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Sirolimus treatment of a PTEN hamartoma tumor syndrome presenting with melena

Background. PTEN hamartoma tumor syndrome (PHTS) is an umbrella term including Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN-related Proteus syndrome (PS), and PTEN-related Proteus-like syndrome. One of the disorders in PHTS spectrum, CS is characterized by macrocephaly, mucocutaneous findings, gastrointestinal system (GIS) polyposis and an increased lifetime risk of GIS, breast, thyroid and other cancers. Case. In this study, we report an adolescent patient presenting with recurrent life-threatening upper GIS bleeding as a result of hamartomatous polyposis. Genetic studies revealed a known pathogenic nonsense mutation confirming the initial diagnosis of CS. Conclusions. Additionally, we describe our therapeutic intervention to improve the patient’s clinical symptoms with sirolimus, which its use is infrequently addressed in the literature for pediatric age group harboring PTEN mutations.

PTEN hamartoma tumor syndrome (PHTS) is an umbrella term including cancer predisposition and overgrowth syndromes such as Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN-related Proteus syndrome (PS) and PTEN-related Proteus-like syndrome. 1 Cowden Syndrome (OMIM 158350) is the first of the PHTS spectrum disorders to be associated with autosomal dominantly inherited germline "Phosphatase and Tensin Homologous) PTEN mutations. [2][3][4] Syndrome is characterized by macrocephaly, mucocutaneous findings such as trichilemmomas, papillomatous papules, acral keratosis and penile freckling, multiple hamartomas in various tissues including gastrointestinal system (GIS) polyposis and an increased lifetime risk for benign and malignant tumors of breast, thyroid, genitourinary and GIS. 2,[5][6][7] Polyps are predominantly hamartomatous, but mixed histologic types including hyperplastic, inflammatory and adenomatous types could also be seen. 1 Polyps may be asymptomatic or complicated by bleeding, obstruction, invagination, and infarction. 8 The prevalence of CS in the general population is thought to be 1 in 200,000, and although the cases are usually diagnosed in the third decade, there are rare cases in early childhood. 8,9 PTEN is a lipid phosphatase, a negative regulator of the phosphatidylinositol-3-kinase (PI3K) / protein kinase B (AKT)/ the mammalian target of rapamycin (mTOR) signaling pathway and plays an active role in cell cycle and apoptosis by controlling phosphoinositol triphosphate levels. 3 Loss of function of this tumor suppressor gene leads to inappropriate activation of the PI3K/AKT /mTOR pathway, uncontrolled cell growth and proliferation causing benign and malignant tumor formations. 3,10 In recent years, successful outcomes have been reported in the treatment of benign and malignant tumors by targeting the inhibition of PTEN-related PI3K/ AKT/mTOR pathway with an mTOR inhibitor, sirolimus. 11,12 Here, we report an adolescent CS patient presenting with life-threatening recurrent upper GIS bleeding. Additionally, we describe our therapeutic intervention to improve the patient's clinical symptoms with sirolimus, which its use is infrequently addressed in the literature for pediatric age group with PTEN mutations.

Case Report
A fourteen-year-old male patient with melena was referred to our hospital for the evaluation of GIS bleeding. From his medical history, it was learned that he had a stomach ache for the last two months, and malaise and darkening of the stool color for the last week.
His parents were first-degree cousins and he had no further significant finding in his family history. On his physical examination, body weight, height and head circumference were compatible with 25p, 10p, and >97 p, respectively. His skin and mucous membranes were pale. He had multiple skin-colored soft papules on his face, axillary region, upper extremity and back (Fig. 1a). Additionally, a hyper-pigmented macule with irregular edges in the glans penis was observed (Fig. 1b). Other system findings were normal. His laboratory examinations revealed iron deficiency anemia; Hgb 7.6 g/dL (9.5-13.3 g/dL), serum iron 24 µg/ dL (45-182 µg/dL), unsaturated iron binding capacity 358 µg/dL (155-300 µg/dL), ferritin 9 ng/mL (23-70 ng/ mL), white blood cell count 11.7 x10 3 /µL, platelet count 339 x10 3 /µL. In his upper GIS endoscopic examination, locally eroded, ulcerated and pedunculated or non-pedunculated polyps with diameters ranging from 0.3-2 cm, localized mostly in the antrumcorpus junction and antrum were observed (Fig.  1c). Colonoscopy showed a decreasing number of polyps from rectum to sigmoid and no polyps were observed in proximal segments of colon. Histopathologic examination demonstrated that the polyps located in the stomach were compatible with mix histology consisting of both hyperplastic and hamartomatous types, and those in the colon were hyperplastic polyps (Fig. 1d).
Genetic studies were performed after obtaining written informed consent from the patient's parent. Genomic DNA of peripheral blood leukocytes was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Libraries were prepared using a capture- Ultrasonography of the thyroid, abdomen and scrotum were normal. Cranial magnetic resonance imaging for the presence of AVmalformations was also normal. Upper GIS bleeding was treated with proton pump inhibitors (PPIs) and octreotide infusion therapy. Occult blood positivity in the stool continued, although his major symptoms regressed. Sirolimus treatment was planned in the patient whose macroscopic bleeding recurred twice during the follow-up, as conventional treatments were insufficient. Sirolimus treatment was initiated at a 0.8 mg/ m² dosage and was administered per oral twice daily. During the treatment, complete blood cell count, hepatic and renal function tests, lipid profile and serum levels of sirolimus were monitored regularly. Serum sirolimus levels were between 6-9 ng/ml during the follow-up. The upper and lower endoscopic examinations were repeated in the third month of the treatment. There was a significant decrease in the number and size of the polyps (Fig. 1e). He continued on a therapeutic dose of sirolimus for a total of 6 months without any major adverse effects. He had only transient mild hypercholesterolemia. Sirolimus was tapered down in two months. The patient was followed for two years and GIS bleeding did not recur.

Discussion
We report an adolescent patient with PHTS whose clinical, laboratory and endoscopic findings were successfully treated with sirolimus. His clinical features (macrocephaly, wart-like papules on his skin and hyperpigmented penile macules) and hamartomatous GIS polyposis were compatible with the clinical diagnostic criteria for PTEN Hamartoma Tumor Syndrome based on the National Comprehensive Cancer Network (NCCN) Guidelines. 13 Clinical diagnosis is also confirmed with the identification of c.388>T p. (Arg130Ter) nonsense variant by molecular studies. PTEN protein consists of two important domains that are required for the tumor suppressor function. First domain is the phosphatase (catalytic) domain that is located between the amino acids 14 to 185, and the second is the C2 lipid membrane-binding domain, which participates in membrane binding, placed in between the amino acids 190 and 350. 14 Exon 5 is a hotspot for germline mutations as it harbors the tyrosine phosphatase signature motif H123CKAGKGR130 and within this loop, the C124 and R130 residues are essential for the physiological function of PTEN in tumor suppression. 15 Identified variant in the present patient is located in signature motif of the catalytic domain at the 130 th position. This variant has been observed in several individuals affected with PHTS. 16 The truncating variant is expected to undergo nonsense-mediated decay, supporting haploinsufficiency as the cause of the clinical features of the present patient.
Main GIS findings of CS are non-neoplastic polyps. Although GIS polyps are predominantly of hamartomatous histopathology, hyperplastic or adenomatous polyps and mixed polyposis with malignancy risk are also reported. 17 Multiple gastric, ileal and colorectal polyps were signs of major GIS involvement in our patient. Although the polyps detected in the patient were of hyperplastic and hamartomatous histopathology, long-term follow-up was continued due to increased risk for the development of malignancies, especially in the colon and thyroid. Thus, detection of PTEN mutation changed the management of the patient not only in terms of follow-up but also in terms of treatment options.
Although polyposis is rare in the pediatric age group, it is diagnosed more frequently with the more widespread use of endoscopic diagnostic examinations. 18 Invasive procedures are often required for the treatment of polyps complicated by severe life-threatening bleeding and anemia. Although our patient's polyps were benign in nature, they caused severe and repetitive GIS bleeding, and profound anemia requiring aggressive transfusion. Polypectomy was not considered as there were too many polyps to be resected. PTEN alterations result in an enhanced PI3K/ AKT/mTOR signaling, causing an uncontrolled cell proliferation and suppression of this pathway represents a rational therapeutic target in PHTS. 10 In recent years significant success has been achieved in the treatment of PHTS patients with sirolimus, an mTOR inhibitor. Beneficial results using sirolimus were first demonstrated in 2008 in both experimental animal models and a 26-month-old male patient with PTEN-related Proteus syndrome (PS). 12,19 In this patient with PS, oral sirolimus treatment at a dose of 0,1 mg/ kg/d for 2 months was effective in reducing the size of hamartomatous masses with prominent clinical improvement. 12 Three years later, another report involving a treatment attempt with sirolimus, demonstrated that a 6-yearold male patient with BRRS regained pain free full mobility as a result of a reduction in the size of vascular masses, with only minor side effects. 20 Clinical improvements reported in abovementioned studies provide a rationale for sirolimus therapy in patients with disorders in the PHTS spectrum. As a result of this rationale, trials have begun to demonstrate the efficacy of sirolimus for patients harboring PTEN mutations with complex vascular anomalies. The majority of studies reported full or partial response to sirolimus such as an amelioration in pain scale scores, an improvement in the appearance and the cutaneous discoloration of the vascular lesions and an increase in patients' performance and quality of life. [21][22][23][24][25] In addition to vascular anomalies, there is a growing number of cases in the literature reporting successful outcomes with sirolimus in various clinical presentations associated with PTEN mutations including oral hamartomatous lesions, thymus hyperplasia, abdominal lipomatosis, isolated infiltrative soft tissue lesions, ganglioneuromatosis, Lhermitte Duclos disease and hypoinsulinemic hypoglycemia. [26][27][28][29][30][31] Moreover, mTOR inhibitors were shown to inhibit polyp formation and prolong the process of the development of dysplasia in mouse models. 32,33 Regarding the efficacy of sirolimus on GIS findings in patients, successful results in decreasing the number and size of polyps have been demonstrated not only for PTEN point mutations but also for intragenic PTEN deletions and larger deletions encompassing both PTEN and BMRP1A. 26,[34][35][36][37] In another recent interventional study by Komiya et al. 11 , a 56-day course of sirolimus treatment was well tolerated in patients with PHTS and was associated with some evidence of improvement in symptoms, skin and GI lesions, cerebellar function, and decreased mTOR signaling. Thus, mTOR inhibitor treatment was initiated in the present study, as a potent role of sirolimus was shown in several studies in patients with PHTS. Table  I summarizes the comprehensive details of the main studies available in the literature to date.
During the treatment, hemoglobin and hematocrit values increased to reference values and significant decrease of the size and number of polyps was observed after a period of 6 months. Red blood cell transfusion was not needed during the 2-year follow-up.  38 No undesirable effects were observed in our patient, except for mildly elevated cholesterol levels, at the 24th week of the treatment.
There is no specific recommendation for the duration of sirolimus treatment in the literature as far as we know. Treatment period differs among previous studies ranging from 56 days to 40 months. 11,39 Due to the rarity of similar cases, information regarding all aspects of sirolimus treatment is limited. Improved post-treatment clinical,

Ethical approval
Genetic studies were performed after obtaining written informed consent from the patient's parent.