The heterogeneous cancer phenotype of individuals with biallelic germline pathogenic variants in CHEK2.

PURPOSE: Females with biallelic CHEK2 germline pathogenic variants (gPVs) more often develop multiple breast cancers than individuals with monoallelic CHEK2 gPVs. This study is aimed at expanding the knowledge on the occurrence of other malignancies. METHODS: Exome sequencing of individuals who developed multiple primary malignancies identified 3 individuals with the CHEK2 (NM_007194.4) c.1100del p.(Thr367MetfsTer15) loss-of-function gPV in a biallelic state. We collected the phenotypes of an additional cohort of individuals with CHEK2 biallelic gPVs (n = 291). RESULTS: In total, 157 individuals (53.4%; 157/294 individuals) developed ≥1 (pre)malignancy. The most common (pre)malignancies next to breast cancer were colorectal- (n = 19), thyroid- (n = 19), and prostate (pre)malignancies (n = 12). Females with biallelic CHEK2 loss-of-function gPVs more frequently developed ≥2 (pre)malignancies and at an earlier age compared with females biallelic for the CHEK2 c.470T>C p.(Ile157Thr) missense variant. Furthermore, 26 males (31%; 26/84 males) with CHEK2 biallelic gPVs developed ≥1 (pre)malignancies of 15 origins. CONCLUSION: Our study suggests that CHEK2 biallelic gPVs likely increase the susceptibility to develop multiple malignancies in various tissues, both in females and males. However, it is possible that a substantial proportion of individuals with CHEK2 biallelic gPVs is missed as diagnostic testing for CHEK2 often is limited to individuals who developed breast cancer.

PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system.

Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821-1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs.

Genotype-phenotype associations in a large PTEN Hamartoma Tumor Syndrome (PHTS) patient cohort.

BACKGROUND: Pathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study. METHODS: Information was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age. RESULTS: At time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5-3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1-2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1-3.5), with trends up to 2-4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3-0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4-0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2. CONCLUSION: The PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants.

Teenage-Onset Colorectal Cancers in a Digenic Cancer Predisposition Syndrome Provide Clues for the Interaction between Mismatch Repair and Polymerase δ Proofreading Deficiency in Tumorigenesis.

Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings' CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient's CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd.

Array genotyping as diagnostic approach in medical genetics.

Genotyping arrays are by far the most widely used genetic tests but are not generally utilized for diagnostic purposes in a medical context. In the present study, we examined the diagnostic value of a standard genotyping array (Illumina Global Screening Array) for a range of indications. Applications included stand-alone testing for specific variants (32 variants in 10 genes), first-tier array variant screening for monogenic conditions (10 different autosomal recessive metabolic diseases), and diagnostic workup for specific conditions caused by variants in multiple genes (suspected familial breast and ovarian cancer, and hypercholesterolemia). Our analyses showed a high analytical sensitivity and specificity of array-based analyses for validated and non-validated variants, and identified pitfalls that require attention. Ethical-legal assessment highlighted the need for a software solution that allows for individual indication-based consent and the reliable exclusion of non-consented results. Cost/time assessment revealed excellent performance of diagnostic array analyses, depending on indication, proband data, and array design. We have implemented some analyses in our diagnostic portfolio, but array optimization is required for the implementation of other indications.

Low-level mosaicism in tuberous sclerosis complex in four unrelated patients: Comparison of clinical characteristics and diagnostic pathways.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by either TSC1 or TSC2 gene mutations. About 15% of TSC patients remain without genetic diagnosis by conventional analysis despite clinical evidence. It is important to identify somatic mosaics, as therapeutic options are now available in patients with TSC1 or TSC2 mutations. Here, we describe the clinical and genetic characteristics of four male TSC patients with low-level mosaicism. Patients presented at ages between 9 months and 32 years. Clinical manifestations varied considerably and included brain lesions in all four patients, cardiac rhabdomyomas in two young patients, skin involvement in two patients, and retinal hamartomas and renal angiomyolipomas in three patients. One patient presented with epileptic seizures and psychomotor delay. Low levels of mosaicism for TSC1 or TSC2 mutation were found in different tissue samples employing next generation sequencing and multiple ligation-dependent probe amplification. The five disease-associated variants, including one second-hit mutation, include three truncating mutations and one deletion in TSC2, and one truncating mutation in TSC1. Sanger sequencing, allele-specific oligonucleotide PCR (ASO-PCR), and droplet digital PCR were used to confirm and quantify the disclosed mutations. Genetic identification of low-level mosaicism for TSC remains challenging but is important for optimal surveillance and management.

Oncotyrol--Center for Personalized Cancer Medicine: Methods and Applications of Health Technology Assessment and Outcomes Research.

BACKGROUND: The Oncotyrol - Center for Personalized Cancer Medicine is an international and interdisciplinary alliance combining research and commercial competencies to accelerate the development, evaluation and translation of personalized healthcare strategies in cancer. The philosophy of Oncotyrol is to collaborate with relevant stakeholders and advance knowledge "from bench to bedside to population and back". Oncotyrol is funded through the COMET Excellence Program by the Austrian government via the national Austrian Research Promotion Agency (FFG). This article focuses on the role of health technology assessment (HTA) and outcomes research in personalized cancer medicine in the context of Oncotyrol. METHODS: Oncotyrol, which currently comprises approximately 20 individual projects, has four research areas: Area 1: Biomarker and Drug Target Identification; Area 2: Assay Development and Drug Screening; Area 3: Innovative Therapies; Area 4: Health Technology Assessment and Bioinformatics. Area 4 translates the results from Areas 1 to 3 to populations and society and reports them back to Area 3 to inform clinical studies and guidelines, and to Areas 1 and 2 to guide further research and development. RESULTS: In a series of international expert workshops, the Oncotyrol International Expert Task Force for Personalized Cancer Medicine developed the Methodological Framework for Early Health Technology Assessment and Decision Modeling in Cancer and practical guidelines in this field. Further projects included applications in the fields of sequential treatment of patients with chronic myeloid leukemia (CML), benefit-harm and cost-effectiveness evaluation of prostate cancer screening, effectiveness and cost-effectiveness of multiple cervical cancer screening strategies, and benefits and cost-effectiveness of genomic test-based treatment strategies in breast cancer. CONCLUSION: An interdisciplinary setting as generated in Oncotyrol provides unique opportunities such as systematically coordinating lab and bench research, product development, clinical studies and decision science/HTA and transparent joint planning of research and development with a partnership of researchers, manufacturers and health policy decision makers. However, generating a joint research and legal framework with numerous partners from different sectors can be challenging, particularly in the starting period of such an endeavor. The journey to translational personalized medicine through multidisciplinary collaborations may still be long and difficult, but it is evident that it must be continued to turn vision into reality.

Screening for MAPK modulators using an in-cell western assay.

Several essential cellular functions are critically influenced by the Ras-Raf-MEK-ERK cascade, and pathological problems have been implicated with loss as well as gain of its activity. Therefore, there is a great demand for chemical probes that act as modulators of the cascade in order to correct medical problems associated with its malfunctions. Here we describe the use of the In-Cell Western assay, a fast and easy method for the detection of ERK activity. This method is useful for the screening of chemical libraries for compounds that modulate the intensity and duration of growth factor-induced MAPK activity. Examples are provided from a screen for MAPK modulators in Vichem Chemie Research´s Nested Chemical Library™.

Ca2+/Calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation.

The long bones of vertebrate limbs originate from cartilage templates and are formed by the process of endochondral ossification. This process requires that chondrocytes undergo a progressive maturation from proliferating to postmitotic prehypertrophic to mature, hypertrophic chondrocytes. Coordinated control of proliferation and maturation regulates growth of the skeletal elements. Various signals and pathways have been implicated in orchestrating these processes, but the underlying intracellular molecular mechanisms are often not entirely known. Here we demonstrated in the chick using replication-competent retroviruses that constitutive activation of Calcium/Calmodulin-dependent kinase II (CaMKII) in the developing wing resulted in elongation of skeletal elements associated with premature differentiation of chondrocytes. The premature maturation of chondrocytes was a cell-autonomous effect of constitutive CaMKII signaling associated with down-regulation of cell-cycle regulators and up-regulation of chondrocyte maturation markers. In contrast, the elongation of the skeletal elements resulted from a non-cell autonomous up-regulation of the Indian hedgehog responsive gene encoding Parathyroid-hormone-related peptide. Reduction of endogenous CaMKII activity by overexpressing an inhibitory peptide resulted in shortening of the skeletal elements associated with a delay in chondrocyte maturation. Thus, CaMKII is an essential component of intracellular signaling pathways regulating chondrocyte maturation.