- Email: [email protected]
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype
Human Pathology (2018) 76, 52–57
www.elsevier.com/locate/humpath
Original contribution
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype☆ Paul N. Manley MD a,⁎, Suzy Abu-Abed MD, PhD, FRCPC b , Richard Kirsch MBChB, PhD, FRCPath(SA) FRCPC c , Andrea Hawrysh MSc d , Nicole Perrier MSc, MD d , Harriet Feilotter PhD, FCCMG, FACMG b , Aaron Pollett MD, FRCPC c , Robert H. Riddell MD, FRCPath, FRCPC c , Lawrence Hookey MD, FRCPC e , Jagdeep S. Walia MBBS, FRCPC, FCCMG a,d,⁎⁎ a
Department of Pathology and Molecular Medicine, Queens University and Kingston General Hospital, Kingston, Ontario, K7L3N6, Canada b Department of Pathology, Royal Columbian Hospital, New Westminster, British Columbia V3L 3W7, Canada c Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada d Division of Medical Genetics in the Department of Pediatrics, Kingston General Hospital, Kingston, Ontario K7L 2V7, Canada e Division of Gastroenterology in the Department of Medicine, Kingston General Hospital, Kingston, Ontario K7L 2V7, Canada Received 1 December 2017; revised 7 February 2018; accepted 14 February 2018
Keywords: Inflammatory fibroid polyp; GIST; PDGFRA; Telocyte; Dysmophology
Summary Germline activating platelet-derived growth factor receptor alpha (PDGFRA) mutations have been described in four families. All the index patients have presented with multiple mesenchymal tumors of the gastrointestinal tract. We identified a fifth family with four first-degree relatives that harbor a PDGFRA exon 18 (D846V) germline mutation. The affected kindred have a unique phenotype including coarse facies and skin, broad hands and feet, and previously undescribed premature tooth loss. While the index patient presented with multiple small bowel inflammatory fibroid polyps (IFPs) and has a gastric gastrointestinal stromal tumor (GIST), no tumors have yet been identified in other family members. We describe the pathology, genetics, the incomplete penetrance and variable expressivity of the familial PDGFRA-mutation syndrome referencing the mouse knock-in Pdgfra model. We speculate on the role of the telocyte, a recently described CD34, PDGFRA+ stromal cell, in the development of inflammatory fibroid polyps and the somatic phenotype. © 2018 Elsevier Inc. All rights reserved.
Abbreviations CM, centimeters; HE, hematoxylin-eosin; HPS, hematoxylin-phloxine-saffron; FFPE, formalin-fixed paraffin-embedded; FNA, fine needle aspiration; GIST, gastrointestinal stromal tumor; IFP, inflammatory fibroid polyp; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PDGFRA, platelet-derived growth factor receptor alpha. ☆
Disclaimer: We have no potential conflicts of interest and no financial contributions related to this work that could have influenced the outcome. ⁎ Corresponding author P.N. Manley, Department of Pathology and Molecular Medicine, Richardson Laboratory Queens University, Kingston, ON K7L 3N6, Canada E-mail addresses: [email protected] (P. N. Manley), [email protected] (J. S. Walia). https://doi.org/10.1016/j.humpath.2018.02.014 0046-8177/© 2018 Elsevier Inc. All rights reserved.
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype
1. Introduction PDGFRA is highly conserved across species [1]. During embryonic development of the mouse, PDGFRA and its ligand PDGF-A, respectively, display tightly regulated epithelial-mesenchymal patterns of expression in the developing gastrointestinal tract, skin and skeleton [2]. In some clinical reports, the gastrointestinal tract develops numerous IFPs in the intestines and several gastrointestinal stromal tumors (GISTs) in the stomach [3,4]. Not surprisingly, constitutively activating PDGFRA mutations both in mouse models and in affected families may also modify the skin and skeletal system. Two of the previously described families with PDGFRA germline mutations also had large hands (Table). Sporadic IFPs and GISTs present as solitary mesenchymal tumors unique to the gastrointestinal tract. GISTs can exhibit malignant behavior, whereas IFPs are benign. Histologically, IFPs are characterized by a submucosal proliferation of stellate-to-spindled cells embedded within a richly vascular, mildly inflamed and variably fibrotic stroma [5]. These spindle tumor cells express CD34 and PDGFRA [6]. Sporadic and familial GISTs most commonly have activating KIT gene mutations. In contrast, only PDGFRA mutations have been observed in IFPs. PDGFRA exon 12, 14, and 18 gain-of-function mutations have been reported in both sporadic and familial IFPs [4,7,8]. Here we present a family with PDGFRA exon 18 mutation with the proband presenting with IFPs and a gastric GIST. The family members positive for the mutation share coarse facial features, large hands and feet, and uniquely amongst all the previously described families, premature loss of teeth.
2. Materials and methods 2.1. Clinical summary The index patient initially presented with abdominal pain and diarrhea at 50 years of age. Intra-operatively, three ileal intussusceptions were identified, and numerous intramural nodules were seen in the small bowel and colon. A single firm
Table
53
nodule was noted in the stomach. One intussuscepted segment of small bowel was resected and the remaining intussusceptions were manually reduced. At 57 years of age, at least five small bowel intussusceptions requiring resection were identified, and hundreds of nodules were palpated throughout the small bowel. The patient was subsequently referred for genetic counseling. Two years later, the patient presented with a jejunal intussusception. Hundreds of small bowel tumors and a few gastric nodules were seen. Four segmental small bowel resections, an appendectomy and a sigmoidectomy were performed. Endoscopic ultrasound-guided fine needle aspiration (FNA) of one of the gastric lesions was completed one year later. The index patient was referred to the Familial Oncology Program (Cancer Genetics, Kingston General Hospital). Following research ethics board approval and informed consent, the proband and several first-degree relatives were interviewed by a clinical geneticist (JW) and genetic counselors (AH, NP). A standard three-generation pedigree was completed as part of the clinical genetics assessment. Dysmorphology examination were performed on all interviewed family members, most of whom agreed to have targeted screening for the PDGFRA mutation identified in the index patient. In addition, upper endoscopy, colonoscopy, and/or small bowel video capsule endoscopy were completed and interpreted by the same gastroenterologist (LH).
2.2. Histochemistry and immunohistochemistry Routine hematoxylin-eosin (HE) and hematoxylin-phloxinesaffron (HPS) stains, as well as immunohistochemical studies were performed on representative tissue sections from formalin-fixed, paraffin-embedded (FFPE) tissue. Primary antibodies were directed against the following antigens: anaplastic lymphoma kinase, beta catenin, CD34, DOG 1, EMA, KIT, S100, and smooth muscle actin. All immunohistochemical studies were performed on automated immunostainers with their reagents (Ventana Medical Systems, Tucson, AZ, USA). The findings were reviewed by multiple pathologists with subspecialty training in gastrointestinal pathology (SA, RK, AP, RR, and PM).
Summary of findings in kindreds with the familial PDGFRA-mutation syndrome
Study
Descent
PDGFRA mutation
IFPs
GISTs
Other
Current
English-Belgian
D846V
Intestinal
Gastric
Ricci et al [8] Carney et al [13]; Pasini et al [3] De Raedt et al [7]
Italian Italian-English-Scottish Luxembourgish
P653L V561D Y555C
Gastric intestinal Gastric intestinal Intestinal
Gastric Gastric None
Chompret et al [4]
French
D846Y
None
Gastric
Broad hands and feet Coarse facies and skin Early loss of teeth Intestinal lipoma Intestinal lipoma Broad hands Coarse facies Large hands
54
2.3. Molecular studies 2.3.1. Preparation of genomic DNA from paraffin-embedded tissue and blood Genomic DNA was extracted from two tumors and uninvolved tissue to screen for possible KIT and PDGFRA germline mutations. Genomic DNA from FFPE tissue was extracted using the QIAmp DNA Mini Kit (Qiagen, Toronto, ON, Canada). In brief, HE-stained sections from the proband’s IFPs were reviewed; areas containing tumor (N70% tumor cells) and background tissue were separately identified. Tumor and background tissue were microdissected from adjacent 10micron thick sections using a scalpel blade and placed in separate microcentrifuge tubes. In addition, genomic DNA was extracted from whole blood from the proband and several first degree relatives using the ArchivePure DNA Purification kit (Inter Medico Technologies, Markham, ON, Canada). Firstdegree family members were consented for a targeted assessment for PDGFRA germline mutation. 2.3.2. PCR amplification and sequencing of genomic DNA Multiple KIT (9, 11, 13, and 17) and PDGFRA (12, 14, and 18) exons were amplified from genomic DNA extracted from the proband’s FFPE tissues. Only PDGFRA exon 18 was amplified from genomic DNA extracted from blood. PCR products from genomic DNA extracted from FFPE tissue and blood were respectively purified using EXOSAP II (Applied Biosystems, Foster, California, USA) and BDx (Becton Dickinson, Franklin Lakes, NJ, USA). Excess primers and unincorporated nucleotides were removed using ExoSAP-it. Purified products were bidirectionally sequenced using the ABI Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, California, USA) on an ABI Verite Thermocycler. Sequencing products were assayed on an ABI 3130XL Automated Sequencer. All sequences were analyzed using ABI Sequencing Analysis v5.2 software, and Mutation Surveyor V.4.0.7. sequencing alignment software.
3. Results 3.1. Pathology Each resected segment of small bowel had a variably thickened submucosa and numerous superimposed well-defined submucosal nodules measuring 0.5 to 9.5 cm in greatest dimension. The nodules invariably involved the submucosa and muscularis propria, and occasionally the subserosa (Figure 1). The appendix and sigmoid colon also had a diffusely thickened submucosa, as well as one submucosal nodule each, measuring 0.8 cm and 3.6 cm respectively. Histologic sections from the small bowel, appendiceal, and sigmoid colon nodules showed similar findings. The nodules were composed of submucosal stellate-to-spindle cell proliferations embedded within a variably fibrotic and minimally
P. N. Manley et al. inflamed stroma that had thick- and thin-walled ectatic vessels (Figure 1). The overlying mucosa was rarely ulcerated, while the underlying muscularis propria ranged from attenuated to completely lost. Intervening segments of bowel wall between the tumor nodules had an expanded submucosa characterized by increased spindle cell cellularity and collagen deposition. No mitotic activity, cytologic atypia, or necrosis was present, and no hyperplasia of the interstitial cells of Cajal was identified. Immunohistochemical studies showed that the spindle cells seen within the expanded submucosa, as well as in the submucosal nodules were positive for CD34 and negative for KIT, DOG1, S100, SMA, EMA, beta-catenin, and ALK (Figure 1). These immunomorphologic findings support a diagnosis of inflammatory fibroid polyp for each of the examined nodules. Finally, the gastric nodule sampled by means of endoscopic ultrasound-guided FNA was morphologically and immunophenotypically (KIT, DOG1 and CD34 positive) diagnostic of a GIST.
3.2. PDGFRA and KIT gene mutation analysis Initial genetic testing was performed on two IFPs and background tissue resected from the index patient’s small bowel at her second surgery. A PDGFRA exon 18 mutation (D846V) was identified in both polyps and the intervening background tissue. One of the polyps had an additional PDGFRA mutation in exon 12 (D576Y). No KIT gene mutations were identified in any of the four exons (9, 11, 13, and 17) tested. Genomic DNA extracted from blood was used to screen for a PDGFRA exon 18 germline mutation in the proband and her family. The index patient, one of her daughters, and two of her brothers tested positive for a D846V germline mutation (Figure 2).
3.3. Clinical findings in the index patient and family Clinical histories from the index patient and first-degree relatives were negative for common forms of hereditary colon cancer and polyposis syndromes. Compared to the mutationnegative family members, those harboring the PDGFRA germline mutation had a coarser face, coarser skin, broader hands and feet (Figure 2) and unexplained premature loss of teeth requiring dentures as early as their 40s. Though the index patient has hundreds of intestinal IFPs, as well as at least one gastric GIST, no polyps or tumors have been identified in any of the family members screened thus far (Figure 2).
4. Discussion We describe the fifth family with a documented germline PDGFRA mutation. Each family’s mutation is unique. They have been identified as single–amino acid substitutions in exons 12 (Y555C and V561D), 14 (P653L), and 18 (D846V
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype
55
Fig. 1 Histopathological description. A, A segmental small bowel resection with multiple polyps ranging from 1.5 to 3 cm and submucosal expansion with loss of mucosal folds. B, A transected 2 cm polyp shows a well-circumscribed homogenous light tan submucosal tumor. The adjacent submucosa is variably expanded by similar tissue. C, HPS-stained slides show a polypoid submucosal hypocellular tumor permeating into the muscularis propria (10×). D, The tumor is formed by a bland spindle-to-stellate cell proliferation set within a loosely fibrous and richly vascular stroma. (40×). E, Occasional vessels surrounded by a concentric spindle cell proliferation (“onion-skinning”) are noted. The background stroma is infiltrated by a mild mixed chronic inflammatory cell infiltrate including mast cells and eosinophils (200×). F, Immunohistochemical studies show the tumor is focally positive for CD34 (200×). me, mesentery; mu, mucosa; mp, muscularis propria; sm, submucosa; arrow, stellate tumor cell; arrowhead, mast cell; asterisk symbol, vessel with onion-skinning.
and D846Y). All are predicted to result in constitutive activation of the tyrosine kinase receptor [3,4,7,8]. The inheritance appears to be autosomal dominant but thus far has displayed incomplete penetrance and variable expressivity. Many mutation carriers develop mesenchymal tumors of the gastrointestinal tract, and some have large hands and/or coarse facies, while others are seemingly phenotypically normal and tumor free (Table). In our family, all mutation-positive members have premature loss of teeth, which has not been
previously described in other families. Ricci et al [8] have named this disorder as the PDGFRA-mutant syndrome. We prefer the name familial PDGFRA-mutation syndrome as PDGFRA mutations are present in sporadic IFPs. GISTs originate from interstitial cells of Cajal, but no precursor cell has been identified for IFPs. We speculate that IFPs may arise from telocytes, a newly described fibroblast-like stromal cell population of many organs including the gastrointestinal tract and skin of humans and laboratory animals [9].
56
P. N. Manley et al. Immunohistochemically, telocytes and the spindle cells of IFPs are CD34 and PDGFRA positive [10]. Telocytes have small cell bodies and extremely long slender prolongations, making them distinct from fibroblasts [11]. Additional studies, including transmission and immunoelectron microscopy, are needed to define whether telocytes are the precursor cell for IFPs. Finally, although our index patient and her mutationpositive female and male kindreds have similar dysmorphic features, mesenchymal tumors of the gastrointestinal tract have only been identified in the index patient (Figure 2). Interestingly, Pdgfra+/Kmutant mice developed by Olson and Soriano recapitulate the phenotype of our index patient and family, as well as other kindreds with the PDGFRA-mutation syndrome [12]. In contrast to knockout mice, which show marked atrophy of mesenchyme-derived tissues, mice with the constitutively activated mutant PDGFRA have an overabundance of mesenchyme formation in several organ systems. Notably, there is diffuse expansion of the gastrointestinal submucosa, which exhibits an increased number of spindled fibroblast-like cells and marked collagen deposition. More than half of the mutant mice also develop a variable number of subcentimeter intestinal polyps that are morphologically similar to IFPs. The Pdgfra+/K mice also exhibit thickened skin due to excess collagen deposition within the dermis and subcutaneous tissues. The Pdgfra+/Kmouse phenotype raises the possibility that the extensive intestinal submucosal thickening, numerous intestinal IFPs and the thickening of the skin in our patient reflect PDGFRA activation of CD34, PDGFRA+ve subepithelial cells, likely the telocyte. Distinct polyps may develop and/or enlarge over time, possibly because additional PDGFRA mutations are gained, as seen in one of the small bowel IFPs from our index patient, or some thickened submucosal areas are elevated by peristaltic waves. Skeletal mesenchymal abnormalities induced by PDGFRA overexpression may contribute to early tooth loss. Remarkably, following relocation of the mutant mouse colony, intestinal polyps could no longer be detected in any of the new Pdgfra+/K mutant mice. This observation indicates that a gene-environment interaction may be affecting susceptibility for developing IFPs (personal communication with Lorin Olson) and may explain the incomplete penetrance and variable expressivity in our family.
Fig. 2 Pedigree, genotype and phenotype. A, Pedigree of the kindred with a PDGFRA exon 18 germline mutation (D846V). Each generation is designated by a Roman numeral and each family member by an Arabic number. Circles denote females, squares male, and slashes deceased family members. Below the symbols are the age and PDGFRA genotype of those who were screened for the D846V mutation. Features indicated are gastric GISTs, intestinal IFPs, coarse facies and skin, broad hands and feet, and negative endoscopy studies. The arrow denotes the index patient (II.2). B, Phenotype of family members. All mutation-positive family members have coarse facial features and broad hands and feet as compared to mutation-negative individuals.
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype
Acknowledgements We thank the family who participated in the study. The study was accomplished through interdepartmental funding.
References [1] Hoch RV, Soriano P. Roles of PDGF in animal development. Development 2003;130:4769-84. https://doi.org/10.1242/dev.00721. [2] Karlsson L, Lindahl P, Heath JK, Betsholtz C. Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis. Development 2000;127:3457-66. [3] Pasini B, Matyakhina L, Bei T, et al. Multiple gastrointestinal stromal and other tumors caused by platelet-derived growth factor receptor alpha gene mutations: a case associated with a germline V561D defect. J Clin Endocrinol Metab 2007;92:3728-32. https://doi.org/10.1210/jc.2007-0894. [4] Chompret A, Kannengiesser C, Barrois M, et al. PDGFRA germline mutation in a family with multiple cases of gastrointestinal stromal tumor. Gastroenterology 2004;126:318-21. [5] Kirsch R. Mesenchymal tumours. In: Riddel R, Jain D, editors. Gastrointestinal Pathology and Its Clinical Implications. 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2014. p. 333-5.
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[6] Liu TC, Lin MT, Montgomery EA, Singhi AD. Inflammatory fibroid polyps of the gastrointestinal tract: spectrum of clinical, morphologic, and immunohistochemistry features. Am J Surg Pathol 2013;37: 586-92. https://doi.org/10.1097/PAS.0b013e31827ae11e. [7] de Raedt T, Cools J, Debiec-Rychter M, et al. Intestinal neurofibromatosis is a subtype of familial GIST and results from a dominant activating mutation in PDGFRA. Gastroenterology 2006;131:1907-12 10.1053/ j.gastro.2006.07.002. [8] Ricci R, Martini M, Cenci T, et al. PDGFRA-mutant syndrome. Mod Pathol 2015;28:954-64. https://doi.org/10.1038/modpathol.2015.56. [9] Ibba-Manneschi L, Rosa I, Manetti M. Telocyte implications in human pathology: An overview. Semin Cell Dev Biol 2016;55:62-9. https:// doi.org/10.1016/j.semcdb.2016.01.022. [10] Vannucchi MG, Traini C, Manetti M, et al. Telocytes express PDGFRα in the human gastrointestinal tract. J Cell Mol Med 2013;17:1099-108. https://doi.org/10.1111/jcmm.12134. [11] Mirancea N. Telocyte - a particular cell phenotype. Infrastructure, relationships and putative functions. Rom J Morphol Embryol 2016; 57:7-21. [12] Olson LE, Soriano P. Increased PDGFRalpha activation disrupts connective tissue development and drives systemic fibrosis. Dev Cell 2009;16: 303-13. https://doi.org/10.1016/j.devcel.2008.12.003. [13] Carney JA, Stratakis CA. Stromal, fibrous, and fatty gastrointestinal tumors in a patient with a PDGFRA gene mutation. Am J Surg Pathol 2008;32:1412-20. https://doi.org/10.1097/PAS. 0b013e31816250ce.
www.elsevier.com/locate/humpath
Original contribution
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype☆ Paul N. Manley MD a,⁎, Suzy Abu-Abed MD, PhD, FRCPC b , Richard Kirsch MBChB, PhD, FRCPath(SA) FRCPC c , Andrea Hawrysh MSc d , Nicole Perrier MSc, MD d , Harriet Feilotter PhD, FCCMG, FACMG b , Aaron Pollett MD, FRCPC c , Robert H. Riddell MD, FRCPath, FRCPC c , Lawrence Hookey MD, FRCPC e , Jagdeep S. Walia MBBS, FRCPC, FCCMG a,d,⁎⁎ a
Department of Pathology and Molecular Medicine, Queens University and Kingston General Hospital, Kingston, Ontario, K7L3N6, Canada b Department of Pathology, Royal Columbian Hospital, New Westminster, British Columbia V3L 3W7, Canada c Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada d Division of Medical Genetics in the Department of Pediatrics, Kingston General Hospital, Kingston, Ontario K7L 2V7, Canada e Division of Gastroenterology in the Department of Medicine, Kingston General Hospital, Kingston, Ontario K7L 2V7, Canada Received 1 December 2017; revised 7 February 2018; accepted 14 February 2018
Keywords: Inflammatory fibroid polyp; GIST; PDGFRA; Telocyte; Dysmophology
Summary Germline activating platelet-derived growth factor receptor alpha (PDGFRA) mutations have been described in four families. All the index patients have presented with multiple mesenchymal tumors of the gastrointestinal tract. We identified a fifth family with four first-degree relatives that harbor a PDGFRA exon 18 (D846V) germline mutation. The affected kindred have a unique phenotype including coarse facies and skin, broad hands and feet, and previously undescribed premature tooth loss. While the index patient presented with multiple small bowel inflammatory fibroid polyps (IFPs) and has a gastric gastrointestinal stromal tumor (GIST), no tumors have yet been identified in other family members. We describe the pathology, genetics, the incomplete penetrance and variable expressivity of the familial PDGFRA-mutation syndrome referencing the mouse knock-in Pdgfra model. We speculate on the role of the telocyte, a recently described CD34, PDGFRA+ stromal cell, in the development of inflammatory fibroid polyps and the somatic phenotype. © 2018 Elsevier Inc. All rights reserved.
Abbreviations CM, centimeters; HE, hematoxylin-eosin; HPS, hematoxylin-phloxine-saffron; FFPE, formalin-fixed paraffin-embedded; FNA, fine needle aspiration; GIST, gastrointestinal stromal tumor; IFP, inflammatory fibroid polyp; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PDGFRA, platelet-derived growth factor receptor alpha. ☆
Disclaimer: We have no potential conflicts of interest and no financial contributions related to this work that could have influenced the outcome. ⁎ Corresponding author P.N. Manley, Department of Pathology and Molecular Medicine, Richardson Laboratory Queens University, Kingston, ON K7L 3N6, Canada E-mail addresses: [email protected] (P. N. Manley), [email protected] (J. S. Walia). https://doi.org/10.1016/j.humpath.2018.02.014 0046-8177/© 2018 Elsevier Inc. All rights reserved.
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype
1. Introduction PDGFRA is highly conserved across species [1]. During embryonic development of the mouse, PDGFRA and its ligand PDGF-A, respectively, display tightly regulated epithelial-mesenchymal patterns of expression in the developing gastrointestinal tract, skin and skeleton [2]. In some clinical reports, the gastrointestinal tract develops numerous IFPs in the intestines and several gastrointestinal stromal tumors (GISTs) in the stomach [3,4]. Not surprisingly, constitutively activating PDGFRA mutations both in mouse models and in affected families may also modify the skin and skeletal system. Two of the previously described families with PDGFRA germline mutations also had large hands (Table). Sporadic IFPs and GISTs present as solitary mesenchymal tumors unique to the gastrointestinal tract. GISTs can exhibit malignant behavior, whereas IFPs are benign. Histologically, IFPs are characterized by a submucosal proliferation of stellate-to-spindled cells embedded within a richly vascular, mildly inflamed and variably fibrotic stroma [5]. These spindle tumor cells express CD34 and PDGFRA [6]. Sporadic and familial GISTs most commonly have activating KIT gene mutations. In contrast, only PDGFRA mutations have been observed in IFPs. PDGFRA exon 12, 14, and 18 gain-of-function mutations have been reported in both sporadic and familial IFPs [4,7,8]. Here we present a family with PDGFRA exon 18 mutation with the proband presenting with IFPs and a gastric GIST. The family members positive for the mutation share coarse facial features, large hands and feet, and uniquely amongst all the previously described families, premature loss of teeth.
2. Materials and methods 2.1. Clinical summary The index patient initially presented with abdominal pain and diarrhea at 50 years of age. Intra-operatively, three ileal intussusceptions were identified, and numerous intramural nodules were seen in the small bowel and colon. A single firm
Table
53
nodule was noted in the stomach. One intussuscepted segment of small bowel was resected and the remaining intussusceptions were manually reduced. At 57 years of age, at least five small bowel intussusceptions requiring resection were identified, and hundreds of nodules were palpated throughout the small bowel. The patient was subsequently referred for genetic counseling. Two years later, the patient presented with a jejunal intussusception. Hundreds of small bowel tumors and a few gastric nodules were seen. Four segmental small bowel resections, an appendectomy and a sigmoidectomy were performed. Endoscopic ultrasound-guided fine needle aspiration (FNA) of one of the gastric lesions was completed one year later. The index patient was referred to the Familial Oncology Program (Cancer Genetics, Kingston General Hospital). Following research ethics board approval and informed consent, the proband and several first-degree relatives were interviewed by a clinical geneticist (JW) and genetic counselors (AH, NP). A standard three-generation pedigree was completed as part of the clinical genetics assessment. Dysmorphology examination were performed on all interviewed family members, most of whom agreed to have targeted screening for the PDGFRA mutation identified in the index patient. In addition, upper endoscopy, colonoscopy, and/or small bowel video capsule endoscopy were completed and interpreted by the same gastroenterologist (LH).
2.2. Histochemistry and immunohistochemistry Routine hematoxylin-eosin (HE) and hematoxylin-phloxinesaffron (HPS) stains, as well as immunohistochemical studies were performed on representative tissue sections from formalin-fixed, paraffin-embedded (FFPE) tissue. Primary antibodies were directed against the following antigens: anaplastic lymphoma kinase, beta catenin, CD34, DOG 1, EMA, KIT, S100, and smooth muscle actin. All immunohistochemical studies were performed on automated immunostainers with their reagents (Ventana Medical Systems, Tucson, AZ, USA). The findings were reviewed by multiple pathologists with subspecialty training in gastrointestinal pathology (SA, RK, AP, RR, and PM).
Summary of findings in kindreds with the familial PDGFRA-mutation syndrome
Study
Descent
PDGFRA mutation
IFPs
GISTs
Other
Current
English-Belgian
D846V
Intestinal
Gastric
Ricci et al [8] Carney et al [13]; Pasini et al [3] De Raedt et al [7]
Italian Italian-English-Scottish Luxembourgish
P653L V561D Y555C
Gastric intestinal Gastric intestinal Intestinal
Gastric Gastric None
Chompret et al [4]
French
D846Y
None
Gastric
Broad hands and feet Coarse facies and skin Early loss of teeth Intestinal lipoma Intestinal lipoma Broad hands Coarse facies Large hands
54
2.3. Molecular studies 2.3.1. Preparation of genomic DNA from paraffin-embedded tissue and blood Genomic DNA was extracted from two tumors and uninvolved tissue to screen for possible KIT and PDGFRA germline mutations. Genomic DNA from FFPE tissue was extracted using the QIAmp DNA Mini Kit (Qiagen, Toronto, ON, Canada). In brief, HE-stained sections from the proband’s IFPs were reviewed; areas containing tumor (N70% tumor cells) and background tissue were separately identified. Tumor and background tissue were microdissected from adjacent 10micron thick sections using a scalpel blade and placed in separate microcentrifuge tubes. In addition, genomic DNA was extracted from whole blood from the proband and several first degree relatives using the ArchivePure DNA Purification kit (Inter Medico Technologies, Markham, ON, Canada). Firstdegree family members were consented for a targeted assessment for PDGFRA germline mutation. 2.3.2. PCR amplification and sequencing of genomic DNA Multiple KIT (9, 11, 13, and 17) and PDGFRA (12, 14, and 18) exons were amplified from genomic DNA extracted from the proband’s FFPE tissues. Only PDGFRA exon 18 was amplified from genomic DNA extracted from blood. PCR products from genomic DNA extracted from FFPE tissue and blood were respectively purified using EXOSAP II (Applied Biosystems, Foster, California, USA) and BDx (Becton Dickinson, Franklin Lakes, NJ, USA). Excess primers and unincorporated nucleotides were removed using ExoSAP-it. Purified products were bidirectionally sequenced using the ABI Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, California, USA) on an ABI Verite Thermocycler. Sequencing products were assayed on an ABI 3130XL Automated Sequencer. All sequences were analyzed using ABI Sequencing Analysis v5.2 software, and Mutation Surveyor V.4.0.7. sequencing alignment software.
3. Results 3.1. Pathology Each resected segment of small bowel had a variably thickened submucosa and numerous superimposed well-defined submucosal nodules measuring 0.5 to 9.5 cm in greatest dimension. The nodules invariably involved the submucosa and muscularis propria, and occasionally the subserosa (Figure 1). The appendix and sigmoid colon also had a diffusely thickened submucosa, as well as one submucosal nodule each, measuring 0.8 cm and 3.6 cm respectively. Histologic sections from the small bowel, appendiceal, and sigmoid colon nodules showed similar findings. The nodules were composed of submucosal stellate-to-spindle cell proliferations embedded within a variably fibrotic and minimally
P. N. Manley et al. inflamed stroma that had thick- and thin-walled ectatic vessels (Figure 1). The overlying mucosa was rarely ulcerated, while the underlying muscularis propria ranged from attenuated to completely lost. Intervening segments of bowel wall between the tumor nodules had an expanded submucosa characterized by increased spindle cell cellularity and collagen deposition. No mitotic activity, cytologic atypia, or necrosis was present, and no hyperplasia of the interstitial cells of Cajal was identified. Immunohistochemical studies showed that the spindle cells seen within the expanded submucosa, as well as in the submucosal nodules were positive for CD34 and negative for KIT, DOG1, S100, SMA, EMA, beta-catenin, and ALK (Figure 1). These immunomorphologic findings support a diagnosis of inflammatory fibroid polyp for each of the examined nodules. Finally, the gastric nodule sampled by means of endoscopic ultrasound-guided FNA was morphologically and immunophenotypically (KIT, DOG1 and CD34 positive) diagnostic of a GIST.
3.2. PDGFRA and KIT gene mutation analysis Initial genetic testing was performed on two IFPs and background tissue resected from the index patient’s small bowel at her second surgery. A PDGFRA exon 18 mutation (D846V) was identified in both polyps and the intervening background tissue. One of the polyps had an additional PDGFRA mutation in exon 12 (D576Y). No KIT gene mutations were identified in any of the four exons (9, 11, 13, and 17) tested. Genomic DNA extracted from blood was used to screen for a PDGFRA exon 18 germline mutation in the proband and her family. The index patient, one of her daughters, and two of her brothers tested positive for a D846V germline mutation (Figure 2).
3.3. Clinical findings in the index patient and family Clinical histories from the index patient and first-degree relatives were negative for common forms of hereditary colon cancer and polyposis syndromes. Compared to the mutationnegative family members, those harboring the PDGFRA germline mutation had a coarser face, coarser skin, broader hands and feet (Figure 2) and unexplained premature loss of teeth requiring dentures as early as their 40s. Though the index patient has hundreds of intestinal IFPs, as well as at least one gastric GIST, no polyps or tumors have been identified in any of the family members screened thus far (Figure 2).
4. Discussion We describe the fifth family with a documented germline PDGFRA mutation. Each family’s mutation is unique. They have been identified as single–amino acid substitutions in exons 12 (Y555C and V561D), 14 (P653L), and 18 (D846V
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype
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Fig. 1 Histopathological description. A, A segmental small bowel resection with multiple polyps ranging from 1.5 to 3 cm and submucosal expansion with loss of mucosal folds. B, A transected 2 cm polyp shows a well-circumscribed homogenous light tan submucosal tumor. The adjacent submucosa is variably expanded by similar tissue. C, HPS-stained slides show a polypoid submucosal hypocellular tumor permeating into the muscularis propria (10×). D, The tumor is formed by a bland spindle-to-stellate cell proliferation set within a loosely fibrous and richly vascular stroma. (40×). E, Occasional vessels surrounded by a concentric spindle cell proliferation (“onion-skinning”) are noted. The background stroma is infiltrated by a mild mixed chronic inflammatory cell infiltrate including mast cells and eosinophils (200×). F, Immunohistochemical studies show the tumor is focally positive for CD34 (200×). me, mesentery; mu, mucosa; mp, muscularis propria; sm, submucosa; arrow, stellate tumor cell; arrowhead, mast cell; asterisk symbol, vessel with onion-skinning.
and D846Y). All are predicted to result in constitutive activation of the tyrosine kinase receptor [3,4,7,8]. The inheritance appears to be autosomal dominant but thus far has displayed incomplete penetrance and variable expressivity. Many mutation carriers develop mesenchymal tumors of the gastrointestinal tract, and some have large hands and/or coarse facies, while others are seemingly phenotypically normal and tumor free (Table). In our family, all mutation-positive members have premature loss of teeth, which has not been
previously described in other families. Ricci et al [8] have named this disorder as the PDGFRA-mutant syndrome. We prefer the name familial PDGFRA-mutation syndrome as PDGFRA mutations are present in sporadic IFPs. GISTs originate from interstitial cells of Cajal, but no precursor cell has been identified for IFPs. We speculate that IFPs may arise from telocytes, a newly described fibroblast-like stromal cell population of many organs including the gastrointestinal tract and skin of humans and laboratory animals [9].
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P. N. Manley et al. Immunohistochemically, telocytes and the spindle cells of IFPs are CD34 and PDGFRA positive [10]. Telocytes have small cell bodies and extremely long slender prolongations, making them distinct from fibroblasts [11]. Additional studies, including transmission and immunoelectron microscopy, are needed to define whether telocytes are the precursor cell for IFPs. Finally, although our index patient and her mutationpositive female and male kindreds have similar dysmorphic features, mesenchymal tumors of the gastrointestinal tract have only been identified in the index patient (Figure 2). Interestingly, Pdgfra+/Kmutant mice developed by Olson and Soriano recapitulate the phenotype of our index patient and family, as well as other kindreds with the PDGFRA-mutation syndrome [12]. In contrast to knockout mice, which show marked atrophy of mesenchyme-derived tissues, mice with the constitutively activated mutant PDGFRA have an overabundance of mesenchyme formation in several organ systems. Notably, there is diffuse expansion of the gastrointestinal submucosa, which exhibits an increased number of spindled fibroblast-like cells and marked collagen deposition. More than half of the mutant mice also develop a variable number of subcentimeter intestinal polyps that are morphologically similar to IFPs. The Pdgfra+/K mice also exhibit thickened skin due to excess collagen deposition within the dermis and subcutaneous tissues. The Pdgfra+/Kmouse phenotype raises the possibility that the extensive intestinal submucosal thickening, numerous intestinal IFPs and the thickening of the skin in our patient reflect PDGFRA activation of CD34, PDGFRA+ve subepithelial cells, likely the telocyte. Distinct polyps may develop and/or enlarge over time, possibly because additional PDGFRA mutations are gained, as seen in one of the small bowel IFPs from our index patient, or some thickened submucosal areas are elevated by peristaltic waves. Skeletal mesenchymal abnormalities induced by PDGFRA overexpression may contribute to early tooth loss. Remarkably, following relocation of the mutant mouse colony, intestinal polyps could no longer be detected in any of the new Pdgfra+/K mutant mice. This observation indicates that a gene-environment interaction may be affecting susceptibility for developing IFPs (personal communication with Lorin Olson) and may explain the incomplete penetrance and variable expressivity in our family.
Fig. 2 Pedigree, genotype and phenotype. A, Pedigree of the kindred with a PDGFRA exon 18 germline mutation (D846V). Each generation is designated by a Roman numeral and each family member by an Arabic number. Circles denote females, squares male, and slashes deceased family members. Below the symbols are the age and PDGFRA genotype of those who were screened for the D846V mutation. Features indicated are gastric GISTs, intestinal IFPs, coarse facies and skin, broad hands and feet, and negative endoscopy studies. The arrow denotes the index patient (II.2). B, Phenotype of family members. All mutation-positive family members have coarse facial features and broad hands and feet as compared to mutation-negative individuals.
Familial PDGFRA-mutation syndrome: somatic and gastrointestinal phenotype
Acknowledgements We thank the family who participated in the study. The study was accomplished through interdepartmental funding.
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