and Other Communication Disorders, National Institutes of Health; NIDCD intramural project ZIA-DC-000075 (CVW); and the Common Fund, Office of the Director, NIH. Funding was also provided to C.E.M. by the Bert L. and N. Kuggie Vallee Foundation, the WorldQuant Foundation, The Pershing Square Sohn Cancer Research Alliance, the National Institutes of Health (1R01MH117406), the Leukemia and Lymphoma Society grants (LLS 923816, Mak, LLS-MCL-982, Chen-Kiang). The NIH had no role in the design of the study; collection, analysis, and interpretation of the data; writing of the manuscript; and decision to submit the article for publication.

SUPPLEMENTARY MATERIALS

Supplementary material associated with this article can be found in the online version at doi:10.1016/j. trsl.2019.08.008.

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