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Purpose: PI3K pathway activation occurs in concomitance with RAS/BRAF mutations in colorectal cancer, limiting the sensitivity to targeted therapies. Several clinical studies are being conducted to test the tolerability and clinical activity of dual MEK and PI3K pathway blockade in solid tumors.Experimental Design: In the present study, we explored the efficacy of dual pathway blockade in colorectal cancer preclinical models harboring concomitant activation of the ERK and PI3K pathways. Moreover, we investigated if TP53 mutation affects the response to this therapy.Results: Dual MEK and mTORC1/2 blockade resulted in synergistic antiproliferative effects in cell lines bearing alterations in KRAS/BRAF and PIK3CA/PTEN. Although the on-treatment cell-cycle effects were not affected by the TP53 status, a marked proapoptotic response to therapy was observed exclusively in wild-type TP53 colorectal cancer models. We further interrogated two independent panels of KRAS/BRAF- and PIK3CA/PTEN-altered cell line– and patient-derived tumor xenografts for the antitumor response toward this combination of agents. A combination response that resulted in substantial antitumor activity was exclusively observed among the wild-type TP53 models (two out of five, 40%), but there was no such response across the eight mutant TP53 models (0%). Interestingly, within a cohort of 14 patients with colorectal cancer treated with these agents for their metastatic disease, two patients with long-lasting responses (32 weeks) had TP53 wild-type tumors.Conclusions: Our data support that, in wild-type TP53 colorectal cancer cells with ERK and PI3K pathway alterations, MEK blockade results in potent p21 induction, preventing apoptosis to occur. In turn, mTORC1/2 inhibition blocks MEK inhibitor–mediated p21 induction, unleashing apoptosis. Clin Cancer Res; 21(24); 5499–510. ©2015 AACR.

Table S1. MiSeq 57-gene panel list

Supplementary Figure and Table Legends. The text describes the panels and tables included as Supplementary Figure and Table Legends

Figure S2. Immunohistochemistry of p53 in tumors from CRC patients treated with MEK- plus PI3K-inhibitors

The MECP2 gene located on Xq28 is one of the most important genes contributing to the spectrum of neurodevelopmental disorders. Therefore, we present our experience in the molecular study of this gene. MECP2 was thoroughly tested for the presence of mutations (sequencing of four exons and rearrangements) in 120 female patients: 28 with classic Rett syndrome, five with atypical Rett syndrome, and 87 with heterogeneous phenotypes with some Rett-like features. Another 120 female patients with intellectual disability of unknown origin were also studied, but in these cases we only tested exons 3 and 4. Finally, 861 healthy controls (519 females and 342 males) were also studied for exon 3 and 4. Eighteen different pathological mutations were found, five of them previously undescribed, and four large deletions detected by multiplex ligation-dependent probe amplification. All were de novo mutations not present in the parents. In conclusion, i) MECP2 is one of the most important genes in the diagnosis of genetic intellectual disability in females; ii) MECP2 must be studied not only in patients with classical/atypical Rett syndrome but also in patients with other phenotypes related to Rett syndrome; and iii) for the new variants, it is important to perform complementary studies, including the analysis of large populations of healthy individuals and the use of in silico programs.

BRCA1-associated breast cancers have been associated to a triple-negative phenotype. The prevalence of BRCA1 germline mutations in young onset TNBC based on informativeness of family history has not been reported. From January 2008 to May 2009 were collected blood and tumor samples from patients with TNBC younger than 50 years and without a family history of breast and ovarian cancer in first- and second-degree relatives. Analysis of BRCA1 germline mutations was made. Age at diagnosis and informativeness of family history (presence of female in first- and second-degree relatives alive until age 45) was collected in all cases. Immunohistochemistry of basal-like features was performed centrally in all available tumors. Seven pathogenic mutations were detected in 92 patients (7.6 %), two of them in patients younger than 35 years (28.6 %) (Fisher's exact test, p = 0.631). Three non-classified variants were detected (3.2 %). Family history was informative in two patients with a pathogenic mutation (28.6 %) and not informative in five (71.4 %) (Fisher's exact test, p = 0.121). Of the seven patients with a pathogenic mutation, four had a basal-like phenotype. Patients with apparently sporadic TNBC younger than 50 years and a non-informative family history are candidates for germline genetic testing of BRCA1.

This is an open access article distributed under the Creative Commons Attribution License.-- et al., Fragile X syndrome is the most common inherited form of intellectual disability. Here we report on a study based on a collaborative registry, involving 12 Spanish centres, of molecular diagnostic tests in 1105 fragile X families comprising 5062 individuals, of whom, 1655 carried a full mutation or were mosaic, three cases had deletions, 1840 had a premutation, and 102 had intermediate alleles. Two patients with the full mutation also had Klinefelter syndrome. We have used this registry to assess the risk of expansion from parents to children. From mothers with premutation, the overall rate of allele expansion to full mutation is 52.5%, and we found that this rate is higher for male than female offspring (63.6% versus 45.6%; P < 0.001). Furthermore, in mothers with intermediate alleles (45-54 repeats), there were 10 cases of expansion to a premutation allele, and for the smallest premutation alleles (55-59 repeats), there was a 6.4% risk of expansion to a full mutation, with 56 repeats being the smallest allele that expanded to a full mutation allele in a single meiosis. Hence, in our series the risk for alleles of < 59 repeats is somewhat higher than in other published series. These findings are important for genetic counselling. © 2014 María-Isabel Tejada et al., Thanks are due to the agreements between GIRMOGEN and the Real Patronato sobre Discapacidad of the Spain’s Ministry for Health, Social Services and Equality (Spain) to carry out this study.

Introduction: Additional nucleotide substitutions in the 3 ´-untranslated region of prothrombin gene could explain some thrombotic events and also adverse pregnancy outcomes. We describe the first case of a homozygous 20209C>T mutation as the cause of deep vein thrombosis in a Spanish patient. Case and methods: The 56-year-old male patient with a partial tear of the Achilles tendon developed calf (tibial) deep vein thrombosis after im- mobilization and was treated with an anticoagulant. To determine if the deep vein thrombosis was of genetic origin, a peripheral blood DNA sample was analysed for the presence of the three most frequent mutations associated with thrombotic events: factor V Leiden (1691G>A), prothrombin (20210G>A) and methylene tetrahydrofolate reductase (677C>T). The presence or absence of the normal allele of prothrombin could not be deter- mined using the PTH-FV-MTHFR StripAssay (Vienna Lab). Results: Comprehensive analysis showed that the patient had a variant interfering with the polymerase chain reaction product, we sequenced the entire prothrombin gene and found that the patient had a homozygous C>T mutation at position 20209; this interfered with the polymerase chain reaction product, which needs a C at this position to be able to bind to the wild-type probe present in the test strip. Conclusion: The homozygous 20209C>T mutation and the presence of the mutation 677C>T in heterozygosity explained the patient’s deep vein thrombosis because the combination of mutations would increase the risk of thrombosis. Suitable genetic counselling should be provided to the pa- tient and first-degree relatives as it important to detect prothrombin gene variants that could increase risk for thrombotic events.

The MECP2 gene located on Xq28 is one of the most important genes contributing to the spectrum of neurodevelopmental disorders. Therefore, we present our experience in the molecular study of this gene. MECP2 was thoroughly tested for the presence of mutations (sequencing of four exons and rearrangements) in 120 female patients: 28 with classic Rett syndrome, five with atypical Rett syndrome, and 87 with heterogeneous phenotypes with some Rett-like features. Another 120 female patients with intellectual disability of unknown origin were also studied, but in these cases we only tested exons 3 and 4. Finally, 861 healthy controls (519 females and 342 males) were also studied for exon 3 and 4. Eighteen different pathological mutations were found, five of them previously undescribed, and four large deletions detected by multiplex ligation-dependent probe amplification. All were de novo mutations not present in the parents. In conclusion, i) MECP2 is one of the most important genes in the diagnosis of genetic intellectual disability in females; ii) MECP2 must be studied not only in patients with classical/atypical Rett syndrome but also in patients with other phenotypes related to Rett syndrome; and iii) for the new variants, it is important to perform complementary studies, including the analysis of large populations of healthy individuals and the use of in silico programs.

Background Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. Methods We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA). Results Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. Conclusions This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice.

et al., It has been demonstrated that monoallelic PALB2 (Partner and Localizer of BRCA2) gene mutations predispose to familial breast cancer. Some of the families reported with germline PALB2 mutations presented male breast cancer as a characteristic clinical feature. Therefore, we wanted to investigate the contribution of germline PALB2 mutations in a set of 131 Spanish BRCA1/BRCA2-negative breast/ovarian cancer families with at least one male breast cancer case. The analysis included direct sequencing of all coding exons and intron/exon boundaries as well as a Multiplex Ligation-dependent Probe Amplification-based analysis of genomic rearrangements. For the first time we have identified a genomic rearrangement of PALB2 gene involving a large deletion from exon 7 to 11 in a breast cancer family. We have also identified several PALB2 variants, but no other obvious deleterious PALB2 mutation has been found. Thus, our study does not support an enrichment of PALB2 germline mutations in the subset of breast cancer families with male breast cancer cases. The identification of intronic and exonic variants indicates the necessity of assessing the implications of variants that do not lead to PALB2 truncation in the pathoghenicity of the PALB2 gene., This study was supported by grants from the Xunta de Galicia (10PXIB 9101297PR) and FMM Foundation given to AV. TC, M de H, and PPS were supported by PS09/00859, RD06/0020/0021 from RTICC, Instituto de Salud Carlos III. JB and AO were supports by the AECC, and RD06/0020/1060 from RTICC ICO: Contract grant sponsor: Asociación Española Contra el Cáncer, Spanish Health Research Fund; Carlos III Health Institute; Catalan Health Institute and Autonomous Government of Catalonia. Contract grant numbers: ISCIIIRETIC RD06/0020/1051, PI10/01422, PI10/ 31488, and 2009SGR290. E.V. grants, CSI004A10-2 (Consejería de Educación, Junta de Castilla y León) and BIO39/VA27/10 (Consejería de Sanidad, Junta de Castilla y León).

Resistance to thyroid hormone (RTH) is a syndrome mostly caused by mutations in thyroid hormone receptor beta gen (THRB). We present five families with RTH phenotype.THRB gene sequencing. In vitro studies to evaluate the mutants response to thyroid hormones and their dominant negative effect. Mechanism of resistance in patients with RTH without THRB mutations quantifying expression of regulator of calcineurin 2 (ZAKI4) and Kruppel-like factor 9 (BTEB) genes in patients fibroblast cultures.THRB mutations were found in three cases: R243Q, R320C, R429Q. Mutants showed a decreased response to T3. R243Q and R320C had a strong dominant negative effect. One subject without THRB mutation showed changes in ZAKI4 and BTEB expression similar to R320C and the other showed expression levels higher than normal controls.Three cases of RTH were caused by THRB heterozygous mutations but in two cases mutations were not found. THRB mutation carriers and one of the patients without mutations share a similar mechanism of resistance and in the other subject RTH is TRβ independent.

9 páginas, 1 figura, 4 tablas.-- et al., [Purpose]: It is not clear that the published estimates of the breast and ovarian cancer penetrances of mutations in BRCA1 and BRCA2 can be used in genetic counseling in countries such as Spain, where the incidence of breast cancer in the general population is considerably lower, the prevalence of BRCA2 mutations seems to be higher, and a distinct spectrum of recurrent mutations exists for both genes. We aimed to estimate these penetrances for women attending genetic counseling units in Spain. [Experimental Design]: We collected phenotype and genotype data on 155 BRCA1 and 164 BRCA2 mutation carrier families from 12 centers across the country. Average age-specific cumulative risks of breast cancer and ovarian cancer were estimated using a modified segregation analysis method. [Results]: The estimated average cumulative risk of breast cancer to age 70 years was estimated to be 52% [95% confidence interval (95% CI), 26-69%] for BRCA1 mutation carriers and 47% (95% CI, 29-60%) for BRCA2 mutation carriers. The corresponding estimates for ovarian cancer were 22% (95% CI, 0-40%) and 18% (95% CI, 0-35%), respectively. There was some evidence (two-sided P = 0.09) that 330A>G (R71G) in BRCA1 may have lower breast cancer penetrance. [Conclusions]: These results are consistent with those from a recent meta-analysis of practically all previous penetrance studies, suggesting that women with BRCA1 and BRCA2 mutations attending genetic counseling services in Spain have similar risks of breast and ovarian cancer to those published for other Caucasian populations. Carriers should be fully informed of their mutation- and age-specific risks to make appropriate decisions regarding prophylactic interventions such as oophorectomy.

A double-blind randomized comparative phase II study of flutrimazole site-release vaginal cream (1, 2 and 4%) with placebo site-release vaginal cream was undertaken in patients with acute vulvovaginal candidosis. Vaginitis was demonstrated by both positive findings on microscopic examination of vaginal smears and positive culture as well as by the presence of clinical signs and symptoms. The vaginal monodose treatment was inserted in the evening at bedtime using a vaginal applicator and, in addition, all four groups of patients received additional topical external cream for application to the vulva twice-daily for 7 days; the placebo group received a placebo cream and the active therapy groups all received a 2% flutrimazole cream. A total of 133 patients who were seen over a 10-month period were screened and randomized: five patients did not take the allocated medication, and four patients whose menstrual period began shortly after study entry were excluded from the study, leaving 124 patients who were randomly allocated to receive a monodose vaginal 1% cream (regimen A, 28 patients), a monodose vaginal 2% cream (regimen B, 32 patients), a monodose vaginal 4% cream (regimen C, 31 patients) or a monodose vaginal placebo cream (regimen D, 33 patients). At the assessment 9 days after the end of therapy the proportion of patients who were cured was 82% in group A, 87.4% in group B, 83.8% in group C and 63.5% in group D. Three patients (10.7%) in group A, four (12.5%) in group B, one (3.2%) in group C and 12 (36.36%) in group D did not respond to the treatment. One patient (3.5%) in group A, and two patients (6.4%) in group C terminated the treatment prematurely due to intolerance. There was a significant association between Candida glabrata and treatment failure (P < 0.04) and C. glabrata and carrier state (P = 0.01) in vagina (chi 2 test, P = 0.01) and vulvovagina (chi 2 test, P = 0.00001). At the assessment 4 weeks after the end of therapy the proportion of cured patients was 60.6% in group A, 78% in group B, 80.6% in group C and 48.4% in group D. Group D (placebo) versus group B (2%) and group C (4%) showed a significant difference (P = 0.01 and P = 0.007, respectively). Although there were no significant differences in clinical and mycological activity between the three active groups, group B (flutrimazole 2% site-release vaginal cream) was chosen for clinical use due to its tolerance profile. Seven patients (25%) in group A, three (9.3%) in group B, two (6.4%) in group C and five (15.1%) in group D relapsed 4 weeks after the end of therapy; the relapse rate was not significantly associated with positive culture results 9 days after treatment. There was a significant association between C. glabrata and the carrier state (P < 0.01). The overall ineffective treatment (includes failures at control 1, relapses at control 2 and premature terminations) was 39% in group A, 21.7% in group B, 16% in group C and 51.3% in group D. There was a significant difference in the overall ineffective treatment when C and D groups were compared with placebo (P = 0.01 and P = 0.003, respectively).