Introduction.
NENs are a heterogeneous group of human neoplasms with a variable clinical behavior, spanning from indolence to frank aggressiveness and malignancy. They arise from various tissues with common embryological origins that share the capacity for producing hormones. Their growth might compromise surrounding structures and they often cause significant morbidity due to their associated syndromes of hormone oversecretion. Although traditionally considered rare, the frequency of NENs has greatly increased in the last decades, due to improved clinical awareness and diagnostic strategies. This group includes lesions such as bronchopulmonary, gastrointestinal, pancreatic, and thymic NENs, medullary thyroid carcinoma (MTC), paragangliomas and pheochromocytomas (PPGLs), parathyroid adenomas and carcinomas, and pituitary neuroendocrine tumors (PitNETs), among others.
Interestingly, NENs are among the human neoplasms with a strongest heritable component. Some NENs are part of autosomal dominant syndromes of multiple endocrine neoplasia, where they present in combination with other characteristic endocrine and non-endocrine tumors in the same individual and/or family. Clinical descriptions of such entities exist in the literature since the mid-twentieth Century, although their genetic causes were not determined until three or four decades ago. In other instances, a single type of tumor, such as MTC, PitNETs, or PPGLs, arises in multiple members of the same family, without other associated features. Occasionally, individuals with apparently sporadic presentation are indeed simplex cases of familial conditions. It is currently known that around 50% of cases of MTC, 40% of PPGLs, 20% of gastrointestinal, pancreatic, bronchopulmonary, and thymic NENs, 15% of parathyroid NENs, and 5-10% of PitNETs are due to germline defects. Aside from heritable genetic defects, alterations at the somatic level determine specific clinical courses in both sporadic and familial NENs.
In the last decade, the widespread use of powerful tools for genetic analyses has resulted in a dramatic increase in the known genetic causes of NENs. These approaches have also yielded previously unsuspected genotype-phenotype associations and have uncovered a great overlap in the clinical presentation of different syndromes of multiple endocrine neoplasia. Genetic tests are key for early diagnosis, tailored clinical management, and genetic counseling. Moreover, genetic analyses on the tumors have uncovered potential biomarkers and therapeutic targets. Yet, platforms for genetic diagnosis are not widely available and data on genetic disease drivers do not accurately represent all human populations. Genetic tests are not widely available in Mexico and are almost non-existent in its public hospitals, which serve the majority of the country's ∼128 million population. Interestingly, Mestizos, which account for most of the Mexican population, are one of the most genetically diverse human groups. Precision medicine strategies require population-specific information, yet, Mexican Mestizos are poorly represented in international genetic databases.
Aims.
Using a precise, robust, and affordable testing platform, this project will describe the repertoire and frequency of germline and somatic genetic causes of NENs in a large prospective cohort of Mexican Mestizos. It will also define the phenotypes and clinical outcomes associated with specific genetic defects and explore the functional consequences of new defects in genes previously associated with these neoplasms. Finally, it will uncover and thoroughly characterize novel genetic associations, including potential druggable targets.
Methods.
Approval for the study has been obtained from the Institutional Review Boards of two reference hospitals: Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ, project 4090), and Hospital de Especialidades de Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social (IMSS, project R-2022-785-011). Under informed consent, DNA samples from blood and fresh or formalin-fixed and paraffin-embedded tumors will be obtained. All experimental methods, except exome sequencing, will be carried out at the Research Support Network of the Universidad Nacional Autónoma de México and INCMNSZ. The study started in August 2022 and is designed as a 15-year prospective cohort with an estimated total of 750 participants. Adult individuals (age ≥18 years at recruitment) with new or previous diagnosis of any type of NEN will be invited to participate, including sporadic, familial, isolated, and syndromic presentations. All participants will be offered the opportunity to obtain the results of their genetic studies. A unique deidentified code will be used to label all samples and data collected for each participant, to protect their personal information.
Details on family history, clinical presentation, biochemical, imaging, histopathological, and previous genetic studies for each participant will be entered into a deidentified electronic clinical database. Data on clinical outcomes will be collected prospectively for a maximum of 15 years. For all cases, a 5 ml peripheral blood sample will be collected in a tube with EDTA at recruitment and stored at 4°C until processing. DNA will be extracted from blood samples within 24 h after collection. Archival formalin-fixed paraffin-embedded (FFPE) tissue samples will be obtained for patients with previous tumor resection, when available. When possible and without interfering with routine histopathological analyses, fresh frozen tissue samples will be obtained from individuals undergoing surgical tumor excision in the course of the study. Frozen tissues will be stored in liquid nitrogen until processing. DNA will be extracted from all tissue samples and RNA will be extracted from blood or tissue samples in selected cases. Sample quality and concentration will be analyzed by spectrophotometry and fluorometry. DNA will be stored at -20 °C and RNA will be stored at -80 °C for the duration of the study.
DNA samples will be analyzed using multiple techniques, consisting of a first step of targeted next generation sequencing (NGS), followed by targeted array-based comparative genomic hybridization (aCGH) and exome sequencing (ES). For NGS, a customized panel including 54 genes with a known association with NENs has been designed, using the Twist Biosciences platform. Libraries from blood DNA from all participants and, when possible, fresh tumor DNA, will be prepared according to the manufacturer's instructions and sequenced in an Illumina MiSeq instrument at an approximate depth of 300x. Sequences will be mapped to the GRCh38/hg38 human genome, PCR duplicates will be marked, and variant calling will be carried out for single nucleotide variants (SNVs), indels, and copy number variants. Variants will be selected based on their frequency (\<0.1% in both general population and Mexican population databases), previous classification (according to the American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines) reported in ClinVar, results from multiple in silico prediction tools, and literature search for additional clinical and experimental data.
For selected cases remaining without genetic diagnosis after the NGS panel, CNV analysis of the same targeted regions will be carried out. For this purpose, a high density aCGH targeting the same regions of interest than the NGS panel has been designed, using the Agilent SureDesign platform. Samples will be processed in a 16 x 25 K array format and the results will be analyzed with the Agilent Genomic Workbench software. Variant filtering and classification will be done in a similar manner as described for SNVs and indels.
All relevant variants classified as pathogenic and likely pathogenic will be subjected to orthogonal validation (Sanger sequencing for SNVs and indels and droplet digital polymerase chain reaction for CNVs). All participants choosing to know their results will receive detailed reports including all relevant variants of uncertain significance (VUS), as well as likely pathogenic, and pathogenic variants with an interpretation of their clinical significance. When clinically indicated, participants will be offered cascade targeted screening of additional family members. Selected VUS, including variants not previously reported, will be subjected to additional evaluations, including search for loss of heterozygosity in tumor tissues, expression assays, coding DNA sequencing and/or breakpoint analyses in blood and/or tumor tissues, or experimental functional validation.
The genetic results will be correlated with the clinical data to determine the frequency and type of genetic defects associated to each phenotype in the study population. Conversely, thorough clinical descriptions of the repertoire of phenotypes associated with each specific genetic defect will be obtained. The influence of particular genotype-phenotype associations on the clinical presentation and clinical outcomes will also be explored. Cases with no genetic diagnosis after targeted NGS and aCGH will be subjected to exome sequencing. Groups of patients with similar phenotypes will be analyzed together to identify disease-specific defects in genes not previously associated with NENs. Novel candidate associations will be thoroughly characterized using custom-designed combinations of experimental approaches plus additional screening in affected families.
Expected results.
Incorporating multiple phenotypes of NENs in the proposed novel platform for general genotyping will significantly improve the probability of variant detection, compared with the currently available genetic tests. In addition to generating a large amount of genetic and clinical data for previously unexplored phenotypes in the study population, this project will very likely also identify novel disease modifiers and founder variants. These data will contribute to the development of population- and genotype-specific strategies for genetic counseling, early diagnosis, clinical follow up, and treatment, thereby improving the future outcomes of individuals with NENs. This strategy will uncover the landscape of germline and somatic NEN-associated defects in an extremely genetically diverse population that is poorly represented in international studies. The results will be compared with data from other populations to better understand the alterations within and outside known driver genes that shape syndromic presentations, tumor behaviors, and inheritance patterns in NENs.
