Mutant Solid Tumors Research Articles

FAT1 Expression and Mutation Status In Adult Acute Lymphoblastic Leukemia

IntroductionFAT1 belongs to the FAT protocadherin family, a drosophila homologous gene involved in development processes. Recently, FAT1 gained large interest as it is mutated in various cancers. Besides the known function of cell-cell interaction and polarity, FAT1 loss of function mutations have been linked to dysregulation of the WNT pathway in solid tumors. In acute lymphoblastic leukemia (ALL), aberrantly high expression of FAT1 was claimed to be associated with inferior outcome in pediatric B-lineage ALL. Herein, we investigated the yet unknown frequency and relevance of FAT1 expression and mutation in a large, homogenously treated cohort of adult ALL patients. Patients and MethodsWe investigated FAT1 expression in diagnostic bone marrow (BM) samples of 112 T-ALL, 122 B-lineage ALL, and additional 63 early T-cell precursor (ETP) ALL patients by real time (RT)-PCR. Patients were enrolled in trials of the German Multicenter Study Group for Adult ALL (GMALL) and outcome was investigated for patients into GMALL trials 06/99 and 07/03. Using the T-cell line BE13 as reference, we defined patients with FAT1 expression higher than BE13 as FAT1pos (0.01-38.5) and patients with a lower expression as FAT1neg (<0.01). We additionally examined peripheral blood (PB), BM, CD34+-, CD3+-cells from healthy donors. FAT1mutation status was investigated in 68 T-ALL patients. For mutation analyses customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to hybridize the targeted regions, followed by 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform. ResultsNormal hematopoietic cells including unselected BM cells, CD34+-progenitors, or CD3+ T-cells from healthy donors lacked FAT1 expression (<0.01) with the one exception of a CD34+-sample (0.03). In contrast, ALL samples aberrantly expressed FAT1: 32% of B-lineage ALL (0.01-38.5) and 54% of T-ALL cases (0.01-31.2) showed elevated FAT1 expression, with a lower frequency in the immature ETP-ALL subgroup (17%; 0.01-15.2). FAT1 expression was associated with a more mature immunophenotype (T-ALL: thymic 74%, mature 45%, early 4%, p<0.001; B-lineage ALL: pre B-ALL 57%, common ALL 26%, pro B-ALL 9%, p=0.04). No significant differences between FAT1pos and FAT1neg patients were observed regarding age and sex. FAT1pos T-ALL patients more frequently showed a white blood cell count (WBC) >30.000/µL at diagnosis compared to FAT1neg T-ALL patients (78% vs. 42%, p<0.01). Whereas no negative prognostic impact was observed for FAT1 expression in B-lineage ALL or T-ALL with respect to overall survival or remission duration, lack of FAT1 expression was associated with primary resistance to induction therapy in T-ALL (T-ALL: FAT1neg 12%, FAT1pos 0%, p=0.04). In addition, we found an unexpected high rate of FAT1 mutations (exclusively missense mutations) with 8 of 68 T-ALL patients (12%). The mutation spectrum, mainly located in the cadherin domains, was similar to the distribution of FAT1 mutations in solid tumors. No difference were observed between FAT1 mutated (FAT1mut) and FAT1 wild-type patients (FAT1wt) with regard to sex, age, WBC, and presentation of antigens associated with an early differentiation stage. FAT1 mutations were more frequent in early T-ALL (3/12, 25%) and in thymic T-ALL (5/41, 12%) than in T-ALL patients with a mature immunophenotype (0/15, 0%). FAT1 expression was more common in FAT1wt T-ALL compared to FAT1mut T-ALL patients (50% vs. 25%). ConclusionThis first comprehensive analysis on FAT1 in a large cohort of adult patients with ALL shows a high frequency of FAT1 expression. Higher FAT1 expression occurred in ALL patients with more mature immunophenotype linking FAT1 to cell-cell adhesion and polarity, thymic homing and interaction with the BM niche. This yet unreported high mutation rate of 12 % in adult T-ALL makes FAT1 to one of the most frequently mutated genes in T-ALL. The link of inactivating FAT1 mutations to aberrant activation of the WNT pathway, as reported in solid tumors, might allow the development of refined treatment options. In summary, these data make FAT1 a promising candidate for disease monitoring, risk stratification and development of targeted therapies. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

Abstract 1131: Novel inhibitor of Notch signaling for the treatment of cancer.

Abstract The Notch pathway is a highly conserved signaling system that plays an important role in development and tissue homeostasis. While Notch mutations are well characterized and implicated in hematological malignancies such as T-cell acute lymphoblastic leukemia, mutations in solid tumors were not reported until recently. With the whole genomic deep sequencing of a large number of samples, deregulated Notch signaling has been implicated in a small percentage of solid tumors such as ovarian, lung, and triple negative breast cancer due to genomic alterations including mutations, amplification, and fusion of Notch pathway components. Inhibition of Notch signaling may provide an attractive targeted cancer therapeutic strategy. We have identified and characterized LY3039478 a novel small molecule that is an exquisitely potent inhibitor of Notch-1 intracellular domain (N1ICD) cleavage with an IC50 of ∼1nM in most of the tumor cell lines tested. We also demonstrate that LY3039478 potently inhibits mutant Notch receptor activity. In a xenograft tumor model, LY3039478 inhibited N1ICD cleavage and expression of Notch-regulated genes in the tumor microenvironment. The inhibition of Notch cleavage also resulted in the induction of apoptosis in a Notch-dependent xenograft model. Using extensive PK/PD data we determined the strength and duration of N1ICD cleavage required for anti-tumor activity which was observed in several xenograft tumors including patient derived tumors representing colon, lung, ovarian, gastric, and breast cancer and glioblastoma. To mitigate the mucoid gasteroentropathy caused by Notch inhibition, PK/PD data were incorporated in devising dosing strategies that identified an optimal intermittent dosing schedule without negatively impacting efficacy. Furthermore, the mucoid gastroentropathy was also mitigated by the prophylactic administration of dexamethasone without negatively impacting the Notch inhibitor mediated efficacy. Mechanistic studies revealed that dexamethasone does not interfere with LY3039478-mediated inhibition of N1ICD cleavage and gene expression but alters the expression of stem cell gene expression in GI tract. In summary, we have characterized an orally bio-available small molecule Notch inhibitor that may provide therapeutic benefit to cancer patients with deregulated Notch signaling. LY3039478 is specifically designed to potently inhibit Notch signaling and is being investigated in Phase I. Citation Format: Mark H. Bender, Hong Gao, Andrew R. Capen, Julia M. Clay, Philip A. Hipskind, Jon K. Reel, Maciej J. Zamek-Gliszczynski, Jason R. Manro, Karim Benhadji, Bharvin K. R. Patel. Novel inhibitor of Notch signaling for the treatment of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1131. doi:10.1158/1538-7445.AM2013-1131

Abstract P6-10-07: Phase I study of BYL719, an alpha-specific PI3K inhibitor, in patients with PIK3CA mutant advanced solid tumors: preliminary efficacy and safety in patients with PIK3CA mutant ER-positive (ER+) metastatic breast cancer (MBC)

Abstract Background: Deregulation of the PI3K pathway occurs in &amp;gt;50% of breast cancers. Mutations in PIK3CA, the gene encoding the p110alpha subunit of PI3K contributes to resistance to endocrine and anti-HER2 therapies. BYL719 is a potent, specific oral inhibitor of the alpha subunit of PI3K, which inhibits wild-type p110alpha and its most common somatic mutations (IC50=5 nM) much more potently than other PI3K isoforms. Methodology: BYL719 has been investigated in a phase I study in patients with PIK3CA mutant advanced solid tumors. BYL719 has a favorable safety and PK profile; the MTD was established at 400 mg once daily (Juric et al., AACR 2012). As of 5 September 2012, 79 patients have been enrolled in this phase I study, among them 20 patients with PIK3CA mutant ER+ MBC. 5 of these patients had ER+/HER2 positive (HER2+) MBC. Presence of PIK3CA mutations was determined by either local or central assessment. Results: 20 patients with ER+ PIK3CA mutant MBC were treated once daily with BYL719 at doses of 30 mg (n = 1), 180 mg (n = 1), 270 mg (n = 1), 400 mg (n = 14) and 450 mg (n = 3). Median age was 55 years (range 40–78). 15/20 patients with ER+ PIK3CA mutant MBC had HER2 negative and 5/20 had HER2+ MBC. Patients had received a median of 5 previous treatment regimens (range 1–11). Most patients had received multiple prior lines of endocrine therapy including tamoxifen and aromatase inhibitors; 9 patients also received fulvestrant and 16 patients received prior chemotherapy. HER2+ patients have been previously treated with trastuzumab and/or lapatinib. Based on a recent analysis of 19 patients (data cut-off 5 July 2012), the safety of BYL719 in patients with ER+ MBC was comparable to the overall study population. Most frequent adverse events (AEs; ≥10%) suspected to be related to BYL719 were hyperglycemia, diarrhea, nausea, fatigue, decreased appetite, vomiting, rash and erythematous rash, dysgeusia, dry mouth, and stomatitis. The majority of AEs were CTCAE grade 1/2 and were clinically well manageable. Most frequent CTCAE grade 3/4 events were hyperglycemia and erythematous rash. 18/20 PIK3CA mutant ER+ MBC patients were treated at potentially effective doses of ≥270 mg/day. 6/18 patients (33%) achieved tumor shrinkage &amp;gt; 20%, among them 2 patients achieved partial responses (PR) by RECIST. Median progression free survival (PFS) analyzed in 17 patients treated at ≥270 mg/day in the ER+ BC patients was 7.3 months (CI [4.6,9.5]), compared to 3.7 months (CI [1.8,5.5]) in 38 patients with other PIK3CA mutant solid tumors (data cut-off 5 July 2012) Conclusion: BYL719 has a favorable safety profile with manageable side effects, which were mainly related to its mechanism of action. BYL719 shows promising preliminary clinical activity as single agent in patients with PIK3CA mutant ER+ MBC, warranting further clinical development. The investigation of BYL719 continues in patients with ER+ MBC as single agent, and in combination with endocrine therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-07.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Updated safety and efficacy results from a phase I/II study of the oral BRAF inhibitor dabrafenib (GSK2118436) combined with the oral MEK 1/2 inhibitor trametinib (GSK1120212) in patients with BRAFi-naive metastatic melanoma.

8510 Background: In preclinical models, the BRAFi/MEKi combination has demonstrated enhanced activity against BRAF-mutant cancer cells compared with either drug alone, delayed emergence of BRAFi resistance, and prevented BRAFi-related proliferative skin lesions. A 3-part study investigating the dabrafenib/trametinib combination was conducted in patients (pts) with V600 BRAF mutant solid tumors. Interim data from the study were previously reported (Infante, ASCO 2011); updated safety and efficacy data are presented. Methods: In Part 2, 125 pts with V600 BRAF mutant solid tumors enrolled, including 77 melanoma pts with no prior BRAFi, and measurable disease according to RECIST 1.1. Pts were treated on 4 escalating dose levels of dabrafenib/trametinib (mg BID/mg QD): 75/1, 150/1, 150/1.5, 150/2. Demographic and efficacy data for the 77 melanoma pts with no prior BRAFi and safety data for all 125 Part 2 pts are reported. Results: Among 77 melanoma pts, median age was 52 years, 61% male, 57% ECOG PS of 0, 91% V600E, 65% M1c stage, 26% prior brain metastases, and 52% LDH &gt; ULN. Confirmed ORR was 56% (95% CI: 44.1%-67.2%) with 4 CR, 39 PR, 29 SD and, 3 PD. Confirmed response rate for each dose level, respectively, was 67% (n=6), 64% (n=22), 48% (n=25), and 54% (n=24). Median PFS (months) for each dose level, respectively, was: 8.7, 8.3, 5.5; PFS is not mature for 150/2. Overall PFS was 7.4 (95% CI: 5.5-9.2). Among the 125 pts, there were 2 grade (G) 5 adverse events (AEs), pneumonia and hyponatraemia. The most common G3/4 AEs were pyrexia (n=6, 5%), fatigue (n=6, 5%) and dehydration (n=6, 5%). Skin toxicity ≥ G2 occurred in 17 (14%) pts. Cutaneous squamous cell carcinoma occurred in 3 (2%) pts and actinic keratoses in 2 (2%). Conclusions: The combination of dabrafenib/trametinib has an acceptable safety profile, with a lower incidence of MEKi-related rash and BRAFi-induced hyperproliferative skin lesions compared with the single agents. The clinical activity of dabrafenib/trametinib observed in pts with V600 BRAF mutant metastatic melanoma is encouraging and will be investigated further in a phase III trial.

Comprehensive Predictive Biomarker Analysis for MEK Inhibitor GSK1120212

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors.

Detection of low prevalence somatic mutations in solid tumors with ultra-deep targeted sequencing

Ultra-deep targeted sequencing (UDT-Seq) can identify subclonal somatic mutations in tumor samples. Early assays' limited breadth and depth restrict their clinical utility. Here, we target 71 kb of mutational hotspots in 42 cancer genes. We present novel methods enhancing both laboratory workflow and mutation detection. We evaluate UDT-Seq true sensitivity and specificity (> 94% and > 99%, respectively) for low prevalence mutations in a mixing experiment and demonstrate its utility using six tumor samples. With an improved performance when run on the Illumina Miseq, the UDT-Seq assay is well suited for clinical applications to guide therapy and study clonal selection in heterogeneous samples.

Identification of fusion genes in breast cancer by paired-end RNA-sequencing

BackgroundUntil recently, chromosomal translocations and fusion genes have been an underappreciated class of mutations in solid tumors. Next-generation sequencing technologies provide an opportunity for systematic characterization of cancer cell transcriptomes, including the discovery of expressed fusion genes resulting from underlying genomic rearrangements.ResultsWe applied paired-end RNA-seq to identify 24 novel and 3 previously known fusion genes in breast cancer cells. Supported by an improved bioinformatic approach, we had a 95% success rate of validating gene fusions initially detected by RNA-seq. Fusion partner genes were found to contribute promoters (5' UTR), coding sequences and 3' UTRs. Most fusion genes were associated with copy number transitions and were particularly common in high-level DNA amplifications. This suggests that fusion events may contribute to the selective advantage provided by DNA amplifications and deletions. Some of the fusion partner genes, such as GSDMB in the TATDN1-GSDMB fusion and IKZF3 in the VAPB-IKZF3 fusion, were only detected as a fusion transcript, indicating activation of a dormant gene by the fusion event. A number of fusion gene partners have either been previously observed in oncogenic gene fusions, mostly in leukemias, or otherwise reported to be oncogenic. RNA interference-mediated knock-down of the VAPB-IKZF3 fusion gene indicated that it may be necessary for cancer cell growth and survival.ConclusionsIn summary, using RNA-sequencing and improved bioinformatic stratification, we have discovered a number of novel fusion genes in breast cancer, and identified VAPB-IKZF3 as a potential fusion gene with importance for the growth and survival of breast cancer cells.

Mitochondrial and Nuclear Genes of Mitochondrial Components in Cancer

Although the observation of aerobic glycolysis of tumor cells by Otto v. Warburg had demonstrated abnormalities of mitochondrial energy metabolism in cancer decades ago, there was no clear evidence for a functional role of mutant mitochondrial proteins in cancer development until the early years of the 21st century. In the year 2000, a major breakthrough was achieved by the observation, that several genes coding for subunits of the respiratory chain (ETC) complex II, succinate dehydrogenase (SDH) are tumor suppressor genes in heritable paragangliomas, fulfilling Knudson’s classical two-hit hypothesis. A functional inactivation of both alleles by germline mutations and chromosomal losses in the tumor tissue was found in the patients. Later, SDH mutations were also identified in sporadic paragangliomas and pheochromocytomas. Genes of the mitochondrial ATP-synthase and of mitochondrial iron homeostasis have been implicated in cancer development at the level of cell culture and mouse experiments. In contrast to the well established role of some nuclear SDH genes, a functional impact of the mitochondrial genome itself (mtDNA) in cancer development remains unclear. Nevertheless, the extremely high frequency of mtDNA mutations in solid tumors raises the question, whether this small circular genome might be applicable to early cancer detection. This is a meaningful approach, especially in cancers, which tend to spread tumor cells early into bodily fluids or faeces, which can be screened by non-invasive methods.

Clonal expansion of different mtDNA variants without selective advantage in solid tumors

In search of tumor-specific mitochondrial DNA (mtDNA) mutations in head and neck squamous cell cancer, we found heteroplasmy in the blood of two individuals, i.e., these individuals carried two alleles of mtDNA. In both cases, the tumor was found to be homoplasmic, i.e., it contained only one of the two mtDNA alleles present in blood. More interestingly, in one case the tumor had acquired the wild-type allele, while in the other case it contained the mutant allele only. Sequencing of the whole 16.5 kb mtDNA showed that the observed heteroplasmic positions in the D-loop region, nucleotides 152 and 16187, respectively, were the only differences between tumor and blood mtDNA genotypes in these individuals. Our findings thus strongly support the hypothesis that accumulation of mtDNA mutations in solid tumors occurs by clonal and random expansion of pre-existing alleles and is not necessary for the metabolic changes generally associated with tumor formation, the Warburg effect.

The Tyrosine Kinase src Is Highly Activated in AML and Plays a Crucial Role in Leukemic Cell Proliferation.

The tyrosine kinase pp60src is involved in several signal transduction pathways such as signalling of hematopoetic growth factors and cytokines. The viral form v-src was the first oncogene described and mutations of regulatory tyrosine residues in cellular src (c-src) have been linked to malignant transformation. However, no mutations in the gene of c-src have been described in leukemia so far, although some data of src mutations in solid tumors have been reported. The current study was undertaken to examine the role of src in acute myeloid leukemia (AML).Blood and bone marrow specimen of patients with newly diagnosed or recurrent AML treated at our institution were sampled. AML cell lines or CD34 positive cells of healthy donors served as positive and negative controls, respectively. RNA was isolated, and RT-PCR was performed using 4 different primer pairs spanning the coding region of c-src. Protein expression and phosphorylation was studied after protein extraction and Western blot analyses using src and phospho-src specific antibodies. The effect of src inhibitors PP1 and PP2 on leukemic cell proliferation was studied in human and murine cell lines. Mutational analyses of the coding region were performed using SSCP/heteroduplex and bi-directionally sequencing.In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Inhibition of src phosphorylation by src-specific inhibitors PP1 and PP2 was detected by Western blot using an antibody specific for phospho-src. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in all cell lines and 60 clinical samples.In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Mutations in the coding sequence of c-src causing constitutive activation could be excluded by mutational analyses of primary AML samples. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML.

P300/CBP and cancer.

p300 and cyclic AMP response element-binding protein (CBP) are adenoviral E1A-binding proteins involved in multiple cellular processes, and function as transcriptional co-factors and histone acetyltransferases. Germline mutation of CBP results in Rubinstein-Taybi syndrome, which is characterized by an increased predisposition to childhood malignancies. Furthermore, somatic mutations of p300 and CBP occur in a number of malignancies. Chromosome translocations target CBP and, less commonly, p300 in acute myeloid leukemia and treatment-related hematological disorders. p300 mutations in solid tumors result in truncated p300 protein products or amino-acid substitutions in critical protein domains, and these are often associated with inactivation of the second allele. A mouse model confirms that p300 and CBP function as suppressors of hematological tumor formation. The involvement of these proteins in critical tumorigenic pathways (including TGF-beta, p53 and Rb) provides a mechanistic route as to how their inactivation could result in cancer.

Targeting mutations in solid tumors: Scientific rationale and novel therapeutic options

Targeting mutations in solid tumors: Scientific rationale and novel therapeutic options

Targeting c-kit mutations in solid tumors: Scientific rationale and novel therapeutic options

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. These tumors span a wide clinical spectrum from benign to malignant and have long been recognized for their nearly absolute resistance to chemotherapy and radiation treatment. Surgery is the primary treatment modality for GISTs, but GISTs represent an incurable malignancy for patients with metastatic or unresectable disease. Thus, novel approaches to the treatment of GISTs were desperately needed. Gastrointestinal stromal tumors are characterized by expression of the transmembrane receptor tyrosine kinase KIT, which is defined by the CD117 antigen and is the product of the c-kit proto-oncogene. Activating or gain-of-function mutations in the c-kit gene have been identified in the majority of GIST cases. The resulting constitutive KIT tyrosine kinase activity was hypothesized to provide growth and survival signals to GIST cells and to be crucial to the pathogenesis of the disease. This hypothesis became testable with the identification of the signal transduction inhibitor imatinib mesylate (formerly STI571, [Gleevec]; Novartis Pharmaceuticals Corp, East Hanover, NJ), which blocks the tyrosine kinase activity of KIT as well as the kinase activity of the normal c-abl gene product, the oncogenic Bcr-Abl chimeric fusion protein of chronic myeloid leukemia, and the platelet-derived growth factor receptor. Preclinical experiments showed rapid inhibition of ligand-independent KIT phosphorylation, decreased cellular proliferation, and induction of apoptosis after exposure of GIST cells to imatinib mesylate in vitro. These results provided the rationale to move forward with clinical testing of imatinib mesylate as an anticancer therapy for GIST. In early 2000, a dramatic clinical and radiographic response to imatinib mesylate was shown in a single patient with advanced, chemotherapy-resistant GIST. The powerful scientific rationale for this proofof-concept study, together with the durable and significant response observed in this first GIST patient treated with imatinib mesylate, have provided the driving force for rapid clinical development of this targeted therapy in this solid tumor indication.

PTEN/ MMAC1 in malignant melanoma and its importance for tumor progression

A novel tumor suppressor gene, PTEN/ MMAC1, on 10q23, displayed a number of mutations in solid tumors as gliomas and breast cancer. Aberrations of the long arm of chromosome 10 have been frequently detected in tumor progression of malignant melanoma of the skin by a variety of methods including cytogenetic analysis, fluorescence in situ hybridization and loss of heterozygosity analysis. Compared to previous studies, which propose an involvement of PTEN/ MMAC1 in malignant melanoma mostly on the basis of data derived from cell lines and metastases, we analyzed a broader spectrum of exclusively patient derived tumor tissue by PCR and direct sequencing analysis of PTEN/ MMAC1. Here, we present data of 25 primary melanomas (8 superficial spreading melanomas, 17 nodular melanomas) and 25 metastases of 41 patients. Neither loss of the complete gene nor a whole exon nor any nonsense mutations could be demonstrated. However, we detected several polymorphisms and some mutations in the introns, and in two metastatic tumors mutations with an amino acid change. Our results obtained from tissue samples underline that mutations of PTEN/ MMAC1 are not an essential event in the onset of malignant melanoma of the skin, but could have an impact on tumor progression.

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors.

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.