Cutaneous Hyperpigmentation and Familial Gastrointestinal Stromal Tumor Associated with Germline KIT Mutation Treated with Imatinib

Background: Familial gastrointestinal stromal tumor (GIST) has been identied with multiple GISTs containing the mutations in germline KIT and PDGFRA. There are only 35 kindreds with germline KIT and 6 with PDGFRA mutations have been reported so far. Familial GIST is often characterized by a series of manifestations, such as multiple lesions, hyperpigmentation, mastocytosis, and dysphagia. Only some kindreds have response to imatinib treatment. Materials and Methods: A 25-year-old Chinese woman went to hospital because of the abdominal pain, and through the computed tomography (CT) scan showed us the multiple tumors in the small intestine. Her father had a history of multifocal GISTs, and referred to the hospital with abdominal pain and tumor recurrences last year. Immuhistochemical analysis of CD117 and DOG-1 were performed on tumor samples from the two patients, while KIT mutational analysis was carried out by direct sequencing on DNA from paran-embedded specimens and saliva sample. Results: Multiple GISTs associated with diffuse interstitial cells of Cajal (ICC) hyperplasia were illustrated in these two patients. These tumors were positive for CD117 and DOG-1. The germline mutation at codon 560 of exon 11 (p.V560G) of the KIT gene were found. The treatment with imatinib resulted in favorable responses in both tumor and cutaneous hyperpigmentation. Conclusions: It is dicult to make a correct diagnosis of familial GIST at rst time for its rarity. This case was nally diagnosed as familial GIST depending on the combination of diffuse ICC hyperplasia, germline KIT mutation, hyperpigmentation and its family history. 9 .This paper describes a case of familial GIST in two related patients (father-daughter relationship), which consulted to our center, presenting with multiple GSTs and cutaneous hyperpigmentation, as well as our detection of a single-point germline mutation of KIT in this family. We also monitor their response to imatinib therapy on GISTs and hypopigmentation related to this syndrome.


Background
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors occurring in the GI tract and originate from the interstitial cells of Cajal 1 . With a wide range of clinical behaviors, prognosis of GISTs varies from indolent disorders with low risks to highly malignant diseases that may metastasize and become lethal 2 . It is known that mutations of the KIT gene have been identi ed in several human tumors, including multiple GISTs 3 . The KIT gene, a proto-oncogene, is of great signi cance in the development of various cell lines, which includes the interstitial cells of Cajal and melanocytes 4 .
Previous researches have demonstrated that the constitutive activation of tyrosine kinase,a main pathogenic event in most GISTs, caused by the mutation within the KIT gene 5 . Although GISTs are mostly sporadic, families with multiple inherited GISTs associated with germline KIT mutation have been found 6 .
A majority of GISTs, including sporadic and familial GISTs, contain gain-of-function mutations of the KIT, which have been identi ed among blood relatives with multiple occurrences of GIST 7 . Those patients who do not harbor germline gain-of-function KIT mutation will have a mutation of PDGFRA or other gene mutations 8 . Reported kindreds of familial GIST have represented variable phenotypic features characterized by early presentation, multiple lesions, involvement of some related family members, cutaneous hyperpigmentation and dysphagia 4 . Because of the rarity, familial GISTs deserve our paying more attention to the results of molecularly targeted therapy with the selective TKI, such as imatinib 4,9 .This paper describes a case of familial GIST in two related patients (father-daughter relationship), which consulted to our center, presenting with multiple GSTs and cutaneous hyperpigmentation, as well as our detection of a single-point germline mutation of KIT in this family. We also monitor their response to imatinib therapy on GISTs and hypopigmentation related to this syndrome.

Case Presentation
Patient Patient 1 A 25-year-old Chinese woman ( :1, Fig. 1) presented to the hospital with abdominal pain and progressive cutaneous hyperpigmentation that developed a few months after birth (Fig. 1A). Many new dark lentiginous macules, similar to cafe-au-lait macules (CALMs), had subsequently appeared over her face, body, limbs (Fig. 1B), and also showed both of her popliteal fossa and hands (Fig. 1C). Multiple tumors in the small intestine were showed under the scanning of the computed tomography (CT). A partial resection of the small intestine for multiple tumors, measuring from 0.5 cm to 4.0 cm, was performed and was diagnosed as GIST in November 2019.

Patient 2
The Patient 1's 50-year-old father ( :1) had a history of GIST in the small intestine 16 years ago, as well as the similar cutaneous hyperpigmentation of skin. He had been admitted to the hospital in 2002 for abdominal pain and dark stools, and then accepted the resection of GISTs. The patient didn't receive imatinib therapy and had no postoperative follow-up. In 2019, he was again referred to the hospital for abdominal pain and was observed and got the results of having tumor recurrences. This patient took the second surgery to remove multiple small intestine tumors (with a maximum diameter of 4.0 cm), which diagnosed as multiple GISTs in July 2019. Some small tumors located in speci c locations were not surgically removed.
Of note, family history showed that his mother ( :2) might die of multiple lesions in abdominal cavity, while the diagnosis was not clear at that time. In addition, she was thought to have the same cutaneous

Genetic Studies
After informing consents were obtained from all patients, genomic DNA (gDNA) was prepared from saliva samples and para n-embedded specimens of tumor tissues of patient 1 and her father patient 2. Furthermore, we analyzed saliva sample obtained from :1, the daughter of patient 1, referred to standard methods (Qiagen, Hilden, Germany). Mutation analysis of the KIT and PDGFRA genomic region and anking sequences (20 bp) were done by polymerase chain reaction (PCR) referred to our previous method 10 .

Histopathology and Immunohistochemistry
All of the tumors re-examined were consisted of spindle-shaped cells in a fascicular and whorl-like pattern ( Fig. 3), and showed high cellularity, light to medium polymorphic but almost no or a low mitotic rate (patient 1: 0 mitosis/50 HPF, patient 2:0-1 mitosis/50 HPF). A diffuse hyperplasia of the ICCs was observed within the myenteric plexus of the small intestine. The tumor was classi ed into the group of GIST with low risk for an aggressive clinical behavior of patient 1 (daughter) and high risk of patient 2 (father) respectively.
Immunohistochemistry showed that great majority tumor cells (spindle-shaped cells) were positive for CD117 (KIT receptor) and DOG-1 in both patients, and the Ki67 index were below 1%.

Mutational Status
KIT and PDGFRA mutations were analyzed using previously described methods. The results showed a similar point mutation at codon 560 in patient 1 and 2, which resulted in the exchange of leucine by proline (p.V560G), indicating a de novo germline KIT mutation. Furthermore, we analyzed saliva sample that obtained from :1 (the daughter of patient 1), and sequence analysis of saliva DNA showed a wildtype sequence in exon 11 of the KIT gene. No additional mutations of KIT exons 9, 13, or 17 as well as PDGFRA exons 12 or 14 were presented in these three family members.

Treatment and Follow-up
Two months after the tumor resection, patient 1 ( :1) was followed up with adjuvant imatinib therapy, 400 mg/d, in case of the recurrence. Three months after treatment initiation, the patient was noticed a decrease in the number of pigmented lesions around her mouth (Fig. 1E), and we can see that the skin on her hands turned to light (Fig. 1D), and the color of her hair, pubic hair, and body hair both changed to white. In August 2020, she underwent CT scan, and there has been no clinical evidence of progression. Previous studies 16 have demonstrated that abnormalities of ICC, the "pacemaker cells of the gut", may cause various motility disorders of the gastrointestinal tract. Miettinen et al. 17 argued that GISTs were derived from transformed neoplastic precursors of the ICC. ICC hyperplasia and multiple cecal physicians were produced in mice transfected with the knock-in mutant KIT (V558del) 18 . In this study, the diffuse ICC hyperplasia, presented in the muscularis propria in the non-tumorous wall of the small bowel, could be found in two patients. Immunohistochemical examination of tumors resected from patients :1 and :1 showed that most tumor cells were positive for DOG-1 and CD117 (KIT protein). What's more, ICC were con rmed to express not only CD117, but also DOG-1. Therefore, data from a transgenic mouse model 19 , together with histologic ndings in previous researches 20 and ours, indicated that activation of germline KIT results in hyperplasia of the ICC cells from which GISTs derive.
Clinically, symptoms such as multiple primary neoplasms (with several affected relatives), dysphagia and abnormal pigmentation 1,13,21 can be observed in familial GISTs. Median age at diagnosis of sporadic GISTs is around 60 years, while tumors of familial GISTs may present at earlier ages than that of sporadic GISTs 21 . The kindred in our study demonstrated some of these traits. It is worth noting that patient :1 and :1(with the same mutation)who were diagnosed with multiple GISTs in their 20 s and 30 s were also noted to have cutaneous pigmentation which developed a few months after birth and increased in size and number with age. : 1, the daughter of patient :1 received genetic counseling and testing for early detection of GIST. Prior researches of familial GISTs have illustrated an association with cutaneous hyperpigmentation 22 .This association results from the role of KIT in development of melanocytes, as well as ICCs from which GISTs derive 23 . One of the dead family member (grandma of :1) in this study in earlier generation ( :2) indicated that "multiple lesions in abdominal cavity" might be the cause of death. The diagnosis was not clear at that time, probably because GISTs had not yet been considered as a unique clinicopathological entity.
Although the effectiveness of imatinib, a tyrosine kinase inhibitor, in the treatment of familial GISTs had not been de ned yet, many individuals with this disease accepted the therapy of imatinib, which might result in resolution of the hyperpigmentation and stable disease 3, 24 . However, not all patients experience pigmentary changes and tumor regression in response to imatinib, nor did all patients experience the same degree of changes 22 . Conca et al. 25 showed GIST in two family members with the L576P mutation (in exon 11) who had a poor response to imatinib since the tumors persisted microscopically. What's more, Divya G et al. 9 reported two siblings of hereditary GISTs with the missense mutation p. (Val559Ala). Both patients were treated with imatinib, but only one patient's condition changed to stable after such treatment; after a stable period of more than one year, progression of the disease could be observed in another. The different responses to imatinib may due to secondary drug resistance, the type of mutation and its location. Patients in our study were treated with adjuvant imatinib (400 mg/ day) after surgery ( :1 after second surgery), within several months of imatinib treatment these hyperpigmentation diminished and the skin tone became lighter. Previous studies have demonstrated that KIT and its ligand stem cell factor (SCF) are considered to play signi cant roles in the development of 4 cell lineages: hematopoietic cells, melanocytes, germ cells, and mast cells 24,26 . As it is known to all, imatinib has been identi ed as the inhibition of KIT, which may leads to downstream inhibition of tyrosinase gene promoter and subsequent melanin synthesis inhibition. In vitro studies 22,27 , researchers have reported that melanocytes with decreased proliferation in broblasts and low tyrosinase activity can be observed in imatinib-treated skin. This indicates that SCF-KIT interaction can regulate the development and survival of melanocytes in the context of GISTs 28 .
Although GIST associated with germline KIT mutations is very rare, it should be taken into consideration when we encounter patients with multiple lesions, cutaneous hyperpigmentation, multiple lentigines, mastocytosis and other typical manifestation. We should emphasize the importance of inquiring about a detailed family history in individuals with abnormal pigmentation, especially any history of GI lesions.
What's more, the possibility of genetic testing and counseling to distinguish individuals with high risk from those with little/no risk is essential. Detecting for the KIT mutation should prior to the start of imatinib therapy, for it can help predicting tumor response. Imatinib as a therapeutic inhibition in these rare disease remains to be determined, mainly because data on therapeutic effects of imatinib in familial GISTs is limited. In addition, careful monitoring for the development of GIST (during and after treatment) is reasonable. The speci c secondary mutations can direct the clinician to select other appropriate second-line treatment of patients with imatinib resistance.

Consent for publication
Written informed consent was obtained from all patients.

Availability of data and materials
All data generated or analyzed during this study are included in this published article

Competing interests
The authors declare that they have no competing interests. Funding