INK4a/Arf is required for suppression of EGFR/DeltaEGFR(2-7)-dependent ERK activation in mouse astrocytes and glioma.

Amplification of the epidermal growth factor receptor (EGFR) or expression of its constitutively activated mutant, DeltaEGFR(2-7), in association with the inactivation of the INK4a/Arf gene locus is a frequent alteration in human glioblastoma. The notion of a cooperative effect between these two alterations has been demonstrated in respective mouse brain tumor models including our own. Here, we investigated underlying molecular mechanisms in early passage cortical astrocytes deficient for p16(INK4a)/p19(Arf) or p53, respectively, with or without ectopic expression of DeltaEGFR(2-7). Targeting these cells with the specific EGFR inhibitor tyrphostin AG1478 revealed that phosphorylation of ERK was only abrogated in the presence of an intact INK4a/Arf gene locus. The sensitivity to inhibit ERK phosphorylation was independent of ectopic expression of DeltaEGFR(2-7) and independent of the TP53 status. This resistance to downregulate the MAPK pathway in the absence of INK4a/Arf was confirmed in cell lines derived from our mouse glioma models with the respective initial genetic alterations. Thus, deletion of INK4a/Arf appears to keep ERK in its active, phosphorylated state insensitive to an upstream inhibitor specifically targeting EGFR/DeltaEGFR(2-7). This resistance may contribute to the cooperative tumorigenic effect selected for in human glioblastoma that may be of crucial clinical relevance for treatments specifically targeting EGFR/DeltaEGFR(2-7) in glioblastoma patients.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Efficacy of cetuximab in metastatic castration-resistant prostate cancer might depend on EGFR and PTEN expression: results from a phase II trial (SAKK 08/07).

PURPOSE: The EGF receptor (EGFR) is overexpressed in the majority of metastatic castration-resistant prostate cancers (mCRPC) and might represent a valid therapeutic target. The combination of docetaxel and cetuximab, the monoclonal antibody against EGFR, has not been tested in patients with prostate cancer. EXPERIMENTAL DESIGN: Patients with mCRPC progressing during or within 90 days after at least 12 weeks of docetaxel were included in this phase II trial. Treatment consisted of docetaxel (75 mg/m(2) every 3 weeks or 35 mg/m(2) on days 1, 8, 15 every 4 weeks) in combination with cetuximab (400 mg/m(2) on day 1 and then 250 mg/m(2) weekly). The primary endpoint was progression-free survival (PFS) at 12 weeks defined as the absence of prostate-specific antigen (PSA), radiographic, or clinical progression. Evaluation of known biomarkers of response and resistance to cetuximab (EGFR, PTEN, amphiregulin, epiregulin) was conducted. RESULTS: Thirty-eight patients were enrolled at 15 Swiss centers. Median age was 68 years and median PSA was 212 ng/mL. PFS at 12 weeks was 34% [95% confidence interval (CI), 19%-52%], PFS at 24 weeks was 20%, and median overall survival (OS) was 13.3 months (95% CI, 7.3-15.4). Seven patients (20%) had a confirmed ≥ 50% and 11 patients (31%) a confirmed ≥ 30% PSA decline. About 47% of enrolled patients experienced grade 3 and 8% grade 4 toxicities. A significantly improved PFS was found in patients with overexpression of EGFR and persistent activity of PTEN. CONCLUSIONS: EGFR inhibition with cetuximab might improve the outcome of patients with mCRPC. A potential correlation between EGFR overexpression, persistent expression of PTEN, and EGFR inhibition should be investigated further.

Quiste dentígero asociado con mesiodens: Exposición de un caso, revisión de la literatura y diagnóstico diferencial.

Dentigerous cyst is one of the most prevalent types of odontogenic cyst and is associated with crown of an unerupted or developing tooth. Dentigerous cysts associated with supernumerary teeth are rare and estimated to constitute 5-6% of all dentigerous cysts. The vast majority, about 90%, are associated with a maxillary mesiodens. The purpose of this article to report the case of an 14-year-old a teenager with a dentigerous cyst associated with an impacted anterior maxillary supernumerary tooth, its dental management and literature review.

Validated SNPs for eGFR and their associations with albuminuria.

Albuminuria and reduced glomerular filtration rate are manifestations of chronic kidney disease (CKD) that predict end-stage renal disease, acute kidney injury, cardiovascular disease and death. We hypothesized that SNPs identified in association with the estimated glomerular filtration rate (eGFR) would also be associated with albuminuria. Within the CKDGen Consortium cohort (n= 31 580, European ancestry), we tested 16 eGFR-associated SNPs for association with the urinary albumin-to-creatinine ratio (UACR) and albuminuria [UACR >25 mg/g (women); 17 mg/g (men)]. In parallel, within the CARe Renal Consortium (n= 5569, African ancestry), we tested seven eGFR-associated SNPs for association with the UACR. We used a Bonferroni-corrected P-value of 0.003 (0.05/16) in CKDGen and 0.007 (0.05/7) in CARe. We also assessed whether the 16 eGFR SNPs were associated with the UACR in aggregate using a beta-weighted genotype score. In the CKDGen Consortium, the minor A allele of rs17319721 in the SHROOM3 gene, known to be associated with a lower eGFR, was associated with lower ln(UACR) levels (beta = -0.034, P-value = 0.0002). No additional eGFR-associated SNPs met the Bonferroni-corrected P-value threshold of 0.003 for either UACR or albuminuria. In the CARe Renal Consortium, there were no associations between SNPs and UACR with a P< 0.007. Although we found the genotype score to be associated with albuminuria (P= 0.0006), this result was driven almost entirely by the known SHROOM3 variant, rs17319721. Removal of rs17319721 resulted in a P-value 0.03, indicating a weak residual aggregate signal. No alleles, previously demonstrated to be associated with a lower eGFR, were associated with the UACR or albuminuria, suggesting that there may be distinct genetic components for these traits.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS Treatment: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer.

Circulating tumor cells (CTCs) are frequently associated with epithelial-mesenchymal transition (EMT).The objective of this study was to detect EMT phenotype through Vimentin (VIM) and Slug expression in cytokeratin (CK)-negative CTCs in non-metastatic breast cancer patients and to determine the importance of EGFR in the EMT phenomenon. In CK-negative CTCs samples, both VIM and Slug markers were co-expressed in the most of patients. Among patients EGFR+, half of them were positive for these EMT markers. Furthermore, after a systemic treatment 68% of patients switched from CK- to CK+ CTCs. In our experimental model we found that activation of EGFR signaling by its ligand on MCF-7 cells is sufficient to increase EMT phenotypes, to inhibit apoptotic events and to induce the loss of CK expression. The simultaneous detection of both EGFR and EMT markers in CTCs may improve prognostic or predictive information in patients with operable breast cancer.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND: Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS: TREATMENT: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS: Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION: First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

A dramatic lung cancer course in a patient with a rare EGFR germline mutation exon 21 V843I: Is EGFR TKI resistance predictable?

We report on the medical history of a Caucasian smoker woman diagnosed with a stage IV NSCLC adenocarcinoma, characterized by a rare epidermal growth factor receptor (EGFR) point mutation in exon 21 codon 843 (p.V843I/c.2527G>A/COSMIC ID 85894). This genetic alteration revealed to be germline, after its presence was demonstrated in chondroblasts from the bone biopsy. While it is the first description of germline V843I mutation without concomitant additional known EGFR activating mutation, we modeled the EGFR ATP catalytic domain in complex with ATP, gefitinib and erlotinib using computer-aided approaches to estimate possible changes in affinity upon the V843I mutation.

Prospective analysis of KRAS, BRAF, and PIK3CA mutations and EGFR copy number in patients (pts) with locally advanced rectal cancer: A translational substudy of a clinical trial (SAKK 41/07) evaluating the effect of neoadjuvant chemoradiation (CRT) with or without panitumumab

Background: Prognostic and predictive markers are of great importance for future study designs and essential for the interpretation of clinical trials incorporating an EGFR-inhibitor. The current study prospectively assessed and validated KRAS, BRAF and PIK3CA mutations in rectal cancer patients screened for the trial SAKK41/07 of concomitant preoperative radio-chemotherapy with or without panitumumab.Methods: Macrodissection was performed on pretreatment formalin fixed paraffin embedded biopsy tissue sections to arrive at a minimum of 50% of tumor cells. DNA was extracted with the Maxwell 16 FFPE Tissue LEV DNA purification kit. After PCR amplification, mutations were identified by pyrosequencing. We prospectively analysed pretreatment biopsy material from 149 rectal cancer pts biopsies for KRAS (exon 2 codon 12 [2-12] and 13 [2-13], exon 3 codon 59 [3-59]) and 61 [3-61], exon 4 codon 117 [4-117] and 146 [4-146]). Sixty-eight pts (KRASwt exon 2, 3 only) were further analysed for BRAF (exon 15 codon 600) and PIK3CA (exon 9 codon 542, 545 and 546, exon 20 codon 1043 [20-1043] and 1047 [20-1047]) mutations, and EGFR copy number by qPCR. For the calculation of the EGFR copy number, we used KRAS copy number as internal reference standard. The calculation was done on the basis of the two standard curves relative quantification method.Results: In 149 screened pts with rectal cancer, the prevalence of KRAS mutations was 36%. Among the 68 pts enrolled in SAKK 41/07 based on initially presumed KRASwt status (exon 2/codons 12+13), 18 pts (26%) had a total of 23 mutations in the RAS/PIK3CA-pathways upon validation analysis. Twelve pts had a KRAS mutation, 7 pts had a PIK3CA mutation, 3 pts had a NRAS mutation, 1 patient a BRAF mutation. Surprisingly, five of these pts had double- mutations, including 4 pts with KRAS plus PIK3CA mutations, and 1 pt with NRAS plus PIK3CA mutations. The median normalized EGFR copy number was 1. Neither mutations of KRAS, BRAF, and PIK3CA, nor EGFR copy number were statistically associated with the primary study endpoint pCR (pathological complete regression).Conclusions: The prevalence of KRAS mutations in rectal and in colon cancer appears to be similar. BRAF mutations are rare; PIK3CA mutations are more common (10%). EGFR copy number is not increased in rectal cancer. A considerable number or KRAS exon 2 wt tumors harbored KRAS exon 3+4 mutations. Further study is needed to determine if KRAS testing should include exons 2-4.

Tolerância de genótipos de cafeeiro ao alumínio em solução nutritiva. I. Crescimento e desenvolvimento da parte aérea e sistema radicular

Para estudar a influência do alumínio no crescimento e desenvolvimento de nove genótipos de café, foi instalado um experimento, em janeiro de 1994, em casa de vegetação do Departamento de Fitotecnia da Universidade Federal de Viçosa, situada na Zona da Mata do Estado de Minas Gerais, a uma altitude média de 651 metros. Para tanto, plantas com dois pares de folhas definitivas foram submetidas a 0 e 0,296 mmol L-1 de alumínio em solução nutritiva, com pH 4,0, durante 115 dias. Após este período, as plantas foram divididas em folhas superiores, folhas inferiores, primeiro par de folhas totalmente expandido, caule e raízes, para a determinação da matéria seca. Avaliaram-se, também, altura das plantas, comprimento da raiz principal, número de raízes secundárias e área foliar do primeiro par de folhas totalmente expandido. A presença do alumínio inibiu tanto o crescimento da parte aérea como das raízes, as quais apresentaram anormalidades típicas de toxidez de alumínio. A redução na matéria seca de raízes foi a característica que permitiu melhor discriminação quanto à tolerância ao alumínio entre os genótipos estudados. Observou-se redução no comprimento da raiz principal, na altura das plantas e na área foliar, bem como aumento no número de raízes secundárias em resposta a aumentos das concentrações de Al na solução nutritiva. As características de crescimento avaliadas permitiram discriminar os genótipos em quatro grupos ou categorias: tolerante (UFV 1359, UFV 2149), moderadamente tolerante (UFV 2145, UFV 2877 e UFV 2163), moderadamente sensível (UFV 3880) e sensível (UFV 2147, UFV 2198 e UFV 2237).

Influência da estrutura do solo na distribuição e na morfologia do sistema radicular do milho sob três métodos de preparo

O estudo da distribuição de raízes no solo é um método adequado para se detectarem as condições adversas ou não ao seu desenvolvimento, bem como para avaliar o efeito das alterações introduzidas por sistemas de preparo do solo. Sendo assim, o objetivo deste trabalho foi avaliar o efeito de métodos de preparo sobre determinadas características do solo e, por conseguinte, sobre o desenvolvimento do sistema radicular do milho. Para tal, foi utilizado um experimento de campo, instalado em um solo Podzólico Vermelho-Escuro com cinco anos de utilização por preparo convencional, reduzido e semeadura direta, na sucessão aveia + trevo/milho. Para observar as modificações estruturais do solo, usou-se o método do perfil cultural adaptado, acompanhado por determinações de densidade do solo, porosidade total, macro e microporosidade e resistência ao penetrômetro. Os métodos da parede do perfil e do monolito (prancha com pregos) foram utilizados para determinar a distribuição e morfologia das raízes. Nos mapas estruturais descritos, observou-se que os modos de organização provocados pelos tratamentos explicaram parcialmente a presença das raízes no perfil. As plantas submetidas à semeadura direta, quando comparadas às do preparo convencional, tiveram o crescimento inicial das raízes diferindo do padrão diagonal normal e, ao final do ciclo, apresentaram raízes com maior raio médio nas profundidades de 10-15 e 25-35 cm e mostraram a densidade de comprimento (cm cm-3) maior na camada superficial (0-5 cm) e menor na camada de 10-15 cm. Os sistemas de preparo não afetaram a massa seca de raízes e o rendimento dos grãos de milho.

Atributos físicos do solo e crescimento radicular de soja em latossolo roxo sob diferentes métodos de preparo do solo

A presença de camadas compactadas no solo, resultantes das operações de preparo, pode causar restrição ao crescimento radicular e, conseqüentemente, reduzir a produção. A determinação de atributos físicos do solo, entre eles a densidade e a resistência, tem sido utilizada para avaliar a compactação, visando a recomendações de manejo. Neste estudo, procurou-se determinar a relação entre esses atributos e o crescimento radicular, em um ensaio com cinco sistemas de preparo de solo e cultura de soja (Glycine max L.), localizado no município de Tarumã (SP), em Latossolo Roxo muito argiloso. As avaliações foram realizadas em janeiro de 1993, sete anos após o início do ensaio. Os sistemas de preparo combinaram preparos de verão e de inverno com grade pesada, arado escarificador e semeadura direta. O tratamento com semeadura direta, no verão e no inverno, apresentou valores elevados de resistência e densidade desde a superfície até 0,3 m, enquanto o tratamento com grade pesada apresentou valores elevados para esses parâmetros entre 0,1 e 0,2 m de profundidade, caracterizando camadas compactadas. Nos tratamentos com escarificação no verão, a resistência e a densidade do solo foram menores, com pequena diferença entre as profundidades analisadas. A semeadura direta apresentou maior densidade de raízes, seguida dos tratamentos com escarificação. Cerca de 50% do sistema radicular ficou concentrado na camada superficial do solo no tratamento com grade pesada, 40% no tratamento com semeadura direta e 30% com escarificador. As análises de regressão e correlação indicaram que, com o aumento da densidade do solo, a resistência e a umidade do solo foram maiores e, em decorrência dos valores obtidos no sistema de semeadura direta, a densidade radicular, nessas condições, também foi maior. A relação entre os parâmetros avaliados indicou que o sistema radicular foi reduzido em profundidade, quando a densidade e resistência foram elevadas na camada de 0,1-0,2 m ou quando foi alto o gradiente de densidade e resistência entre as camadas de 0,1-0,2 e 0-0,1 m. Os valores de resistência e densidade de um solo, tomados isoladamente, não puderam ser considerados como indicadores do crescimento de raízes.

Crescimento aéreo e radicular da soja em solos compactados e inoculados com estirpes de Bradyrhizobium japonicum

Um experimento com soja, utilizando amostras de um Latossolo Vermelho-Escuro álico textura média foi realizado em casa de vegetação da Universidade Federal de Uberlândia (MG), de julho/96 a fevereiro/97, visando avaliar o comportamento de estirpes de Bradyrhizobium japonicum inoculadas e nativas do solo e efeitos de níveis de compactação subsuperficial na produção de matéria seca da parte aérea (PMSPA), de raízes no anel superior da coluna e na absorção e concentração de nitrogênio na parte aérea da soja. Os tratamentos dispostos em fatorial 2 x 3 x 4 corresponderam à brometização e não-brometização do solo, presença de três estirpes de Bradyrhizobium japonicum (nativa do solo, SEMIA 5079 e SEMIA 5080 inoculadas na semente) e de quatro níveis de compactação (densidades de: 1,15; 1,30; 1,45 e 1,60 kg dm-3), aplicados com prensa hidráulica no solo do anel central de uma coluna formada pela sobreposição de três anéis de PVC de 15 cm de diâmetro. Quarenta e sete dias após a emergência, as plantas foram cortadas, secas e pesadas, e as raízes do anel superior coletadas e quantificadas. A PMSPA foi crescente com a brometização do solo e inoculação com a estirpe SEMIA 5080, dentro das densidades de até 1,30 kg dm-3. Para valores maiores do que este, as estirpes estudadas não diferiram entre si. A compactação promoveu reduções lineares na produção de matéria seca da parte aérea no solo não-brometizado e parabólico, e maior produção na densidade de 1,35 kg dm-3 no solo brometizado. A concentração de nitrogênio na parte aérea foi significativamen-te menor com a inoculação da estirpe SEMIA 5079 no solo brometizado e não diferiu entre si no solo não brometizado. Maior produção de raízes na camada superficial ocorreu na ausência de brometização do solo e de inoculação com Bradyrhizobium japonicum. Com a brometização, a maior produção ocorreu com a inoculação da estirpe SEMIA 5079. A compactação produziu efeito linear crescente na produção de raízes no solo não brometizado e parabólico, com maior produção na densidade de 1,43 kg dm-3 no solo brometizado.