Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-ß and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-ß and Daxx-γ show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-ß and Daxx-γ display a distinct distribution pattern. Furthermore, Daxx-ß and Daxx-γ show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-ß/-γ to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-ß and Daxx-γ are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.

EGFR-specific T cell frequencies correlate with EGFR expression in head and neck squamous cell carcinoma.

BACKGROUND: In head and neck squamous cell carcinoma (HNSCC), expression levels of the epidermal growth factor receptor (EGFR) correlate with poor prognosis and decreased survival rates. As the mechanisms responsible for cellular immune response to EGFR in vivo remain unclear, the frequency and function of EGFR-specific cytotoxic T cells (CTL) was determined in HNSCC patients. METHODS: The frequency of CTL specific for the HLA-A2.1-restricted EGFR-derived YLN peptide (YLNTVQPTCV) and KLF peptide (KLFGTSGQKT) was determined in 16 HLA-A2.1+ HNSCC patients and 16 healthy HLA-A2.1+ individuals (NC) by multicolor flow cytometry. Patients' results were correlated to EGFR expression obtained by immunohistochemistry in corresponding tumor sections. Proliferation and anti-tumor activity of peptide-specific CTL was demonstrated by in vitro stimulation with dendritic cells pulsed with the peptides. RESULTS: Frequency of EGFR-specific CTL correlated significantly with EGFR expression in tumor sections (p = 0.02, r2 = 0.6). Patients with elevated EGFR scores (> 7) had a significantly higher frequency of EGFR-specific CTL than NC and patients with low EGFR scores (< 7). EGFR-specific CTL from cancer patients were expanded ex vivo and produced IFN-γ upon recognition of EGFR+ target cells. CONCLUSION: EGFR expressed on HNSCC cells induces a specific immune response in vivo. Strategies for expansion of EGFR-specific CTL may be important for future immunotherapy of HNSCC patients.

Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines.

Many metastatic cancers recapitulate the epithelial-to-mesenchymal transition (EMT) resulting in enhanced cell motility and invasiveness. The EMT is regulated by several transcription factors, including the zinc finger protein SNAI2, also named Slug, which appears to exert additional functions during development and cancer progression. We have studied the function of SNAI2 in prostate cancer cells. Quantitative RT-PCR analysis showed strong SNAI2 expression particularly in the PC-3 and PC3-16 prostate carcinoma cell lines. Knockdown of SNAI2 by specific siRNA induced changes in EMT markers and inhibited invasion of both cell lines into a matrigel matrix. SNAI2 siRNA-treated cells did not tolerate detachment from the culture plates, likely at least in part due to downregulation of integrin alpha6beta4. SNAI2 knockdown disturbed the microtubular and actin cytoskeletons, especially severely in PC-3 cells, resulting in grossly enlarged, flattened, and sometimes multinuclear cells. Knockdown also decreased cell proliferation, with a prominent G0/G1 arrest in PC3-16. Together, our data imply that SNAI2 exerts strong effects on the cytoskeleton and adhesion of those prostate cancer cells that express it and is necessary for their proliferation and invasiveness.

Changes in cortical cytoskeletal and extracellular matrix gene expression in prostate cancer are related to oncogenic ERG deregulation.

BACKGROUND: The cortical cytoskeleton network connects the actin cytoskeleton to various membrane proteins, influencing cell adhesion, polarity, migration and response to extracellular signals. Previous studies have suggested changes in the expression of specific components in prostate cancer, especially of 4.1 proteins (encoded by EPB41 genes) which form nodes in this network. METHODS: Expression of EPB41L1, EPB41L2, EPB41L3 (protein: 4.1B), EPB41L4B (EHM2), EPB41L5, EPB49 (dematin), VIL2 (ezrin), and DLG1 (summarized as "cortical cytoskeleton" genes) as well as ERG was measured by quantitative RT-PCR in a well-characterized set of 45 M0 prostate adenocarcinoma and 13 benign tissues. Hypermethylation of EPB41L3 and GSTP1 was compared in 93 cancer tissues by methylation-specific PCR. Expression of 4.1B was further studied by immunohistochemistry. RESULTS: EPB41L1 and EPB41L3 were significantly downregulated and EPB41L4B was upregulated in cancer tissues. Low EPB41L1 or high EPB41L4B expression were associated with earlier biochemical recurrence. None of the other cortical cytoskeleton genes displayed expression changes, in particular EPB49 and VIL2, despite hints from previous studies. EPB41L3 downregulation was significantly associated with hypermethylation of its promoter and strongly correlated with GSTP1 hypermethylation. Protein 4.1B was detected most strongly in the basal cells of normal prostate epithelia. Its expression in carcinoma cells was similar to the weaker one in normal luminal cells. EPB41L3 downregulation and EPB41L4B upregulation were essentially restricted to the 22 cases with ERG overexpression. Expression changes in EPB41L3 and EPB41L4B closely paralleled those previously observed for the extracellular matrix genes FBLN1 and SPOCK1, respectively. CONCLUSIONS: Specific changes in the cortical cytoskeleton were observed during prostate cancer progression. They parallel changes in the expression of extracellular matrix components and all together appear to be associated with oncogenic ERG overexpression. We hypothesize that these alterations may contribute to the increased invasivity conferred to prostate cancer cells by ERG deregulation.

Squamous cell carcinoma arising from a localized vulval lesion of Hailey-Hailey disease after tacrolimus therapy.

Hailey-Hailey disease (HHD) is a rare, autosomal dominant intraepidermal blistering disorder characterized by recurrent vesicles and erosions affecting mostly the intertriginous areas. We report a case of HHD affecting exclusively the vulva from which an invasive squamous cell carcinoma developed after tacrolimus therapy.

Expression profile of the polycomb group protein enhancer of Zeste homologue 2 and its prognostic relevance in renal cell carcinoma.

PURPOSE: EZH2 increases the proliferation rate and apoptosis resistance of renal cell carcinoma cell lines. To date its clinical impact on the outcome in patients with renal cell carcinoma is not known. To our knowledge this is the first study of the association of EZH2 expression with histopathological features and disease outcomes in a large cohort of patients who underwent surgery for renal cell carcinoma. MATERIALS AND METHODS: Real-time reverse transcriptase-polymerase chain reaction was done to quantify EZH2 expression in malignant and adjacent benign renal tissue from a cohort of 119 patients with clear cell renal cell carcinoma. Median followup was 51 months. Immunohistochemistry was performed in a subset of samples. The impact of EZH2 expression on clinicopathological tumor features and outcome was investigated. RESULTS: EZH2 was over expressed in renal cell carcinoma compared to adjacent benign renal parenchyma (median 57.02, range 0 to 368.11 vs 0, range 0 to 280.87, p <0.001). Immunohistochemistry showed concordant results and revealed EZH2 protein predominantly located in the nucleus. EZH2 expression was not associated with histopathological tumor features and patient characteristics. High EZH2 levels predicted a lower disease recurrence rate on univariate and multivariate analysis (p = 0.047 and 0.037, respectively). CONCLUSIONS: These data support a role of EZH2 expression for renal cell carcinoma tumorigenesis rather than tumor progression. Contrary to previous EZH2 findings in cases of various human malignancies, high tumor EZH2 levels appear to indicate less aggressive tumor phenotypes with a favorable prognosis in renal cell carcinoma cases. Our findings suggest that EZH2 provides not only a potential therapeutic target, but also a molecular parameter for outcome prediction in patients with renal cell carcinoma.

Malignant advanced granulosa cell tumor of the adult testis: case report and review of the literature.

Testicular granulosa cell tumors (GCTs) are very rare neoplasms. Although adult GCTs are thought to have a relatively indolent course, several reports have demonstrated the malignant potential of these lesions. In case of distant metastases, the overall survival is very short. To date, there is no well-established treatment for these tumors owing to poor results and very rapid progression. A 55-year-old male patient was diagnosed with a testicular GCT with distant lung metastases. He underwent surgical treatment with orchiectomy and adjuvant polychemotherapy (cisplantine, etoposide, and bleomycine) as well as metastasectomy of the right lung. We report the first case of a successfully treated testicular GCT with bipulmonary metastases at initial diagnosis. Thirty-nine months after treatment, the patient is alive with no evidence of disease. We subsequently reviewed all reported cases of an adult GCT in the published literature (25 published cases). This review will summarize all reported cases and discuss treatment options. The current case suggests that a combination of varying treatment modalities could be a promising and reasonable way to manage malignant advanced GCT of the adult testis.

Characteristics of testicular dysgenesis syndrome and decreased expression of SRY and SOX9 in Frasier syndrome.

Frasier syndrome (FS) is characterized by chronic renal failure in early adulthood, varying degrees of gonadal dysgenesis, and a high risk for gonadal germ cell malignancies, particularly gonadoblastoma. Although it is known to arise from heterozygous splice mutations in intron 9 of the Wilms' tumor gene 1 (WT1), the mechanisms by which these mutations result in gonadal dysgenesis in humans remain obscure. Here we show that a decrease in WT1 + KTS isoforms due to disruption of alternative splicing of the WT1 gene in a FS patient is associated with diminished expression of the transcription factors SRY and SOX9 in Sertoli cells. These findings provide the first confirmation in humans of the results obtained by others in mice. Consequently, Sertoli cells fail to form the specialized environment within the seminiferous tubules that normally houses developing germ cells. Thus, germ cells are unable to fully mature and are blocked at the spermatogonial-spermatocyte stage. Concomitantly, subpopulations of the malignant counterpart of primordial germ cells/gonocytes, the intratubular germ cell neoplasia unclassified type (ITGCN), are identified. Furthermore, dysregulated Leydig cells produce insufficient levels of testosterone, resulting in hypospadias. Collectively, the impaired spermatogenesis, hypospadias and ITGCN comprise part of the developmental disorder known as 'testicular dysgenesis syndrome' (TDS), which arises during early fetal life. The data presented here show that critical levels of WT1 + KTS, SRY and SOX9 are required for normal Sertoli cell maturation, and subsequent normal spermatogenesis. To further study the function of human Sertoli cells in the future, we have established a human cell line.

[Radiotherapy of soft tissue sarcoma--part of a multidisciplinary strategy]. / Radiotherapie der Weichteilsarkome--Teil einer multidisziplinären Strategie.

The management of soft tissue sarcoma has evolved from a solitary surgical treatment to an interdisciplinary multimodal approach including radiotherapy. These fundamental changes are the result of increased knowledge in tumor biology, radiation sensitivity and the improvement in modern radiation therapy techniques. A successful effective therapy regimen strongly depends on distinct preoperative diagnostics, preoperative conception of the surgical intervention and an experienced oncological team. Of significant importance for the prognosis is early diagnosis as well as tumor excision with a wide negative margin. However, even after complete wide resection in sano, the use of postoperative radiotherapy can further improve local control and should therefore be applied to the majority of patients. Consequently, radiotherapy should only be omitted in cases in which the tumor has been excised with a very wide negative margin; this implies, however, high quality of surgery and distinct histopathological analysis. Patients with non- or questionable resectable tumors, should be referred for pre-operative radiotherapy in order to improve the surgical results. Recent studies have underlined the efficiency of modern radiotherapy regimens. The different radiotherapy regimens will be highlighted against the background of tumor stage and tumor resectibility.

Factor interaction analysis for chromosome 8 and DNA methylation alterations highlights innate immune response suppression and cytoskeletal changes in prostate cancer.

BACKGROUND: Alterations of chromosome 8 and hypomethylation of LINE-1 retrotransposons are common alterations in advanced prostate carcinoma. In a former study including many metastatic cases, they strongly correlated with each other. To elucidate a possible interaction between the two alterations, we investigated their relationship in less advanced prostate cancers. RESULTS: In 50 primary tumor tissues, no correlation was observed between chromosome 8 alterations determined by comparative genomic hybridization and LINE-1 hypomethylation measured by Southern blot hybridization. The discrepancy towards the former study, which had been dominated by advanced stage cases, suggests that both alterations converge and interact during prostate cancer progression. Therefore, interaction analysis was performed on microarray-based expression profiles of cancers harboring both alterations, only one, or none. Application of a novel bioinformatic method identified Gene Ontology (GO) groups related to innate immunity, cytoskeletal organization and cell adhesion as common targets of both alterations. Many genes targeted by their interaction were involved in type I and II interferon signaling and several were functionally related to hereditary prostate cancer genes. In addition, the interaction appeared to influence a switch in the expression pattern of EPB41L genes encoding 4.1 cytoskeleton proteins. Real-time RT-PCR revealed GADD45A, MX1, EPB41L3/DAL1, and FBLN1 as generally downregulated in prostate cancer, whereas HOXB13 and EPB41L4B were upregulated. TLR3 was downregulated in a subset of the cases and associated with recurrence. Downregulation of EPB41L3, but not of GADD45A, was associated with promoter hypermethylation, which was detected in 79% of carcinoma samples. CONCLUSION: Alterations of chromosome 8 and DNA hypomethylation in prostate cancer probably do not cause each other, but converge during progression. The present analysis implicates their interaction in innate immune response suppression and cytoskeletal changes during prostate cancer progression. The study thus highlights novel mechanisms in prostate cancer progression and identifies novel candidate genes for diagnostic and therapeutic purposes. In particular, TLR3 expression might be useful for prostate cancer prognosis and EPB41L3 hypermethylation for its detection.

Genomic and expression analysis of the 3q25-q26 amplification unit reveals TLOC1/SEC62 as a probable target gene in prostate cancer.

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined.

Rac upregulates tissue inhibitor of metalloproteinase-1 expression by redox-dependent activation of extracellular signal-regulated kinase signaling.

The Rho-like GTPase Rac regulates distinct actin cytoskeleton changes required for adhesion, migration and invasion of cells. Tiam1 specifically activates Rac, and Rac has been shown to affect several signaling pathways in a partly cell-type-specific manner. Recently, we demonstrated that Rac activation inhibits Matrigel invasion of human carcinoma cells by transcriptional upregulation of tissue inhibitor of metalloproteinase-1. The purpose of the present study was to identify key mediators of Tiam1/Rac-induced tissue inhibitor of metalloproteinase-1 expression. Mutational analysis of the human tissue inhibitor of metalloproteinase-1 promoter revealed a major role for a distinct activating protein-1 site at -92/-86 and a minor role for an adjacent polyoma enhancer A3 site. Moreover, Rac activation induced the generation of reactive oxygen species and subsequent reactive oxygen species-dependent activation of extracellular signal-regulated kinase 1,2. In contrast, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activities were not affected. In line with this, Tiam1/Rac-induced tissue inhibitor of metalloproteinase-1 expression as well as Tiam1/Rac-induced binding of nuclear extracts to the activating protein-1 site at -92/-86 were inhibited by catalase and by specific inhibitors of the extracellular signal-related kinase-1,2 activators, mitogen-activated protein kinase kinase-1 and mitogen-activated protein kinase kinase-2 (PD098059, U0126). In conclusion, Rac-induced transcriptional upregulation of tissue inhibitor of metalloproteinase-1 is mediated by reactive oxygen species-dependent activation of extracellular signal-related kinase-1,2 and by transcription factors of the activating protein-1 family.

Epigenetic control of CTCFL/BORIS and OCT4 expression in urogenital malignancies.

Aberrant hypomethylation in many cancers reactivates retrotransposons and selected single-copy genes such as cancer-testis antigens. Genes reactivated in this manner have recently been postulated to include CTCFL/BORIS, a presumptive testis-specific chromatin regulator, and OCT4/POU5F1, a transcriptional activator in pluripotent cells. We found both genes expressed at high levels in testis and at much lower levels in normal prostate tissue. In prostate and bladder carcinoma cell lines and cancer tissues expression remained largely unchanged, but individual prostate carcinomas showed modestly increased CTCFL expression compared to normal tissues. OCT4 expression was significantly decreased in cancer tissues. Promoter methylation in both genes paralleled expression levels. CTCFL, but not OCT4 was dramatically induced in cancer cell lines by 5-aza-2'-deoxycytidine, but neither gene by the histone deacetylase inhibitor suberoylanilide hydroxamic acid. Thus, CTCFL and OCT4 resemble cancer-testis antigens in being selectively hypomethylated and expressed in male germ cells but differ in lacking significant reexpression and hypomethylation in prostate carcinomas. DNA methylation appears the crucial mechanism in the control of CTCFL transcription, but less decisive in that of OCT4. These findings imply that inhibitors of DNA methylation used for cancer treatment may induce CTCFL expression. Immunohistochemistry demonstrated nuclear localization of CTCFL in developing spermatocytes, and cytoplasmatic localization in spermatogonia, Leydig cells, and epithelial prostate cells. Teratocarcinoma cell lines showed nuclear, and 5-aza-2'-deoxycytidine-treated prostate cancer lines nuclear or cytoplasmatic localization. These different localizations might indicate additional control of CTCFL function via intracellular compartmentation.

Hip arthroscopy for excision of osteoid osteoma and for the application of a collagen cartilage implant: Case report in a professional athlete, and literature review.

BACKGROUND: Osteoid osteoma is an osteoblastic benign bone tumor usually affecting young adolescents. Intra-articular lesions are not common; usually the diagnosis is delayed. A lot of studies report difficulties and complications in the management of osteoid osteoma of the hip joint using imaging guided techniques or open surgical procedures. Only few published cases have described that it can be treated precisely using hip arthroscopy. Additionally, the use of hip arthroscopy to apply the Chondrofiller®, an acellular collagen matrix for the management of articular cartilage defects of the hip joint, has not yet been described. CASE PRESENTATION: This report presents an osteoid osteoma of the femoral neck. A 20-year-old female professional basketball player presented with pain in the left groin since more than 12 months. On magnetic resonance and computed tomography imaging, an osteoid osteoma was suggested. The lesion was successfully removed using arthroscopy. During surgery, a concomitant grade 4 cartilage lesion on the femoral head was detected. For the treatment of this severe defect we used the Chondrofiller®, which is a new acellular collagen implant for auto-regeneration of articular cartilage. This matrix was filled into the prepared and dried defect using CO2 arthroscopy. After the hardening of the matrix the surgery was finished. The patient was pain free shortly after the operation and returned to sports within 16 weeks. Return to high-performance sports 8 months after surgery was without of any sign of complaints. CONCLUSIONS: This article demonstrates that hip arthroscopy is a valuable tool for biopsy and excision of intra-articular osteoid osteoma affecting the hip joint, as well as for addressing other concomitant pathologies such as a severe synovitis or cartilage defects. CO2 arthroscopy provided good conditions for the drying and filling of the cartilage defect with the Chondrofiller®.

Hypomethylation of the XIST gene promoter in prostate cancer.

In a process denoted "global hypomethylation" repetitive DNA sequences like LINE-1 retrotransposons become hypomethylated in human cancers, including a subset of prostate carcinomas. It is less well known to what extent single-copy sequences are affected by this phenomenon. Therefore, we have analyzed methylation and expression of the XIST gene by bisulfite sequencing and real-time RT-PCR. The promoter of this single-copy gene is strongly methylated in normal male cells, including leukocytes and normal prostate. In prostate cancer tissues and particularly in cell lines, partial hypomethylation was observed paralleling that of LINE-1 sequences. Weak XIST expression was found in normal prostate tissues, but none in leukocytes. Only slight increases in expression of this gene were found in cancer tissues and cell lines. Our data suggest that hypomethylation in prostate cancer is indeed "global," affecting repeat and unique sequences in parallel. Detection of partially hypomethylated XIST alleles in prostate cancer tissues might be useful for the identification of cases with pronounced hypomethylation, which tend to be more aggressive.

Epigenetic inactivation of the placentally imprinted tumor suppressor gene TFPI2 in prostate carcinoma.

BACKGROUND: Imprinted genes are often arranged in clusters epigenetically controlled by differentially methylated regions (DMR) containing bivalent histone modifications. Both DNA hypermethylation and hypomethylation in cancer can therefore disturb imprinted gene expression. We have studied expression, DNA methylation and histone modifications of TFPI2, a presumed tumor suppressor, and that of other genes in the 7q21 imprinted gene cluster in prostate cancer. MATERIALS AND METHODS: TFPI, TFPI2, SGCE and PON2 expression were assessed by qRT-PCR in prostate cancer tissues and cell lines. DNA methylation and histone modifications were investigated by bisulphite sequencing and chromatin immunoprecipatation. RESULTS: TFPI2 was highly variably expressed in cancer tissues, in contrast to TFPI, and did not correlate to unchanged SGCE and significantly elevated PON2 expression. TFPI2 expression variations were unrelated to global DNA hypomethylation, but were associated with promoter methylation. PC3 cells with high expression retained normal methylation and bivalent histone modifications at DMR and promoter, whereas low-expressing LNCaP cells presented aberrant DNA methylation and more repressive histone modifications. CONCLUSION: Epigenetic disturbances in the 7q21 cluster affect imprinted genes in a non-coordinate manner suggesting an unstable epigenetic state prone to selection for specific expression changes.

Emerging therapeutic options in fulminant invasive rhinocerebral mucormycosis.

OBJECTIVE: Five cases of rhinocerebral mucormycosis (RCM) with different courses of illness and outcomes also due to different therapeutical strategies including Posaconazole as a new therapeutic option are described. Predisposing conditions for RCM are diabetes mellitus and immunosuppression. Diagnosis is often delayed because patients complain about nonspecific symptoms of acute rhinosinusitis, and initial CT imaging is unimpressive. Progressive disease, however, leads to early orbital and cerebral invasion. Due to the lack of typical clinical signs, diagnosis relies on histopathology. Therapy consists of the management of predisposing factors, radical surgical intervention and systemic antifungal therapy. METHODS: We describe five cases of RCM with different comorbidities and outcomes. RESULTS: RCM remains a diagnostic and therapeutic challenge because it begins with nonspecific symptoms and ends as fulminant disease with high mortality. Here, systemic treatment with Posaconazole appears to be a more effective alternative to amphotericin. CONCLUSION: If a patient is suspected having RCM, improvement of predisposing diseases, radical surgical debridement and effective systemic antifungal therapy must be instituted immediately.

Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases.

PURPOSE: Epidermal growth factor receptor (EGFR) antibody therapy is established in patients with wild-type KRAS colorectal carcinoma; however, up to 50% of these patients do not respond to this therapy. To identify the possible causes of this therapy failure, we searched for mutations in different EGFR-dependent signaling proteins and analyzed their distribution patterns in primary tumors and corresponding metastases. EXPERIMENTAL DESIGN: Tumor tissues, macrodissected from tumor centers, invasion fronts (n = 100), lymph nodes (n = 55), and distant metastases (n = 20), respectively, were subjected to DNA extraction and mutation analysis of KRAS, BRAF, and PIK3CA. RESULTS: Activating mutations were detected in 41% (KRAS), 7% (BRAF), and 21% (PIK3CA) of the primary tumors. By comparing tumor centers and invasion fronts, the intratumoral heterogeneity of KRAS, BRAF, and PIK3CA mutations was observed in 8%, 1%, and 5% of primary tumors, respectively. Heterogeneity between primary tumors and lymph node metastases was found in 31% (KRAS), 4% (BRAF), and 13% (PIK3CA) of the cases. Heterogeneity between primary tumors and distant metastases was present in two patients (10%) for KRAS and one patient for PIK3CA (5%), but not for BRAF. Discordant results between primary tumors and metastases could markedly be reduced by testing the additional tumor samples. CONCLUSIONS: Failure of EGFR antibody therapy in patients with wild-type KRAS colorectal cancer may result from activating BRAF or PIK3CA mutations and false-negative sequencing results caused by intratumoral heterogeneity. Due to the particularly high rates of heterogeneity between primary tumors and lymph node metastases, the latter are least suitable for diagnostic mutation analysis.