Paulina Orellana

Activating PIK3CA somatic mutation in congenital unilateral isolated muscle overgrowth of the upper extremity

Congenital unilateral overgrowth of the upper extremity affecting only the muscle tissue is rare. We describe on the clinical, histopathological, and neuroimaging findings in a 6‐year‐old girl with a congenital, non‐progressive muscle enlargement of the entire left upper limb with an ipsilateral hand deformity. No cutaneous stigmata or additional features were detected. Sanger sequencing for the AKT1, PIK3CA, and PTEN genes identified an activating c.3140A>G, p.H1047R mutation in the PIK3CA gene from the affected muscle DNA. We demonstrate that isolated congenital muscular upper limb overgrowth with aberrant hand muscles is another condition related genetically to the PIK3CA‐related overgrowth spectrum.

Síndrome de Lynch: selección de pacientes para el estudio genético mediante análisis de inestabilidad microsatelital e inmunohistoquímica

BACKGROUND Selection of patients with Lynch Syndrome (LS) for a genetic study involves the application of clinical criteria. To increase the rate of identification of mutations, the use of molecular studies as Microsatellite Instability (MSI) and Immunohistochemistry (IHC) in the tumor has been proposed. AIM To demonstrate the usefulness of MSI and IHC in the detection of mutations in patients with LS. MATERIAL AND METHODS From our Familial Colorectal Cancer Registry, families suspected of LS were selected according to Amsterdam or Bethesda clinical criteria. Screening of germline mutations of MLH1, MSH2 and MSH6 genes was performed. In addition, analysis of MSI and IHC were performed in colorectal tumors. RESULTS A total of 35 families were studied (19 met Amsterdam and 16 met Bethesda criteria). Twenty one families harbored a germline alteration in MLH1, MSH2 or MSH6 (18 Amsterdam and 3 Bethesda). In these families, eighteen different alterations were found, 15 of which were mutations and 3 corresponded to variants of uncertain pathogenicity. On the other hand, 80% of the tumors showed positive microsatellite instability (27 MSI-high and 1 MSI-low), and immunohistochemical testing showed that 77% of tumors had the loss of a protein. Correlation between results of tumor molecular studies and the finding of germline nucleotide change showed that IHC and MSI predicted mutations in 81 and 100% of patients, respectively. CONCLUSIONS MSI and IHC can efficiently select patients with a high probability of carrying a mutation in DNA repair genes.

Programa de detección de neoplasias colorrectales en población mayor de 50 años

BACKGROUND Mortality from colorectal cancer (CCR) in Chile has nearly doubled over the past 15 years. International studies have shown that CCR screening programs based on fecal occult blood test (FOBT) reduce CCR mortality. AIM To analyze the results from a CCR screening model in people over 50 years. MATERIAL AND METHODS Between 2007 and 2009, a prospective multicenter study was performed in seven major Chilean cities. FOBT using an immunological method, was measured in asymptomatic subjects aged 50 years or more, without risk factors. In patients with a positive FOBT, with symptoms or with family risk factors, a colonoscopy was indicated. RESULTS A total of 6348 subjects were assessed, FOBT was performed in 4938 of them, with a compliance of 77%. The result was positive in 9.6%. A total of 2359 colonoscopies were ordered, with an overall compliance of 50.1%. Of the 1184 colonoscopies performed, adenomas and high risk adenomas were found in 304 (26%) and 75 (6%) patients, respectively. Thirteen patients were diagnosed with stage I and IICCR. Three of these lesions were excised endoscopically and 10 surgically. The detection rate of polyps, high risk adenomas and cancer was 75, 12 and 2 per 1000 screened individuals, respectively. CONCLUSIONS This program allowed the early detection of an important number of high risk colon lesions, and all patients with CCR were diagnosed at early stages.

EGFR pathway subgroups in Chilean colorectal cancer patients, detected by mutational and expression profiles, associated to different clinicopathological features

Colorectal cancer is a multistep process affecting several signaling pathways including EGFR (epidermal growth factor receptor), a therapeutic target for metastatic disease. Our aim was to characterize the mutational and expression profiles of the EGFR pathway in colorectal tumors and to integrate these results according to five previously defined groups. We screened seven genes for mutations (KRAS-BRAF-PIK3CA-PIK3R1-AKT1-MAP2K1-PTEN) and six proteins (EGFR-p110α-p85α-PTEN-phosphoAKT-phosphoMEK1) by immunohistochemistry, PTEN deletion, and MSI. At least one mutated gene was observed in 68% of tumors (KRAS 45%, PIK3CA 21%, BRAF 14%, and PTEN 7%). PTEN deletion was observed in 10.7% of tumors and 19.6% were MSI-High. In all, 54% of tumors showed a high EGFR expression, 48% p110α, 4.4% phosphoAKT, and 22% phosphoMEK1; and 43% showed low PTEN expression and 22% p85α. In total, five groups of tumors were defined based on MSI, BRAF, and KRAS mutations. Three groups gather mainly early-stage tumors, whereas a fourth group is mostly conformed by advanced tumors. We described here that 71.4% of tumors from one group have a mutated PI3K/PTEN pathway, in comparison to other groups having 32%, 27%, and 25%. In addition, the five groups are differentiated by molecular features such as EGFR, p85α, p110α, and PTEN, showing variable expression among tumor groups. In conclusion, alterations on the EGFR pathway were found in a high percentage of colorectal cancer patients. Using the integration of diverse molecular markers, we ratified previous classification in an ethnic group having relevant genetic differences and living in a different environmental background, adding complementary molecular targets related to therapy.

Differential expression profile of CXCR3 splicing variants is associated with thyroid neoplasia. Potential role in papillary thyroid carcinoma oncogenesis

Papillary thyroid cancer (PTC) is the most prevalent endocrine neoplasia. The increased incidence of PTC in patients with thyroiditis and the frequent immune infiltrate found in PTC suggest that inflammation might be a risk factor for PTC development. The CXCR3-ligand system is involved in thyroid inflammation and CXCR3 has been found upregulated in many tumors, suggesting its pro-tumorigenic role under the inflammatory microenvironment. CXCR3 ligands (CXCL4, CXCL9, CXCL10 and CXCL11) trigger antagonistic responses partly due to the presence of two splice variants, CXCR3A and CXCR3B. Whereas CXCR3A promotes cell proliferation, CXCR3B induces apoptosis. However, the relation between CXCR3 variant expression with chronic inflammation and PTC development remains unknown. Here, we characterized the expression pattern of CXCR3 variants and their ligands in benign tumors and PTC. We found that CXCR3A and CXCL10 mRNA levels were increased in non-metastatic PTC when compared to non-neoplastic tissue. This increment was also observed in a PTC epithelial cell line (TPC-1). Although elevated protein levels of both isoforms were detected in benign and malignant tumors, the CXCR3A expression remained greater than CXCR3B and promoted proliferation in Nthy-ori-3-1 cells. In non-metastatic PTC, inflammation was conditioning for the CXCR3 ligands increased availability. Consistently, CXCL10 was strongly induced by interferon gamma in normal and tumor thyrocytes. Our results suggest that persistent inflammation upregulates CXCL10 expression favoring tumor development via enhanced CXCR3A-CXCL10 signaling. These findings may help to further understand the contribution of inflammation as a risk factor in PTC development and set the basis for potential therapeutic studies.

Mutación homocigota en la línea germinal del gen MUTYH en una paciente chilena con poliposis adenomatosa familiar

Recently, MUTYH mutations have been reported to predispose to the development of polyposis. However, polyposis caused by mutations in MUTYH has been characterized as an autosomal recessive hereditary disease, different from the autosomal dominant pattern observed in polyposis caused by APC mutations. We report a 41-year-old female consulting for anemia. Colonoscopy detected multiple sessile polyps and a cecal carcinoma. The patient was operated and in the surgical piece, the tumor invaded serosa and there was lymph node involvement. Approximately 100 polyps were found. The patient received 5-fluorouracil, as adjuvant therapy. The patient had a sister (of a total of 12 brothers) with a colorectal carcinoma. The genetic study identified a homozygous mutation of the MUTYH gene, called c.340T > C, that produces an amino acid change of tyrosine for histidine called p.Y114H. The sister with colorectal cancer was a heterozygous carrier of this mutation.

Abstract 2114: Deletions/Duplications detection by MLPA in Chilean families with hereditary colorectal cancer: Lynch syndrome and FAP

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The main variants of colorectal cancer hereditary are Familial Adenomatous Polyposis (FAP) and Lynch Syndrome, which account for approximately 5% of total cases of colorectal cancer. Point mutations in APC and MMR genes are responsible of about 85% and 50% of Chilean FAP (classic phenotype) and Lynch Syndrome (Amsterdam criteria) families, respectively. Different molecular strategies are available for the detection of mutations in these genes. Studies in other populations have identified deletions and duplications of one or more consecutive exons account for around 50% and 10-20% of the total alterations in MMR and APC genes, respectively. The aim of our work was the detection of genomic deletion/duplication through Multiplex Ligation-Dependent Probe Amplification (MLPA) in Lynch Syndrome and FAP patients in Chile. In this study, we analized alterations in MLH1 and MSH2 in 26 Lynch Syndrome families (5 Amsterdam and 21 Bethesda families), and in APC in 10 FAP families (6 classic and 4 attenuated), all of them non carriers of point mutations in these genes. We identified 3 different alterations in the MLH1 in four Amsterdam families, all of them are 0.5 times deletion, which are located: exon 1 in one family, exon 19 in two families, and exons 14 and 15 in one family. In the MSH2, in one Amsterdam family we detected a 0.5 time deletion of exon 2. In the APC, we found 2 different alterations in three classic FAP families: in one family we detected a 0.5 times deletion of the whole gene including promoter region, and in two families we found a 1.5 time amplification of exons 1, 2 and 3. These alterations were confirmed in other relatives of these families and in two independent MLPA analysis. The deletion of exon 19 in MLH1 and the amplification of exons 1, 2 and 3 in APC have not been previously described in other populations. In conclusion, genomic deletions/duplications were only detected in 5 Lynch Syndrome families that fulfilled Amsterdam criteria, and in 3 FAP families with classic phenotype. The detection of different mutations involved in these syndromes, allows the development of prevention strategies of these diseases. Financed by Cleveland Clinic Foundation and Las Condes Clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2114.

Abstract 2230: Gene mutations and deletions inactivates PTEN tumor suppressor in Chilean colon cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction. Tumor suppressor PTEN acts as a negative regulator of the PI3K/AKT/mTOR pathway, thus controlling cell survival and proliferation. Mutations or deletions inactivating PTEN function lead to over-activation of the PI3K/AKT/mTOR pathway in cancer. PTEN gene alterations have been described in different types of tumors. In colon cancer these mutations range from 1% to 29% of the cases. The aim of this study was to determine the prevalence of PTEN mutations and/or deletions in Chilean colon cancer patients. Methods: ninety-one colon cancer patients were recruited for this study. Genomic DNA from normal and tumor tissue was obtained, and the deletion of PTEN locus was determined by PCR amplification of three STR markers flanking PTEN, and two intragenic markers. To search point mutations we amplified exons 7 and 8 by PCR, and sequenced. PTEN expression was evaluated through immunohistochemistry. Results: We observed deletion events in 9.8% (9/91) of tumors, and inactivating mutations in 5.5% (5/91). Two tumors presented a deletion in one allele and a mutation in the other. Immunohistochemistry detected a very weak expression of PTEN in 55/76 tumors. Conclusions: 15.4 % (12/91) of tumors have PTEN gene inactivated. We observed that deletion was a frequent mechanism for PTEN inactivation in Chilean colon cancer patients. Note: This abstract was not presented at the meeting. Citation Format: Gonzalo Encina, Karin Alvarez, Paulina Orellana, Ana Maria Wielandt, Cynthia Villarroel, Daniela Simian, Luis Contreras, Udo Kronberg, Francisco Lopez, Pilar Carvallo. Gene mutations and deletions inactivates PTEN tumor suppressor in Chilean colon cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2014-2230

Abstract 2232: Next generation sequencing of the EGFR signaling pathway in colon cancer tumors from Chilean patients

Introduction: The Epidermal Growth Factor Receptor (EGFR) signaling pathway regulates key cell functions like proliferation and survival. This pathway presents two signaling cascades, RAS/RAF/MAPK and PIK3/PTEN/AKT/mTOR. Mutations in different components of the EGFR pathway have been described in different cancers. This pathway is a therapeutic target for monoclonal antibodies and tyrosine kinase inhibitors based therapies. Additionally, in advanced colorectal cancer patients, somatic KRAS mutations are response predictors for biological therapy with anti-EGFR antibody. The sensitivity of the mutations detection methods is key to a proper application of personalized therapeutic strategies. Traditionally, somatic mutations are detected by Sanger sequencing and real-time PCR. We determined the presence of mutations in specific genes of the EGFR pathway in colon cancer tumors from Chilean patients. Methods: DNA was extracted from twenty-eight fresh frozen tumor samples. DNA libraries were created using SureSelect (Agilent) designed for the analysis of EGFR, KRAS, BRAF, MAP2K1, PIK3CA, PIK3R1 and PTEN genes. Next generation sequencing was performed in MiSeq sequencer (Illumina) at Sistemas Genomicos (Spain). Results: Sequencing coverage varied between 100 and 250 readings between tumors, which assures the real presence of the changes. This technique allows detecting mutations that are present in a smaller percentage of tumoral cells, as low as 20% in the sample. Pathogenic mutations were identified in 19 out of the 28 tumors. Mutations were found in KRAS (p.Gly12Asp, p.Gly12Arg, p.Gly12Val and p.Gly12Cys), BRAF (p.Val600Glu and p.Gly606Glu), PIK3CA (p.Glu542Lys, p.Glu545Lys, p.Glu545Gln, p.Ala1035Val, p.Asp1045Val and p.His1047Arg) and PTEN (p.Lys267ArgfsX9). Five tumors presented mutations in both branches of the EGFR signaling pathway (KRAS-PIK3CA, BRAF-PIK3CA and BRAF-PTEN). Three allelic variants were identified in MAP2K1 and PIK3R1. Mutations that were identified in KRAS, BRAF and PIK3CA genes corresponded to gain of function mutations while a change found in PTEN gene corresponded to a loss of function mutation. Finally, mutations in PIK3CA and KRAS of 6 tumors were not previously detected by Sanger sequencing. Discussion: Mutations were found in 68% of analyzed tumors. Eighteen percent of the tumors presented mutations in two genes. Forty-six percent of mutations were identified in KRAS gene. Three allelic variants of unknown significance were detected which have not been described in cancer mutations nor in SPNs databases. Therefore, it is necessary to perform functional studies to determine the effect on protein function. Next generation sequencing allowed us to obtain more reliable and reproducible results, and was able to detect mutations present in a low percentage of tumoral cells in the sample, maybe due to tumoral heterogeneity or contamination with normal cells. FONDECYT 1111020 Citation Format: Karin Alvarez, Paulina Orellana, Cynthia Villarroel, Gonzalo Encina, Daniela Simian, Camila Estay, Maki Kobayashi, Hiroshi Kawashi, Udo Kronberg, Francisco Lopez, Pilar Carvallo. Next generation sequencing of the EGFR signaling pathway in colon cancer tumors from Chilean patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2014-2232

Abstract 2645: Genomic deletions and point mutations in the STK11 gene in Peutz-Jeghers Chilean families

Background: Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant hereditary disease characterized by mucocutaneous pigmentation and gastrointestinal hamartomatous polyposis. Individuals with PJS are at increased risk for development of various neoplasms. The genetic susceptibility for PJS has been associated with germline mutation in the serine/threonine kinase 11 (STK11) gene. The aim of the present study was to characterize the genotype and phenotype of Chilean PJS patients. Methods: Mutation screening of 13 patients from 8 PJS families was performed by using single strand conformation polymorphism (SSCP) analysis and DNA sequencing, covering the entire coding region and the splice-site boundaries. In addition, multiplex ligation-dependent probe amplification (MLPA) assay was used for the identification of large exonic deletions or duplications. Results: We identified seven different pathogenic mutations in STK11 gene in 7 unrelated families. Point mutations correspond to a small deletion (c.109delC) in exon 1 that generates a premature stop codon and two intronic mutations localized in splice site of intron 4 (c.597+2 T>A) and intron 6 (c.862-2 A>G). The pathogenic effect on splicing of intronic changes was demonstrated in mRNA of STK11 from lymphocytes of patients. By MLPA, we identified four large deletions ranging from one exon to the whole gene. Breakpoints were identified in two of these patients. Three point mutations (43%, 3/7) may be considered as novel. Conclusions: Different germline mutations in STK11 were detected. A combination of sensitive techniques may improve a high STK11 mutation detection frequency (88%) in PJS Chilean families. Funded by Clinica Las Condes Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2645. doi:1538-7445.AM2012-2645