Germline Heterozygous Variants in SEC23B Are Associated with Cowden Syndrome and Enriched in Apparently Sporadic Thyroid Cancer.

Cancer-predisposing genes associated with inherited cancer syndromes help explain mechanisms of sporadic carcinogenesis and often inform normal development. Cowden syndrome (CS) is an autosomal-dominant disorder characterized by high lifetime risks of epithelial cancers, such that ∼50% of affected individuals are wild-type for known cancer-predisposing genes. Using whole-exome and Sanger sequencing of a multi-generation CS family affected by thyroid and other cancers, we identified a pathogenic missense heterozygous SEC23B variant (c.1781T>G [p.Val594Gly]) that segregates with the phenotype. We also found germline heterozygous SEC23B variants in 3/96 (3%) unrelated mutation-negative CS probands with thyroid cancer and in The Cancer Genome Atlas (TCGA), representing apparently sporadic cancers. We note that the TCGA thyroid cancer dataset is enriched with unique germline deleterious SEC23B variants associated with a significantly younger age of onset. SEC23B encodes Sec23 homolog B (S. cerevisiae), a component of coat protein complex II (COPII), which transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Interestingly, germline homozygous or compound-heterozygous SEC23B mutations cause an unrelated disorder, congenital dyserythropoietic anemia type II, and SEC23B-deficient mice suffer from secretory organ degeneration due to ER-stress-associated apoptosis. By characterizing the p.Val594Gly variant in a normal thyroid cell line, we show that it is a functional alteration that results in ER-stress-mediated cell-colony formation and survival, growth, and invasion, which reflect aspects of a cancer phenotype. Our findings suggest a different role for SEC23B, whereby germline heterozygous variants associate with cancer predisposition potentially mediated by ER stress "addiction."

A Hyalinized Trichilemmoma of the Eyelid in a Teenager.

A 16-year-old African American male, the youngest patient to date, presented with a well-circumscribed upper eyelid lesion. On excision, the dermal nodule was contiguous with the epidermis, displayed trichohyalin-like bodies in an expanded outer root sheath, and was composed chiefly of small cellular clusters separated by a prominent network of periodic acid Schiff -positive hyaline bands of basement membrane material. The tumor cells were positive for high molecular weight cytokeratins (CK) 5/6, CK14, and CK34ßE12 and were negative for CK7, carcinoembryonic antigen and epithelial membrane antigen. Negative S100, glial fibrillary acidic protein, and smooth muscle actin immunoreactions ruled out a myoepithelial lesion. The Ki-67 proliferation index was <10%. The diagnosis was a hyalinized trichilemmoma, contrasting with the more common lobular type. As an isolated lesion, trichilemmoma does not portend Cowden syndrome.

A clinical scoring system for selection of patients for PTEN mutation testing is proposed on the basis of a prospective study of 3042 probands.

Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome are allelic, defined by germline PTEN mutations, and collectively referred to as PTEN hamartoma tumor syndrome. To date, there are no existing criteria based on large prospective patient cohorts to select patients for PTEN mutation testing. To address these issues, we conducted a multicenter prospective study in which 3042 probands satisfying relaxed CS clinical criteria were accrued. PTEN mutation scanning, including promoter and large deletion analysis, was performed for all subjects. Pathogenic mutations were identified in 290 individuals (9.5%). To evaluate clinical phenotype and PTEN genotype against protein expression, we performed immunoblotting (PTEN, P-AKT1, P-MAPK1/2) for a patient subset (n = 423). In order to obtain an individualized estimation of pretest probability of germline PTEN mutation, we developed an optimized clinical practice model to identify adult and pediatric patients. For adults, a semiquantitative score-the Cleveland Clinic (CC) score-resulted in a well-calibrated estimation of pretest probability of PTEN status. Overall, decreased PTEN protein expression correlated with PTEN mutation status; decreasing PTEN protein expression correlated with increasing CC score (p < 0.001), but not with the National Comprehensive Cancer Network (NCCN) criteria (p = 0.11). For pediatric patients, we identified highly sensitive criteria to guide PTEN mutation testing, with phenotypic features distinct from the adult setting. Our model improved sensitivity and positive predictive value for germline PTEN mutation relative to the NCCN 2010 criteria in both cohorts. We present the first evidence-based clinical practice model to select patients for genetics referral and PTEN mutation testing, further supported biologically by protein correlation.

mTOR signaling in neural stem cells: from basic biology to disease.

The mammalian target of rapamycin (mTOR) pathway is a central controller of growth and homeostasis, and, as such, is implicated in disease states where growth is deregulated, namely cancer, metabolic diseases, and hamartoma syndromes like tuberous sclerosis complex (TSC). Accordingly, mTOR is also a pivotal regulator of the homeostasis of several distinct stem cell pools in which it finely tunes the balance between stem cell self-renewal and differentiation. mTOR hyperactivation in neural stem cells (NSCs) has been etiologically linked to the development of TSC-associated neurological lesions, such as brain hamartomas and benign tumors. Animal models generated by deletion of mTOR upstream regulators in different types of NSCs reproduce faithfully some of the TSC neurological alterations. Thus, mTOR dysregulation in NSCs seems to be responsible for the derangement of their homeostasis, thus leading to TSC development. Here we review recent advances in the molecular dissection of the mTOR cascade, its involvement in the maintenance of stem cell compartments, and in particular the implications of mTOR hyperactivation in NSCs in vivo and in vitro.

Dysplastic cerebellar gangliocytoma lhermitte-duclos disease imaging and magnetic resonance spectroscopy.

Lhermitte-Duclos disease (LDD) is a rare, benign, slow-growing, unilateral mass of the cerebellar cortex. Our case is that of a 71-year-old male with a superior cerebellar lesion consistent with LDD on imaging and Magnetic Resonance Spectroscopy (MRS). It has been reported that MRS can be a valuable diagnostic addition, as it allows for a non-invasive diagnosis and analysis to distinguish a benign lesion, such as an intraparenchymal lesion, and in our case, from a true neoplastic lesion.

Trichilemmomas show loss of PTEN in Cowden syndrome but only rarely in sporadic tumors.

BACKGROUND: Trichilemmoma (TL) can occur as a solitary sporadic lesion usually on the face or as multiple facial lesions almost invariably associated with Cowden syndrome (CS). CS is a multisystem disorder caused by a germline inactivating mutation in PTEN (10q23.31), a tumor suppressor gene. We sought to identify PTEN loss by immunohistochemistry (IHC) in sporadic and CS-associated TL to determine whether IHC is a useful tool to assess an individual for CS. METHODS: Six TL biopsies associated with CS and 33 biopsies without CS were retrieved. IHC for PTEN was performed. RESULTS were scored as positive (reactivity in TL cells) or negative (no reactivity in TL cells); normal squamous epithelium and vascular endothelium served as internal positive controls. RESULTS: Complete PTEN loss was noted in 5/6 (83%) CS-associated TL and 1/33 (3%) sporadic (non-CS) TL. CONCLUSION: Demonstration of complete PTEN loss in TL by IHC is strongly suggestive of association with CS, but retention of PTEN staining does not entirely exclude CS. Therefore, PTEN IHC in TLs may be helpful in screening TL for association with CS, but should be used in context with other established clinical criteria, and possibly germline PTEN genotyping to confirm a diagnosis of CS.

Cowden syndrome: report of two cases with immunohistochemical analysis for PTEN expression.

Cowden syndrome (CS) is a rare autosomal dominant disorder characterized by multisystem hamartomatous growths and carcinomas. CS is linked to germ line mutations in PTEN (phosphatase and tensin homolog) located on chromosome 10q23.3. PTEN acts as a tumor suppressor to negatively control cellular growth and survival via the PI3K/AKT signaling pathway. Presented here are 2 patients with multiple, persistent, and asymptomatic papules on the face and the upper body, histologically consistent with trichilemmomas. Diagnosis of CS was made in each case based on the established diagnostic criteria and confirmed using immunohistochemistry directed against PTEN. We propose that the assessment of PTEN expression levels can aid in the identification of patients with CS.

Germline and somatic cancer-associated mutations in the ATP-binding motifs of PTEN influence its subcellular localization and tumor suppressive function.

Germline and somatic PTEN mutations are found in Cowden syndrome (CS) and multiple sporadic malignancies, respectively. PTEN function appears to be modulated by subcellular compartmentalization, and mislocalization may affect function. We have shown that cellular ATP levels affect nuclear PTEN levels. Here, we examined the ATP-binding capabilities of PTEN and functional consequences, relevant to cancer-associated mutations. PTEN mutation analysis of CS patients and sporadic colorectal carcinomas and comparative aminoacid analysis were utilized to identify mutations in ATP-binding motifs. The ability of wild-type (WT) or mutant PTEN to bind ATP was assessed by ATP-agarose-binding assays. Subcellular fractionation, western blotting, confocal microscopy and growth assays were used to determine relative nuclear-cytoplasmic localization and function. Somatic colorectal carcinoma-derived PTEN missense mutations were associated with nuclear mislocalization. These mutations altered cellular proliferation, apoptosis and anchorage-dependent growth. Examination of PTEN's amino acid sequence revealed these mutations resided in previously undescribed ATP-binding motifs (c.60-73; c.122-136). In contrast to WT PTEN, both cancer-associated somatic and germline-derived PTEN missense mutations, which lie within the ATP-binding motifs, result in mutant PTEN that does not bind ATP efficiently. We also show that CS patients with germline ATP-binding motif-mutations had nuclear PTEN mislocalization. Of four unrelated patients with functional germline ATP-binding domain mutations, all three female patients had breast cancers. Germline and somatic mutations within PTEN's ATP-binding domain play important pathogenic roles in both heritable and sporadic carcinogenesis by PTEN nuclear mislocalization resulting in altered signaling and growth. Manipulation of ATP may represent novel therapies in tumors with such PTEN alterations.

Enhanced lymphocyte interferon (IFN)-γ responses in a PTEN mutation-negative Cowden disease kindred.

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-γ can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-γ secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-γ responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-γ secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-γ secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-γ that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.

Familial thyroid cancer: a review.

Thyroid carcinomas can be sporadic or familial. Familial syndromes are classified into familial medullary thyroid carcinoma (FMTC), derived from calcitonin-producing C cells, and familial non-medullary thyroid carcinoma, derived from follicular cells. The familial form of medullary thyroid carcinoma (MTC) is usually a component of multiple endocrine neoplasia (MEN) IIA or IIB, or presents as pure FMTC syndrome. The histopathological features of tumors in patients with MEN syndromes are similar to those of sporadic tumors, with the exception of bilaterality and multiplicity of tumors. The genetic events in the familial C-cell-derived tumors are well known, and genotype-phenotype correlations well established. In contrast, the case for a familial predisposition of non-medullary thyroid carcinoma is only now beginning to emerge. Although, the majority of papillary and follicular thyroid carcinomas are sporadic, the familial forms are rare and can be divided into two groups. The first includes familial syndromes characterized by a predominance of non-thyroidal tumors, such as familial adenomatous polyposis and PTEN-hamartoma tumor syndrome, within others. The second group includes familial syndromes characterized by predominance of papillary thyroid carcinoma (PTC), such as pure familial PTC (fPTC), fPTC associated with papillary renal cell carcinoma, and fPTC with multinodular goiter. Some characteristic morphologic findings should alert the pathologist of a possible familial cancer syndrome, which may lead to further molecular genetics evaluation.

Characterization of a novel PTEN mutation in MDA-MB-453 breast carcinoma cell line.

BACKGROUND: Cowden Syndrome (CS) patients with germ line point mutations in the PTEN gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant PTEN alleles are predisposed to malignant conversion. This article will characterize the biochemical and biological properties of a mutant PTEN protein found in a commonly used metastatic breast cancer cell line. METHODS: The expression of PTEN in human breast carcinoma cell lines was evaluated by Western blotting analysis. Cell line MDA-MB-453 was selected for further analysis. Mutation analysis of the PTEN gene was carried out using DNA isolated from MDA-MB-453. Site-directed mutagenesis was used to generate a PTEN E307K mutant cDNA and ectopic expressed in PC3, U87MG, MCF7 and Pten(-/-) mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in Sf9 baculovirus expression system. Lipid phosphatase and ubiquitination assays were carried out to characterize the biochemical properties of PTEN E307K mutant. The intracellular localization of PTEN E307K was determined by subcellular fractionation experiments. The ability of PTEN E307K to alter cell growth, migration and apoptosis was analyzed in multiple PTEN-null cell lines. RESULTS: We found a mutation in the PTEN gene at codon 307 in MDA-MB-453 cell line. The glutamate (E) to lysine (K) substitution rendered the mutant protein to migrate with a faster mobility on SDS-PAGE gels. Biochemically, the PTEN E307K mutant displayed similar lipid phosphatase and growth suppressing activities when compared to wild-type (WT) protein. However, the PTEN E307K mutant was present at higher levels in the membrane fraction and suppressed Akt activation to a greater extent than the WT protein. Additionally, the PTEN E307K mutant was polyubiquitinated to a greater extent by NEDD4-1 and displayed reduced nuclear localization. Finally, the PTEN E307K mutant failed to confer chemosensitivity to cisplatinum when re-expressed in Pten(-/-) MEFS. CONCLUSIONS: Mutation at codon 307 in PTEN C2 loop alters its subcellular distribution with greater membrane localization while being excluded from the cell nucleus. This mutation may predispose breast epithelial cells to malignant transformation. Also, tumor cells harboring this mutation may be less susceptible to the cytotoxic effects of chemotherapeutics.

Red flags for early recognition of adult patients with PTEN Hamartoma Tumour Syndrome.

Patients with PTEN Hamartoma Tumour Syndrome (PHTS) are at increased risk of developing cancer. Many adult PHTS patients are not recognized as such and do not receive the cancer surveillance they need. Our aim was to define phenotypic characteristics that can easily be assessed and manifest by early adulthood, and hence could serve as red flags (i.e. alerting signals) for early recognition of adult patients at high risk of PHTS. Phenotypic characteristics including macrocephaly, multinodular goitre (MNG), and oral features were examined in 81 paediatric and 86 adult PHTS patients by one of two medical experts during yearly surveillance visits at our Dutch PHTS expert centre between 1997 and 2020. MNG was defined as signs of thyroid nodules and/or goitre. Oral features included gingival hypertrophy, high palate (adults only) and oral papillomas. Based on the characteristics' prevalence in different age groups, combinations of phenotypic characteristics were defined and evaluated on their potential to recognize individuals with PHTS. Macrocephaly was present in 100% of paediatric and 67% of adult patients. The prevalence of MNG was ∼50% in paediatric and gradually increased to >90% in adult patients. Similar percentages were observed for any of the oral features. Scoring two out of three of these characteristics yielded a sensitivity of 100% (95%CI 94-100%) in adults. The presence of the combination macrocephaly, MNG, or multiple oral features could serve as a red flag for general practitioners, medical specialists, and dentists to consider further assessment of the diagnosis PHTS in adults. In this way, recognition of adult PHTS patients might be improved and cancer surveillance can be offered timely.

Molecular apocrine differentiation is a common feature of breast cancer in patients with germline PTEN mutations.

INTRODUCTION: Breast carcinoma is the main malignant tumor occurring in patients with Cowden disease, a cancer-prone syndrome caused by germline mutation of the tumor suppressor gene PTEN characterized by the occurrence throughout life of hyperplastic, hamartomatous and malignant growths affecting various organs. The absence of known histological features for breast cancer arising in a PTEN-mutant background prompted us to explore them for potential new markers. METHODS: We first performed a microarray study of three tumors from patients with Cowden disease in the context of a transcriptomic study of 74 familial breast cancers. A subsequent histological and immunohistochemical study including 12 additional cases of Cowden disease breast carcinomas was performed to confirm the microarray data. RESULTS: Unsupervised clustering of the 74 familial tumors followed the intrinsic gene classification of breast cancer except for a group of five tumors that included the three Cowden tumors. The gene expression profile of the Cowden tumors shows considerable overlap with that of a breast cancer subgroup known as molecular apocrine breast carcinoma, which is suspected to have increased androgenic signaling and shows frequent ERBB2 amplification in sporadic tumors. The histological and immunohistochemical study showed that several cases had apocrine histological features and expressed GGT1, which is a potential new marker for apocrine breast carcinoma. CONCLUSIONS: These data suggest that activation of the ERBB2-PI3K-AKT pathway by loss of PTEN at early stages of tumorigenesis promotes the formation of breast tumors with apocrine features.

PTEN Mouse Models of Cancer Initiation and Progression.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently mutated, deleted, and functionally inactivated tumor suppressor genes in human cancer. PTEN is found mutated both somatically and in the germline of patients with PTEN hamartoma tumor syndrome (PHTS). PTEN encodes a dual lipid and protein phosphatase that dephosphorylates the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), in turn negatively regulating the oncogenic PI3K-AKT pathway, a key proto-oncogenic player in cancer development and progression. Because of importance of PTEN in tumorigenesis, a large number of sophisticated genetically engineered mouse models (GEMMs) has been designed to elucidate the underlying mechanisms by which the "PTEN pathway" promotes tumorigenesis, while simultaneously providing a well-tailored system for the identification of novel therapies and offering platforms for new drug discoveries. This review summarizes the major cancer mouse models through which the PTEN pathway has been genetically deconstructed, and outlines the rapid development of GEMMs toward more detailed functional and tissue-specific analysis.

Three adolescents with Lhermitte-Duclos disease.

Lhermitte-Duclos disease (LDD) is a rare, benign cerebellar disorder, characterized by replacement of granular cells and Purkinje cells by an overgrowth of cerebellar ganglion cells. LDD can be associated with Cowden's syndrome (CS). We studied three adolescent LDD patients, including two with CS. MRI revealed the typical tiger-stripe pattern in all patients. CS was diagnosed on the basis of secondary mucocutaneous lesions and a PTEN/MMAC1 gene mutation. Adolescent-onset LDD appears to have the same phenotype and genotype as adult-onset LDD. Total resection of the lesion is recommended as an initial management strategy. We strongly recommend that patients with LDD undergo genetic screening to find out as early as possible whether CS has developed.

PTEN-opathies: from biological insights to evidence-based precision medicine.

The tumor suppressor phosphatase and tensin homolog (PTEN) classically counteracts the PI3K/AKT/mTOR signaling cascade. Germline pathogenic PTEN mutations cause PTEN hamartoma tumor syndrome (PHTS), featuring various benign and malignant tumors, as well as neurodevelopmental disorders such as autism spectrum disorder. Germline and somatic mosaic mutations in genes encoding components of the PI3K/AKT/mTOR pathway downstream of PTEN predispose to syndromes with partially overlapping clinical features, termed the "PTEN-opathies." Experimental models of PTEN pathway disruption uncover the molecular and cellular processes influencing clinical phenotypic manifestations. Such insights not only teach us about biological mechanisms in states of health and disease, but also enable more accurate gene-informed cancer risk assessment, medical management, and targeted therapeutics. Hence, the PTEN-opathies serve as a prototype for bedside to bench, and back to the bedside, practice of evidence-based precision medicine.

PTEN hamartoma tumor syndromes.

The PTEN hamartoma tumor syndromes (PHTS) are a collection of rare clinical syndromes characterized by germline mutations of the tumor suppressor PTEN. These syndromes are driven by cellular overgrowth, leading to benign hamartomas in virtually any organ. Cowden syndrome (CS), the prototypic PHTS syndrome, is associated with increased susceptibility to breast, thyroid, and endometrial cancer. PTEN is located on chromosome 10q22-23 and negatively regulates the prosurvival PI3K/Akt/mTOR pathway through its lipid phosphatase activity. Loss of PTEN activates this pathway and leads to increased cellular growth, migration, proliferation, and survival. Clinical management of patients with PHTS, particularly those with CS, should include early and frequent screening, surveillance, and preventive care for associated malignancies. Concomitant with improved understanding of the biology of PTEN and the PI3K/Akt/mTOR pathway, inhibitors of this pathway are being developed as anticancer agents. These medications could have applications for patients with PHTS, for whom no medical options currently exist.

Rapamycin treatment for a child with germline PTEN mutation.

BACKGROUND: A 9-month-old boy with Proteus syndrome and a de novo germline mutation in the tumor suppressor PTEN was referred to a specialist centre for management. Over the first years of life, the patient developed life-threatening respiratory dysfunction and malnutrition because of progressive growth of hamartomas affecting the chest, mediastinum, abdomen and pelvis. INVESTIGATIONS: Physical examination, CT scans of the mediastinum, pelvis and abdomen, measurement of serum insulin-like growth factor binding protein-2, and investigation of the effect of the PTEN mutation on phosphatidylinositol 3-kinase/mammalian target of rapamycin signaling in an in vitro cell model. DIAGNOSIS: PTEN hamartoma tumor syndrome, specifically Proteus syndrome. MANAGEMENT: Oral rapamycin.

Bcl-2, CD34 and CD10 expression in basaloid follicular hamartoma, vellus hair hamartoma and neurofollicular hamartoma demonstrate full follicular differentiation.

Generalized basaloid follicular hamartoma syndrome (GBFHS) is a rare, recently-described, autosomal-dominantly inherited disorder that presents with disseminated milia, palmoplantar pitting, hypotrichosis and basaloid follicular hamartomas (BFH). BFH is a benign adnexal tumor that resembles basal cell carcinoma (BCC). In this study, we report two cases of GBFHS and stain BFH, a vellus hair hamartoma (VHH) and a neurofollicular hamartoma (NH) with CD34, bcl-2 and CD10 to characterize and compare the staining patterns of these follicular tumors. Standard immunohistochemistry labeling with CD34, bcl-2 and CD10 was performed on paraffin-embedded, formalin-fixed tissue sections of five BFH (four for CD10), one VHH and one NH. CD34 stromal staining was observed in all specimens. Bcl-2 stained the outermost cell layers of the basaloid nests in all specimens. CD10 stained the peritumoral stroma of all specimens. The BFH, NFH and the VHH showed CD10 staining of matrical cells. CD34 and CD10 stain peritumoral stroma of BFH, VHH and NH. Bcl-2 stains the outermost cell layer of these tumors. CD10 was also observed to stain matrical cells. These results show the similarities in differentiation between these benign follicular neoplasms and trichoepithelioma.