A Novel Kindred with Familial Gastrointestinal Stromal Tumors Caused by a Rare KIT Germline Mutation (N655K): Clinico-Pathological Presentation and TKI Sensitivity

Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the gastrointestinal tract, are characterized by activating mutations in KIT or PDGFRA genes. The vast majority of GISTs are sporadic, but rare hereditary forms have been reported, often featuring multifocality and younger age of onset. We here report the identification of a novel kindred affected by familial GIST caused by a KIT germline mutation in exon 13 (N655K). No family affected by hereditary GIST due to this KIT variant has been reported in literature so far. We were able to track the mutation in three members of the family (proband, mother, and second-degree cousin), all affected by multiple GISTs. Due to its rarity, the N655K variant is poorly characterized. We conducted in vitro drug sensitivity assays that indicated that most tyrosine kinase inhibitors (TKIs) currently included in the therapeutic armamentarium for GISTs have a limited inhibitory activity toward this mutation. However, when compared to a classical imatinib-resistant KIT mutation (T670I), N655K was slightly more sensitive to imatinib, and encouraging responses were observed with last-generation TKIs.


Introduction
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and are thought to arise from the interstitial cells of Cajal (ICCs) [1,2]. The majority of GISTs (about 75%) harbor activating mutations in the KIT gene, most commonly in exon 11 and 9. Mutations in PDGFRA are detected in about 10-15% of GISTs. The fraction of GISTs devoid of KIT or PDGFRA mutations (~15%) is driven by rarer genetic alterations [1][2][3][4].

Molecular Analysis
DNA was extracted from FFPE tumor specimens by QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany). DNA from peripheral blood mononuclear cells was extracted with an EZ1 biorobot (QIAGEN).

Engineering of Ba/F3 Cells for the Expression of KIT Mutants
The murine interleukin-3 (IL-3) dependent Ba/F3 cell line is a cell model widely used in kinase studies. In fact, when ectopically expressed, certain tyrosine kinases can relieve Ba/F3 cells from IL-3 dependency, while tyrosine kinase inhibitors antagonize this effect [7,8].
Human KIT wild-type cDNA was cloned into the pLVX IRES ZsGreen viral vector (Clontech, Mountain View, CA, USA) and KIT exon 11 (W557_K558del), exon 13 (N655K), and exon 14 (T670I) mutant alleles were generated by PCR. Correct mutation introduction was checked by sequencing. To generate KIT lentiviral particles, human embryonic kidney HEK 293T cells (ATCC) were transfected with vectors encoding KIT mutants. HEK 293T cells were cultured in DMEM medium supplemented with 10% FBS and gentamicin (8 µg/mL). Ba/F3 cells were infected with HEK 293T-derived lentiviral supernatants in the presence of polybrene (8 µg/mL). Ectopically KIT expressing cells were selected by IL-3 withdrawal. Lentiviral infection efficiency was monitored in situ by green fluorescence. Expression of the ectopic KIT alleles was checked by Western blot. Briefly, cells were lysed in RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA). Protein lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Protran Whatman, Merck, Darmstadt, Germany). Expression analyses were performed with the following antibodies: anti c-KIT (H300; Santa Cruz Biotechnology) and anti-GAPDH (MAB374, Chemicon International, Temecula, CA, USA), for total protein loading normalization. A ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA) was used for visualization. Ba/F3 cells expressing comparable levels of ectopic KIT mutants were used in TKI cell viability assays.

TKI Cell Viability Assay
Ba/F3 cells engineered to express the test mutation (KIT exon 13 N655K), an imatinib-sensitive mutation (KIT exon 11 W557_K558del), or an imatinib-resistant mutation (KIT exon 14 T670I) were seeded on a 48-well plate at a density of 7500 cells/mL. The following day, cells were exposed to the indicated doses of TKIs for 72 h, as previously described [7]. TKIs (Selleckchem, Munich, Germany) were dissolved in DMSO (dimethyl sulfoxide) as a vehicle. Drug dosage ranges were selected based on literature data and were as follows: imatinib (0-1000 nM), sunitinb (0-36 nM), regorafenib (0-480 nM), cabozantinib (0-24 nM), avapritinib (0-250 nM), and ripretinib (0-80 nM). Cell viability was evaluated by Trypan Blue staining and expressed as percentage of cells surviving the treatment, normalized to vehicle (DMSO)-treated cells. Four replicates per dose were evaluated. Results were confirmed on at least two independent Ba/F3 cell infections.

Ethical Approval
All procedures followed were in accordance with the Helsinki Declaration and with institutional and national ethical standards. The study was approved by the CRO Aviano institutional review board (IRB-04-2017). Written informed consent to be included in the study was obtained from patients.

Results
In March 2019, a 52-year-old woman was referred for genetic counseling after a diagnosis of multifocal GIST. She displayed diffuse freckles on her arms, legs, and trunk, and an atypical junctional nevus was surgically removed in February 2019. She had a history of estrogen-receptor-positive intraductal breast carcinoma diagnosed at the age of 43. She was also under surveillance for a thyroid nodule.
GIST was an incidental finding during bilateral salpingo-oophorectomy for uterine myomas and ovarian cyst (November 2018). Multiple nodules, the largest of 6 cm, were detected at the small bowel. At histopathological examination, these nodules showed spindle cells arranged in fascicles, and interlacing bundles with eosinophilic cytoplasm and elongated nuclei. Immunohistochemistry showed positivity for KIT/CD117 and DOG1, weak/focal reactivity for SMA and CD34, negativity for desmin and S100 ( Figure 1A). Mitotic index was low (1 per 50 high-power fields). Diagnosis was of GIST of intermediate risk of progression according to Miettinen [9]. The diagnostic workup included a 18FDG-PET (fluorine-18-fluorodeoxyglucose positron emission tomography) that showed two lesions at the small bowel and one at the gastric fundus. Upper gastrointestinal endoscopy and ultrasound endoscopy showed a 2 cm submucosal nodule at the gastric fundus ( Figure 1A). A second surgery was performed in January 2019 for resection of the gastric tumor and multiple small bowel nodules (size range 0.5-2 cm). Pathological examination corroborated the diagnosis of multinodular GIST. Molecular analysis, performed using a comprehensive GIST-specific NGS panel, revealed in both 2018 and 2019 tumor specimens a heterozygous T-to-G transversion at codon 655 of KIT exon 13 (c.1965T > G) resulting in an N655K amino acid change ( Figure 1B). Besides the N655K KIT mutation, no other pathogenic mutation in GIST driver genes (PDGFRA, BRAF, SDHA, SDHB, SDHC, SDHD, HRAS, KRAS, NRAS, SPRED1, NF1, NF2, and TP53) was found.
surgery was performed in January 2019 for resection of the gastric tumor and multiple small bowel nodules (size range 0.5-2 cm). Pathological examination corroborated the diagnosis of multinodular GIST. Molecular analysis, performed using a comprehensive GIST-specific NGS panel, revealed in both 2018 and 2019 tumor specimens a heterozygous T-to-G transversion at codon 655 of KIT exon 13 (c.1965T > G) resulting in an N655K amino acid change ( Figure 1B). Besides the N655K KIT mutation, no other pathogenic mutation in GIST driver genes (PDGFRA, BRAF, SDHA, SDHB, SDHC, SDHD, HRAS, KRAS, NRAS, SPRED1, NF1, NF2, and TP53) was found.
Regarding family history ( Figure 1C), the index case has a healthy sister (47 years old) with freckles on the trunk. Their mother died in 2012 at the age of 74 of metastatic GIST. She presented with multifocal intestinal GIST (positive for KIT/CD117, DOG1, and CD34; negative for S100, SMA, and desmin) and hepatic metastasis at diagnosis. Sequencing of tumor DNA revealed the same KIT N655K mutation detected in the proband ( Figure 1D). Regarding family history ( Figure 1C), the index case has a healthy sister (47 years old) with freckles on the trunk. Their mother died in 2012 at the age of 74 of metastatic GIST. She presented with multifocal intestinal GIST (positive for KIT/CD117, DOG1, and CD34; negative for S100, SMA, and desmin) and hepatic metastasis at diagnosis. Sequencing of tumor DNA revealed the same KIT N655K mutation detected in the proband ( Figure 1D).
In 2000, the maternal second-degree cousin of the index case (II-1 at pedigree) was diagnosed at the age of 59 with multiple gastric and small-bowel mesenchymal malignant neoplasms compatible with GIST. The patient received two lines of combination chemotherapy for sarcomas (epidoxorubicine plus ifosfamide first, followed by CYVADIC, a combination of cyclophosphamide, vincristine, doxorubicin, and dacarbazine). Five years later (January 2006), he was referred to our center where PET/CT scan showed multifocal abdominal and hepatic increased uptake. Histological revision of the primary tumor supported the diagnosis of KIT/CD117-positive, DOG1-positive, GIST (immunohistochemistry was negative for CD34, S100, SMA, and desmin). Numerous microscopic GIST-like lesions (microGISTs) were also identified alongside the intestinal wall. Molecular analysis revealed a KIT exon 13 N655K mutation ( Figure 1E). He started imatinib (400 mg daily). The disease progressed slowly until January 2007 when he underwent surgery for multiple abdominal masses. The patient was shifted to sunitinib for 15 months and, on progression, a rechallenge with imatinib at 800 mg allowed a progression-free interval of 9 months. A last line of treatment with nilotinib did not provide benefit and was stopped after 2 months.
The index case's uncle (mother's brother) and the maternal grandmother died of abdominal malignancy of unknown site and pathology (NOS). Colon cancer appeared as a recurrent event in the proband's paternal branch.
In light of multifocal presentation, family history, and the recurrence of the same rare KIT exon 13 mutation in the neoplasms of the family, genetic testing was proposed to the proband. Analysis of peripheral blood DNA highlighted the presence of the same KIT N655K mutation detected in the tumors thus supporting its germline origin ( Figure 1B).
Due to the intermediate risk of recurrence and the fact that KIT exon 13-mutated sporadic GISTs are in general scarcely responsive to imatinib, after curative surgery, the index case was put on close surveillance with alternating CT scan and 18FDG-PET every six months. A papillary thyroid carcinoma, follicular variant, was diagnosed in January 2020.
Genetic counseling was considered for the proband's first-degree relatives. The sister refused genetic testing. Thus, annual abdominal ultrasound and upper-gastrointestinal endoscopy were suggested. The proband's children were under age 18 and were therefore considered not eligible for genetic testing.
In sporadic GISTs, the KIT exon 13 N655K detected in this family is very uncommon. Thus, it is poorly characterized [7,8]. To address its sensitivity to TKIs approved or under trial for GIST treatment, we performed in vitro cytotoxicity assays. Specifically, the response to imatinib, sunitinib, regorafenib, avapritinib, cabozantinib, and ripretinib of Ba/F3 cells engineered to express the N655K mutation was compared with that of cells expressing a prototypical imatinib-sensitive (KIT exon 11 W557_K558del) and an imatinib-resistant (KIT exon 14 T670I) mutation ( Figure 1F). Compared to the canonical KIT exon 11 sensitizing mutation (W557_K558del), the N655K mutation displayed a lower sensitivity to all TKIs tested. Compared to the reference resistant mutation (KIT exon 14 T670I), N655K appeared less responsive to sunitinib, regorafenib, and cabozantinib; it was instead slightly more sensitive to imatinib, especially at higher dosages, in keeping with previous results [7]. In addition, last-generation TKIs such as avapritinib and ripretinib [10] demonstrated a certain degree of activity toward this mutation ( Figure 1F).

Discussion
Very few cases of KIT/PDGFRA-associated familial GIST syndromes have been reported so far. A PubMed search retrieved 51 reports, 45 describing hereditary GISTs due to germline KIT mutations, most of which involving exon 11, and 6 reports of familial GIST associated with germline PDGFRA mutations (Table 1) . Here, we report a new kindred with familial GISTs caused by a KIT exon 13 germline mutation (N655K). In sporadic GISTs, KIT exon 13 mutations, which affect the ATP-binding pocket of the kinase, are uncommon and usually arise as secondary/imatinib-resistance events [1,2]. Among KIT exon 13 mutations, N655K is extremely rare, accounting for less than 0.1% of all GIST-associated KIT variants recorded in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. With the only exception of a single, undetailed entry in the ClinVar database, to the best of our knowledge, there are no literature reports implicating this mutation in hereditary GISTs. Owing to its rarity, the N655K variant is poorly characterized and scanty literature data exist about its sensitivity to TKIs [7,8].
Our in vitro assay results indicate that N655K conveys a limited sensitivity to most TKIs approved for the treatment of GISTs. However, compared to a canonical resistance mutation (T670I), N655K was slightly more sensitive to imatinib, especially at higher dosages, a finding that is somehow in line with the response observed in the proband's second-degree cousin. Always with reference to the T670I resistance mutation, N655K appeared more sensitive also to avapritinib and ripretinib. These latter are new-generation TKIs that have been proven to be effective in controlling a wide range of KIT and PDGFRA mutations, including classical resistant mutations [10].
The limited number of kindreds reported so far prevents genotype-phenotype correlations and the presentation pattern of these tumor forms and the management of patients carrying GIST-predisposing gene mutations still need to be defined [2,5].
As far as the treatment is concerned, there is currently no evidence supporting a differential therapeutic approach for GISTs developing in a background of germline KIT/PDGFRA mutations compared to sporadic GISTs, although tumor multiplicity and, hence, surgery-requiring complications (e.g., intestinal hemorrhages and occlusions) are more likely to occur in the former.
Definitively, when a familial GIST syndrome is suspected, based on early age of onset, tumor multifocality, and family history, genotyping is highly recommended and, if positive, counseling and predictive genetic testing should be offered to all first-degree relatives. Moreover, it is worth bearing in mind that individuals affected by hereditary GISTs seem to have an increased risk for other tumor types [2,5]. Indeed, our index case also developed breast and papillary thyroid cancers.
In our opinion, GIST-predisposing-mutation carriers should undergo close surveillance for early detection of cancer. This is even more important when the genetic setting makes the tumor poorly amenable to pharmacological inhibition, as in the family presented here, and the earliest diagnosis of neoplastic growth would increase the chance of curative surgery. Unfortunately, no specific guidelines exist on how to manage these subjects. In particular, consensus recommendations for clinical surveillance are lacking and whether regular esophagogastroduodenoscopy (EGD) or 18FDG-PET examinations are useful options remain open questions.
In this context, single case reports such as the one described here are fundamental to build up a body of information that may lay down the ground for the development of evidence-based guidelines.

Conclusions
In this paper we report the identification and characterization of a new kindred affected by multiple GISTs due to a rare KIT germline N655K mutation. Very few cases of syndromic KIT/PDGFRA-associated familial GISTs have been described so far. Case studies such as ours may help in defining the presentation pattern of these tumor forms and contribute to the formulation of clinical practice guidelines. In addition, in vitro evaluation of drug sensitivity may provide a basis for treatment personalization.

Conflicts of Interest:
The authors declare no conflict of interest.