Whole genome sequencing as a first-tier diagnostic test for infants in neonatal intensive care units: A pilot study in Brazil.

In this pilot study, we aimed to evaluate the feasibility of whole genome sequencing (WGS) as a first-tier diagnostic test for infants hospitalized in neonatal intensive care units in the Brazilian healthcare system. The cohort presented here results from a joint collaboration between private and public hospitals in Brazil considering the initiative of a clinical laboratory to provide timely diagnosis for critically ill infants. We performed trio (proband and parents) WGS in 21 infants suspected of a genetic disease with an urgent need for diagnosis to guide medical care. Overall, the primary indication for genetic testing was dysmorphic syndromes (n = 14, 67%) followed by inborn errors of metabolism (n = 6, 29%) and skeletal dysplasias (n = 1, 5%). The diagnostic yield in our cohort was 57% (12/21) based on cases that received a definitive or likely definitive diagnostic result from WGS analysis. A total of 16 pathogenic/likely pathogenic variants and 10 variants of unknown significance were detected, and in most cases inherited from an unaffected parent. In addition, the reported variants were of different types, but mainly missense (58%) and associated with autosomal diseases (19/26); only three were associated with X-linked diseases, detected in hemizygosity in the proband an inherited from an unaffected mother. Notably, we identified 10 novel variants, absent from public genomic databases, in our cohort. Considering the entire diagnostic process, the average turnaround time from enrollment to medical report in our study was 53 days. Our findings demonstrate the remarkable utility of WGS as a diagnostic tool, elevating the potential of transformative impact since it outperforms conventional genetic tests. Here, we address the main challenges associated with implementing WGS in the medical care system in Brazil, as well as discuss the potential benefits and limitations of WGS as a diagnostic tool in the neonatal care setting.

Low-pass whole genome sequencing is a reliable and cost-effective approach for copy number variant analysis in the clinical setting.

INTRODUCTION: Next generation sequencing technology has greatly reduced the cost and time required for sequencing a genome. An approach that is rapidly being adopted as an alternative method for CNV analysis is the low-pass whole genome sequencing (LP-WGS). Here, we evaluated the performance of LP-WGS to detect copy number variants (CNVs) in clinical cytogenetics. MATERIALS AND METHODS: DNA samples with known CNVs detected by chromosomal microarray analyses (CMA) were selected for comparison and used as positive controls; our panel included 44 DNA samples (12 prenatal and 32 postnatal), comprising a total of 55 chromosome imbalances. The selected cases were chosen to provide a wide range of clinically relevant CNVs, the vast majority being associated with intellectual disability or recognizable syndromes. The chromosome imbalances ranged in size from 75 kb to 90.3 Mb, including aneuploidies and two cases of mosaicism. RESULTS: All CNVs were successfully detected by LP-WGS, showing a high level of consistency and robust performance of the sequencing method. Notably, the size of chromosome imbalances detected by CMA and LP-WGS were compatible between the two different platforms, which indicates that the resolution and sensitivity of the LP-WGS approach are at least similar to those provided by CMA. DISCUSSION: Our data show the potential use of LP-WGS to detect CNVs in clinical diagnosis and confirm the method as an alternative for chromosome imbalances detection. The diagnostic effectiveness and feasibility of LP-WGS, in this technical validation study, were evidenced by a clinically representative dataset of CNVs that allowed a systematic assessment of the detection power and the accuracy of the sequencing approach. Further, since the software used in this study is commercially available, the method can easily be tested and implemented in a routine diagnostic setting.

A Benchmark of In-House Homologous Recombination Repair Deficiency Testing Solutions for High-Grade Serous Ovarian Cancer Diagnosis.

Homologous recombination deficiency (HRD) has become an important prognostic and predictive biomarker for patients with high-grade serous ovarian cancer who may benefit from poly-ADP ribose polymerase inhibitors (PARPi) and platinum-based therapies. HRD testing provides relevant information to personalize patients' treatment options and has been progressively incorporated into diagnostic laboratories. Here, we assessed the performance of an in-house HRD testing system deployable in a diagnostic clinical setting, comparing results from two commercially available next-generation sequencing (NGS)-based tumor tests (SOPHiA DDMTM HRD Solution and AmoyDx® (HRD Focus Panel)) with the reference assay from Myriad MyChoice® (CDx). A total of 85 ovarian cancer samples were subject to HRD testing. An overall strong correlation was observed across the three assays evaluated, regardless of the different underlying methods employed to assess genomic instability, with the highest pairwise correlation between Myriad and SOPHiA (R = 0.87, p-value = 3.39 × 10-19). The comparison of the assigned HRD status to the reference Myriad's test revealed a positive predictive value (PPV) and negative predictive value (NPV) of 90.9% and 96.3% for SOPHiA's test, while AmoyDx's test achieved 75% PPV and 100% NPV. This is the largest HRD testing evaluation using different methodologies and provides a clear picture of the robustness of NGS-based tests currently offered in the market. Our data shows that the implementation of in-house HRD testing in diagnostic laboratories is technically feasible and can be reliably performed with commercial assays. Also, the turnaround time is compatible with clinical needs, making it an ideal alternative to offer to a broader number of patients while maintaining high-quality standards at more accessible price tiers.

BRCA1 and BRCA2 germline mutation analysis from a cohort of 1267 patients at high risk for breast cancer in Brazil.

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country.

Chromosomal microarray analyses from 5778 patients with neurodevelopmental disorders and congenital anomalies in Brazil.

Chromosomal microarray analysis (CMA) has been recommended and practiced routinely since 2010 both in the USA and Europe as the first-tier cytogenetic test for patients with unexplained neurodevelopmental delay/intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. However, in Brazil, the use of CMA is still limited, due to its high cost and complexity in integrating the results from both the private and public health systems. Although Brazil has one of the world's largest single-payer public healthcare systems, nearly all patients referred for CMA come from the private sector, resulting in only a small number of CMA studies in Brazilian cohorts. To date, this study is by far the largest Brazilian cohort (n = 5788) studied by CMA and is derived from a joint collaboration formed by the University of São Paulo and three private genetic diagnostic centers to investigate the genetic bases of neurodevelopmental disorders and congenital abnormalities. We identified 2,279 clinically relevant CNVs in 1886 patients, not including the 26 cases of UPD found. Among detected CNVs, the corresponding frequency of each category was 55.6% Pathogenic, 4.4% Likely Pathogenic and 40% VUS. The diagnostic yield, by taking into account Pathogenic, Likely Pathogenic and UPDs, was 19.7%. Since the rational for the classification is mostly based on Mendelian or highly penetrant variants, it was not surprising that a second event was detected in 26% of those cases of predisposition syndromes. Although it is common practice to investigate the inheritance of VUS in most laboratories around the world to determine the inheritance of the variant, our results indicate an extremely low cost-benefit of this approach, and strongly suggest that in cases of a limited budget, investigation of the parents of VUS carriers using CMA should not be prioritized.

An Apparently Balanced Complex Chromosome Rearrangement Involving Seven Breaks and Four Chromosomes in a Healthy Female and Segregation/Recombination in Her Affected Son.

Duplication of the distal 1q and 4p segments are both characterized by the presence of intellectual disability/neurodevelopmental delay and dysmorphisms. Here, we describe a male with a complex chromosome rearrangement (CCR) presenting with overlapping clinical findings between these 2 syndromes. In order to better characterize this CCR, classical karyotyping, FISH, and chromosomal microarray analysis were performed on material from the patient and his parents, which revealed an unbalanced karyotype with duplications at 1q41q43 and 4p15.2p14 in the proband. The rearrangements, which were derived from a maternal balanced karyotype, included an insertion of a segment from the long to the short arm of chromosome 1, a balanced translocation involving chromosomes 14 and 18, and an insertion of a segment from the short arm of chromosome 4 into the derived chromosome 14. This study aimed to better define the clinical history and prognosis of a patient with this rare category of chromosomal aberration. Our results suggest that the frequency of CCR in the general population may be underestimated; when balanced, they may not have a phenotypic effect. Moreover, they emphasize the need for cytogenetic techniques complementary to chromosomal microarray for proper genetic counseling.

A novel variant in the COX15 gene causing a fatal infantile cardioencephalomyopathy: A case report with clinical and molecular review.

The cytochrome c-oxidase (COX) enzyme, also known as mitochondrial complex IV (MT-C4D), is a transmembrane protein complex found in mitochondria. COX deficiency is one of the most frequent causes of electron transport chain defects in humans. Therefore, high energy demand organs and tissues are affected in patients with mutations in the COX15 gene, with variable phenotypic expressiveness. We describe the case of a male newborn with hypertrophic cardiomyopathy and serum and cerebrospinal fluid hyperlacticaemia, whose exome sequencing revealed two variants in a compound heterozygous state: c.232G > A; p.(Gly78Arg), classified as likely pathogenic, and c.452C > G; p.(Ser151Ter), as pathogenic; the former never previously described in the literature.

Congenital afibrinogenemia in a patient with vascular abnormalities and a novel variant: clinical-molecular description and literature review.

: The objective is to report a patient with congenital afibrinogenemia and vascular abnormalities and also review the clinical and molecular issues. The female proband, diagnosed with congenital afibrinogenemia, was admitted at a hospital due to a hemorrhagic shock. Angiotomography revealed ectasias from ascending branch to the abdominal aorta, with multiple calcifications and atheroma. Clinical exome identified a homozygous novel pathogenic variant in FGG gene. In our review the main symptom, at diagnosis, was umbilical cord bleeding and the degree of clinical involvement varied from asymptomatic to severe. The FGA gene was the most affected and possible hot spots were observed. Variants considered as loss of function were the most frequent. The association of vascular abnormalities in a patient with congenital afibrinogenemia alerts for a closer follow-up of vascular issues in these patients.

Hip, Knee, and Ankle Functional Demand During Habitual and Fast-Pace Walking in Younger and Older Women.

This study compared functional demand (FD) between older and younger women walking at habitual and fast speed and determined strength thresholds necessary to maintain FD below 80%. FD was calculated by expressing walking flexion and extension joint torques of the ankle, knee, and hip as a percentage of maximal strength. Young women had an average FD of 65% across joint actions and speeds, whereas older women had FD of 90%. In older women, the greatest FD occurred in the hip musculature. The hip, knee, and ankle extensor strengths required to maintain FD below 80% were 1.66, 1.86, and 0.57 Nm/kg, and flexor strengths were 1.24, 0.49, and 0.69 Nm/kg, respectively. Older women have limited functional reserve to increase gait speed and rely heavily on available hip strength during walking. This study identifies strength targets for the hip, knee, and ankle that will assist in maintaining FD of older women at a sustainable level.

Comparação dos parâmetros de força e propriocepção entre indivíduos com e sem instabilidade funcional de tornozelo / Comparación entre los parámetros de fuerza y propiopercepción entre sujetos con y sin inestabilidad funcional del tobillo / Comparison of strength and proprioception parameters between subjects with and without functional ankle instability

Após a entorse de tornozelo, 40% dos indivíduos continuam a relatar uma sensação de instabilidade articular que está relacionada à disfunção músculo-esquelética denominada instabilidade funcional do tornozelo (IFT). Contudo, o mecanismo como ocorre esta disfunção músculo-esquelética ainda permanece desconhecido. Nesse sentido, o conhecimento das alterações músculo-esqueléticas que ocorrem em indivíduos com IFT pode ser um fator importante para traçar intervenções preventivas mais efetivas. Dessa forma, o objetivo deste estudo foi comparar o pico de torque (PT) concêntrico de inversão (INV) e eversão (EVE), a razão convencional (EVE/INV) e o reposicionamento articular passivo em indivíduos com e sem IFT em atletas recreacionais do gênero feminino. A amostra foi composta por 22 voluntárias na faixa etária entre 18 e 25 anos que foram divididas em grupo controle e grupo com IFT. A avaliação do torque foi realizada em um dinamômetro isocinético com cinco contrações máximas concêntricas a velocidades de 60 e 120 graus.s-1 no movimento de INV e EVE e reposicionamento articular passivo com ângulo-alvo de 10° e 20° de inversão. Foram analisados os dados de PT, razão convencional e média do erro absoluto do ângulo-alvo. A análise estatística foi feita com o teste t-Student para amostras independentes. Foi encontrado que indivíduos com IFT apresentaram diminuição da força eversora comparados aos indivíduos controle, bem como desequilíbrio da razão muscular, que podem aumentar a predisposição deste grupo a entorses de tornozelos.

Después del esguince de tobillo, el 40% de los sujetos siguen informando que hay una sensación de inestabilidad articular que se relaciona con la disfunción del musculo-esquelética llamada inestabilidad funcional del tobillo (IFT). Sin embargo, todavía no se conoce el mecanismo que es responsable por esta disfunción musculo-esquelética. En este sentido, el conocimiento de las alteraciones musculo-esqueléticas que ocurren en personas con IFT puede ser un factor importante para planear intervenciones preventivas más eficaces. De esta manera, esta investigación tiene como objetivo comparar el momento de torque (PT) de inversión concéntrica (INV) y eversión (EVE), la razón convencional (EVE/INV) y el reposicionamiento pasivo de las articulaciones en personas con y sin IFT en atletas recreacionales del género femenino. La muestra está compuesta por 22 voluntarios en la franja etaria entre 18 y 25 años, los cuales se dividieron en un grupo control y un grupo con IFT. La evaluación del torque se realizó en un dinamómetro isocinético con cinco contracciones máximas concéntricas con velocidades de 60 y 120 grados.s-1 en un movimiento de INV y EVE y reposicionamiento pasivo de la articulación con ángulo objetivo de 10° y 20° de inversión. Se analizó los datos del PT, de la razón convencional y la media del error absoluta del ángulo objetivo. Para el análisis, se utilizó la prueba t-Student para muestras independientes. Si comparados con las personas control se observó que las personas con IFT presentaron una disminución de la fuerza eversora y también un desequilibrio de la razón muscular, lo que puede aumentar la susceptibilidad de éstos a los esguinces de tobillos.

After an ankle sprain 40% of individuals continue to report a sensation of joint instability that is related to musculoskeletal dysfunction called functional ankle instability (FAI). However, a mechanism like this musculoskeletal dysfunction occurs remains unknown. In this sense, the knowledge of musculoskeletal changes that occur in individuals with FAI may be an important factor to plot more effective preventive interventions. Thus, the aim of this study was to compare peak torque (PT) concentric inversion (INV) and eversion (EVE), conventional ratio (EVE/ INV) and passive joint repositioning in individuals with and without FAI in recreational athletes females. The sample consisted of 22 volunteers aged 18-25 years who were divided into control group and the group with FAI. The torque rating was performed on an isokinetic dynamometer with five maximal concentric contractions at speeds of 60 and 120 graus.s-1 in INV and EVE movement and passive joint repositioning to target angle of 10° and 20° of inversion. PT data, conventional ratio and average absolute error of the target angle were analyzed. Statistical analysis was performed using the Student t-test for independent samples. Found that individuals with LFS showed decreased evertor strength compared to control subjects, as well as unbalanced muscle ratio, which may increase the susceptibility of this group to ankle sprains.

Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer.

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

A órtese não influencia no desempenho de indivíduos com instabilidade do tornozelo / The orthosis had no influence on the performance of individuals with ankle instability

Introdução: A instabilidade funcional do tornozelo (IFT) é uma das consequências das entorses de tornozelo, que podem gerar déficits de funcionalidade. Um dos métodos utilizados para avaliar esses déficits são testes funcionais (TF), além disso, pode-se prevenir a entorse usando órteses de tornozelo, no entanto, ambos os métodos ainda são controversos na literatura. Objetivo: Comparar o desempenho de indivíduo com e sem IFT em testes funcionais com e sem o uso da órtese de tornozelo. Método: Vinte e duas voluntárias na faixa etária entre 18 e 25 anos participaram do estudo e foram divididas em grupo controle e grupo com IFT. As mulheres realizaram cinco testes funcionais com e sem o uso de órtese de tornozelo. Resultados: Não foi encontrada diferença significativa entre os grupos e nem entre as condições. Conclusão: A órtese de tornozelo não influenciou no desempenho de indivíduos com e sem IFT em testes funcionais.

Introduction: Functional ankle instability (FAI) is a consequence of ankle sprains, which can generate functional deficits. One of the methods used to evaluate these deficits are the functional tests (FT), in addition, one method of preventing ankle sprains are the orthosis, however, both methods are still controversial. Objective: To compare the performance of individuals with and without IFT in functional tests with and without the use of ankle orthosis. Method: Twenty-two volunteers aged 18 to 25 years participated in the study and were divided into a control group and a group with IFT. The subjects performed five functional tests with and without the use of ankle orthosis. Results: No significant difference was found between groups and between conditions. Conclusion: The ankle orthosis had no influence on the performance of individuals with and without FAI in functional tests.

Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models.

The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER)-negative cells. Basal cell-origin tumours displayed similar histological phenotypes, regardless of the depleted gene. In contrast, luminal ER-negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER-negative and, strikingly, ER-positive invasive ductal carcinomas. Molecular profiling demonstrated that luminal ER-negative cell-origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal-like, luminal B and 'normal-like'. Furthermore, a subset of these tumours resembled the 'claudin-low' tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell of origin and driver genetic aberrations, but also multiple mammary tumour subtypes, including both ER-positive and -negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour aetiology.

Adenoid cystic carcinomas constitute a genomically distinct subgroup of triple-negative and basal-like breast cancers.

Adenoid cystic carcinoma (AdCC) is a rare form of triple-negative and basal-like breast cancer that has an indolent clinical behaviour. Four breast AdCCs were recently shown to harbour the recurrent chromosomal translocation t(6;9)(q22-23;p23-24), which leads to the formation of the MYB-NFIB fusion gene. Our aims were (i) to determine the prevalence of the MYB-NFIB fusion gene in AdCCs of the breast; (ii) to characterize the gene copy number aberrations found in AdCCs; and (iii) to determine whether AdCCs are genomically distinct from histological grade-matched or triple-negative and basal-like invasive ductal carcinomas of no special type (IDC-NSTs). The presence of the MYB-NFIB fusion gene was investigated in 13 AdCCs of the breast by fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR (RT-PCR), and MYB and BRCA1 RNA expression was determined by quantitative RT-PCR. Fourteen AdCCs, 14 histological grade-matched IDC-NSTs, and 14 IDC-NSTs of triple-negative and basal-like phenotype were microdissected and subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). The MYB-NFIB fusion gene was detected in all but one AdCC. aCGH analysis demonstrated a relatively low number of copy number aberrations and a lack of recurrent amplifications in breast AdCCs. Contrary to grade-matched IDC-NSTs, AdCCs lacked 1q gains and 16q losses, and in contrast with basal-like IDC-NSTs, AdCCs displayed fewer gene copy number aberrations and expressed MYB and BRCA1 at significantly higher levels. Breast AdCCs constitute an entity distinct from grade-matched and triple-negative and basal-like IDC-NSTs, emphasizing the importance of histological subtyping of triple-negative and basal-like breast carcinomas.

Nottingham Prognostic Index in triple-negative breast cancer: a reliable prognostic tool?

BACKGROUND: A breast cancer prognostic tool should ideally be applicable to all types of invasive breast lesions. A number of studies have shown histopathological grade to be an independent prognostic factor in breast cancer, adding prognostic power to nodal stage and tumour size. The Nottingham Prognostic Index has been shown to accurately predict patient outcome in stratified groups with a follow-up period of 15 years after primary diagnosis of breast cancer. Clinically, breast tumours that lack the expression of Oestrogen Receptor, Progesterone Receptor and Human Epidermal growth factor Receptor 2 (HER2) are identified as presenting a "triple-negative" phenotype or as triple-negative breast cancers. These poor outcome tumours represent an easily recognisable prognostic group of breast cancer with aggressive behaviour that currently lack the benefit of available systemic therapy. There are conflicting results on the prevalence of lymph node metastasis at the time of diagnosis in triple-negative breast cancer patients but it is currently accepted that triple-negative breast cancer does not metastasize to axillary nodes and bones as frequently as the non-triple-negative carcinomas, favouring instead, a preferentially haematogenous spread. Hypothetically, this particular tumour dissemination pattern would impair the reliability of using Nottingham Prognostic Index as a tool for triple-negative breast cancer prognostication. METHODS: The present study tested the effectiveness of the Nottingham Prognostic Index in stratifying breast cancer patients of different subtypes with special emphasis in a triple-negative breast cancer patient subset versus non- triple-negative breast cancer. RESULTS: We demonstrated that besides the fact that TNBC disseminate to axillary lymph nodes as frequently as luminal or HER2 tumours, we also showed that TNBC are larger in size compared with other subtypes and almost all grade 3. Additionally, survival curves demonstrated that these prognostic factors are equally important to stratify different survival outcomes in non-TNBC as in TNBC. We also showed that the NPI retains the ability to stratify and predict survival of TNBC patients. CONCLUSION: The importance of this study relies on the need of prognostication improvements on TNBC, showing, at a clinical standpoint, that Nottingham Prognostic Index is as a truthful prognostic tool in TNBC.

Microarrays in the 2010s: the contribution of microarray-based gene expression profiling to breast cancer classification, prognostication and prediction.

Breast cancer comprises a collection of diseases with distinctive clinical, histopathological, and molecular features. Importantly, tumors with similar histological features may display disparate clinical behaviors. Gene expression profiling using microarray technologies has improved our understanding of breast cancer biology and has led to the development of a breast cancer molecular taxonomy and of multigene 'signatures' to predict outcome and response to systemic therapies. The use of these prognostic and predictive signatures in routine clinical decision-making remains controversial. Here, we review the clinical relevance of microarray-based profiling of breast cancer and discuss its impact on patient management.

Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype.

BACKGROUND AND AIM: The study of CD44/CD24 and ALDH1 expression is the most accurate method to identify cancer stem cells (CSC) from breast cancer populations. However, the overlap between CD44(+)CD24(-/low) and ALDH1(high) CSC phenotypes in breast cancer seems to be very small, as well as their distribution among intrinsic breast cancer subtypes. Due to this discrepancy, it is imperative to improve the understanding of breast CSC marker distribution. METHODS: 466 invasive breast carcinomas and eight breast cancer cell lines were analysed for the expression of CD44, CD24 and ALDH1, to evaluate their distribution among the distinct molecular subtypes. RESULTS: Basal-like tumours (76.5%) contained the higher percentage of cells with the CSC phenotype CD44(+)CD24(-/low) (p<0.0001). From ALDH1-positive cases, 39.4% were also basal-like tumours (p<0.0001). The analysis of breast cancer cell lines indicated that luminal cell lines are mainly enriched in a CD44(-/low)CD24(+) cell population, basal/mesenchymal breast cancer cell lines are enriched in the CD44(+)CD24(-/low) phenotype, whereas the remaining basal/epithelial cell lines are mainly positive for both markers. ALDH1 activity was mainly found in HER-OE and basal/epithelial breast cancer cell. CONCLUSIONS: CD44(+)CD24(-/low) and ALDH1(+) phenotypes seem to identify CSC with distinct levels of differentiation. It seems that the paramount method and biomarkers that identify breast CSC within the distinct molecular subtypes need to be better explored, because it is pivotal to translate the CSC concept to clinical practice. In the future, the recognition of reliable markers to distinguish the CSC pool in each molecular subtype will be decisive for the development of specific target therapies.

Genomic analysis reveals the molecular heterogeneity of ovarian clear cell carcinomas.

PURPOSE: Ovarian clear cell carcinomas (OCCC) are a drug-resistant and aggressive type of epithelial ovarian cancer. We analyzed the molecular genetic profiles of OCCCs to determine whether distinct genomic subgroups of OCCCs exist. EXPERIMENTAL DESIGN: Fifty pure primary OCCCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). Unsupervised hierarchical clustering using Ward's linkage analysis was performed to identify genomic subgroups of OCCCs. Survival analysis was performed using Kaplan-Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. Differentially amplified regions between genomic subgroups of OCCCs were identified using a multi-Fisher's exact test. RESULTS: Hierarchical cluster analysis revealed two distinct clusters of OCCCs with different clinical outcomes. Patients from cluster-1 had a significantly shorter median progression-free survival (PFS) than those from cluster-2 (11 vs. 65 months, P = 0.009), although estimates for ovarian cancer-specific survival (OCS) did not reach statistical significance (P = 0.065). In multivariate analysis, suboptimal debulking surgery and genomic cluster were independently prognostic for PFS. Recurrently amplified genomic regions with a significantly higher prevalence in cluster-1 than cluster-2 OCCCs were identified and validated. HER2 gene amplification and protein overexpression was observed in 14% of OCCCs, suggesting that this may constitute a potential therapeutic target for a subgroup of these tumors. CONCLUSIONS: OCCCs constitute a heterogeneous disease at the genomic level despite having similar histological features. The pattern of genomic aberrations in subgroups of OCCCs is of clinical significance. We have identified recurrently amplified regions that may harbor potential therapeutic targets for subgroups of OCCCs.

Oncogenic mutations in gastric cancer with microsatellite instability.

AIM: Mitogen-activated protein kinase (MAPK) cascade and phosphatidylinositol 3-kinase (PI3K) survival pathways are frequently activated in the progression of gastrointestinal malignancies. In this study, we aimed to determine the frequency of gene mutations in members of these pathways--Epithelial Growth Factor Receptor (EGFR), KRAS, BRAF, PIK3CA and MLK3 in a series of 63 gastric carcinomas with high levels of microsatellite instability (MSI). METHODS: Gene mutation analysis was performed by PCR amplification followed by direct sequencing. In selected tumour cases, EGFR expression was evaluated by immunohistochemistry. Association studies between molecular data and clinicopathologic characteristics were performed. RESULTS: Mutations in EGFR (3'-untranslated region [UTR] polyA repeat), KRAS, PIK3CA and MLK3 genes occurred in 30 (47.6%), 11 (17.5%), 9 (14.3%) and 2 (3.2%) of the MSI gastric cancer (GC) cases, respectively. No BRAF or EGFR hotspot mutations were identified. Overall, mutations in at least one of these genes were found in 55.6% (35/63) of gastric carcinomas. From those mutant cases 40.0% (14/35) of them had concomitant gene mutations, always involving EGFR polyA deletions. Interestingly, we observed significant associations between oncogenic mutations and female gender (p = 0.046) old age of diagnosis (p = 0.001) and intestinal subtype (p = 0.043). CONCLUSION: Our results show that MSI gastric carcinoma frequently shows activation of EGFR-MAPK and PI3K pathways. Within all alterations found, deletions of the A13 repeats of EGFR were common, suggesting this molecular event as an important biomarker for stratification of GC patients for treatment with EGFR inhibitors.

MSI phenotype and MMR alterations in familial and sporadic gastric cancer.

Microsatellite instability (MSI) is a major pathway involved in gastric carcinogenesis occurring in 20% of gastric cancer (GC). However, it is not clear whether MSI phenotype preferentially occurs in the sporadic or familial GC, when stringent inclusion criteria are used. The aim of this study was to compare the frequency of MSI and hypermethylation of MLH1 promoter in a large series of familial GC patients (non-HNPCC and non-CDH1-related) and sporadic cases. Additionally, we analysed the immunoexpression of MMR proteins in a fraction of cases. Overall, the frequency of familial GC was 7.1%, and the frequency of hereditary tumours was 4.6%. MSI phenotype and MLH1 hypermethylation frequencies were not statistical different between familial and sporadic GC settings. Further, the MSI phenotype was not associated with any clinico-pathological features studied in the familial GC setting, whereas in the sporadic setting, it was associated with older age, female gender and intestinal histotype. Using our stringent Amsterdam-based clinical criteria to select familial GC (number of cases, age of onset), we verified that sporadic and familial cases differed in gender but shared histopathological features. We verified that the frequency of MSI was similar in familial and sporadic GC settings, demonstrating that this molecular phenotype is not a hallmark of familial GC in contrast to what is verified in HNPCC. Moreover, we observed that the frequency of MLH1 hypermethylation is similar in sporadic and familial cases suggesting that in both settings MSI is not associated to MMR genetic alterations but in contrast to epigenetic deregulation.