Gene mutations in myelodysplasia.

The myelodysplastic syndrome is a paradigm of human preleukaemia. Normal haemopoiesis is progressively displaced by an abnormal clone derived from a mutated stem cell. The initial mutation is unknown but its occurrence may be related to the overall load of random mutations which are a consequence of both intrinsic DNA defects and external mutagens. Evolution of the pathological population is marked by an increasing load of genetic lesions at the molecular and cytogenetic levels. Ras mutations can be detected in the blood of about 50% of MDS patients. Fms mutations are less common but these lesions can be found both in patients and in haematologically normal subjects who have previously received cytotoxic therapy suggesting that they can occur early in the preleukaemic process. Clonal haemopoiesis in the absence of either ras or fms mutations can occur in these subjects. The data suggest the inability of mutant ras or fms genes alone to produce observable preleukaemic changes but that subjects with these mutations may be predisposed to future MDS. Ras mutations are a common accompaniment of a wide variety of malignancies and experimental transfection of the mutant gene can induce a malignant phenotype in cultured cells. There are many possible mechanisms for this transformation which may be relevant in a clinical context. Experimentally observed effects include a direct influence on the cell cycle, the induction of drug resistance and the stimulation of autocrine growth factor production. It may eventually be possible to define which gene mutations are important in conferring a malignant state, which determine phenotype and which are of incidental significance.     

Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ hybridization.

PURPOSE: Chromogenic in situ hybridization (CISH) is a new modification of the fluorescence in situ hybridization (FISH) technique for detection of oncogene amplification in archival tumor samples. In CISH, the oncogene probe is detected using a peroxidase reaction, allowing use of transmitted light microscopy. We compared detection of HER-2/neu amplification by CISH with a Food and Drug Administration-approved two-color FISH test in an interlaboratory setting. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tumor samples from 197 breast cancers were analyzed for HER-2 amplification by CISH. Two-color FISH (PathVysion) CISH of 17 centromere was done if the observer considered it necessary to ascertain amplification status in tumors with borderline HER-2 CISH copy numbers. RESULTS: Paired CISH/FISH results were available from 192 (97%) of 197 cases, no clear difference in success rates of either method was observed. Centromere 17 CISH was considered necessary in seven tumors. CISH and two-color FISH results were concordant in 180 cases (93.8%). There were 92 and 88 tumors found HER-2 amplified and nonamplified, respectively, by both methods. Eight tumors were amplified by CISH but not by FISH, and four tumors exhibited the opposite condition (kappa coefficient 0.875). In 7 of 12 cases differences between the two methods could have related to a lack of CISH chromosome 17 information. The remaining cases were explained by difficult histology (ductal carcinoma in situ, poor representativity, dense lymphocytic infiltration, or intratumoral heterogeneity). CONCLUSIONS: These results indicate that CISH could provide an accurate and practical alternative to FISH for clinical diagnosis of HER-2/neu oncogene amplification in archival formalin-fixed breast cancer samples.     

RAS signaling pathways, mutations and their role in colorectal cancer

Two of the main cellular pathways in which the RAS protein operates are the mitogen-activated protein kinases (MAPK) and phosphoinositide-3 kinase (PI3K) pathways. In a normal cell, these are important in controlling several functions, such as cell growth and survival. It becomes self-evident that these events will be disrupted in a malignant cell with a deregulated MAPK or PI3K pathway. Mutations in genes involved in these pathways and interacting with RAS, as well as RAS itself will be discussed. The second part of this review concentrates on how crucial RAS signaling is in colorectal cancer progression, with references to treatment response and prognosis when RAS or other related mutations are present.     

HER-2/neu amplification detected by fluorescence in situ hybridization in fine needle aspirates from primary breast cancer.

BACKGROUND: The HER-2/neu gene is amplified in 20-30% of human breast cancers and has been shown to have prognostic and predictive value for treatment with chemotherapy, hormone therapy and antibodies against the HER-2/neu domain (trastuzumab). The aim of our study was to evaluate the reliability of HER-2/neu determination by fluorescence in situ hybridization (FISH) on fine-needle aspirates (FNAs) from primary breast cancer patients by comparison with the results obtained by FISH and immunohistochemistry (IHC) on the corresponding histological sections. MATERIALS AND METHODS: HER-2/neu amplification was determined by FISH on 66 breast cancer FNAs. Twenty-three and 36 corresponding formalin-fixed, paraffin-embedded sections were assayed by FISH and by IHC, respectively, in order to detect HER-2/neu amplification and HER-2/neu protein expression. RESULTS: Twenty-seven per cent (18/66) of breast cancer FNAs showed amplification of HER-2/neu by FISH. Paired results by FISH cytology and FISH histology were available in 22 cases. Concordance was 91% (20/22). Paired results by FISH cytology and IHC were available in 36 cases. Concordance was 92% (33/36). Eighteen of 66 breast cancer FNAs were also submitted to flow cytometric DNA analysis. None of the diploid cases showed HER-2/neu amplification by FISH. Six out of the eight aneuploid cases were amplified and two were polysomic. CONCLUSIONS: HER-2/neu gene amplification can be reliably estimated by FISH on breast cancer FNAs and a good correlation has been found between FISH and IHC results from the corresponding histological sections.     

KRAS mutations detected by the amplification refractory mutation system-Scorpion assays strongly correlate with therapeutic effect of cetuximab

Background:

We aimed to compare the sensitive and quality-controlled KRAS testing with direct sequencing and to assess the impact on decision making of treatment.

Patients and methods:

We analysed genomic DNA isolated from macrodissected formalin-fixed paraffin-embedded specimens by direct sequencing and an amplification refractory mutation system–Scorpion assay (ARMS/S) method. Cetuximab was administered to patients identified as having wild-type (WT) KRAS using direct sequencing. Therapeutic effects were evaluated according to their KRAS status as determined by ARMS/S.

Results:

Among the 159 patients, the overall mutation rate was determined to be 37.0% by direct sequencing and 44.0% by ARMS/S. For the patients diagnosed as WT by direct sequencing and treated with cetuximab (n=47), a response rate of 16.0% was observed for 38 ARMS/S WT patients, whereas 9 ARMS/S mutant (MUT) patients failed to respond. The ARMS/S WT patients showed significant improvement in progression-free survival (PFS) and overall survival (OS) compared with ARMS/S MUT patients (PFS median 5.0 vs 1.7 months, hazards ratio (HR)=0.29, P=0.001; OS median 12.1 vs 3.8 months, HR=0.26, P=0.001).

Conclusion:

Sensitive and quality-controlled KRAS testing may provide improved predictive power to determine the efficacy of anti-epidermal growth factor antibodies.     

Evaluation of the reliability of chromosomal imbalances detected by combined use of universal DNA amplification and comparative genomic hybridization.

Comparative genomic hybridization (CGH) analysis of microscopic tumor samples is allowed by universal DNA amplification using degenerate oligonucleotide primed-PCR (DOP-PCR). To evaluate the reliablity of DOP-PCR CGH, we performed DOP-PCR CGH and standard CGH in parallel using DNAs extracted from 10 malignant tumors of the hepatobiliary tract and pancreas. Similar results were obtained by both methods with a few exceptions, indicating that DOP-PCR CGH provides cytogenetic information equivalent to that obtained from standard CGH. We also investigated the sensitivity of DOP-PCR CGH using sequential dilutions of DNA from microdissected tumor cells. DOP-PCR using 100 to 800 pg of template DNA yielded successful CGH results. However, less than 50 pg of template DNA was not suitable because of the small amount of generated DNA. These findings suggest that DOP-PCR CGH is applicable for CGH analysis of tiny specimens which are too small for standard CGH. Accordingly, DOP-PCR CGH analysis may become a useful method in clinical laboratory examination.     

PTPN11, RAS and FLT3 mutations in childhood acute lymphoblastic leukemia

PTPN11, the gene which encodes protein tyrosine phosphatase SHP-2, plays an important role in regulating intracellular signaling. Germline mutations in PTPN11 were first observed in Noonan syndrome, while somatic mutations were identified in hematological myeloid malignancies. Recently, PTPN11 mutations have been reported in children with acute lymphoblastic leukemia (ALL). In the present study, we investigated the prevalence of mutations in PTPN11, RAS and FLT3 in samples from 95 Japanese children with ALL. We observed exon 3 and 8 missense mutations of PTPN11 in 6 children with B precursor ALL. One patient with Down syndrome and ALL had PTPN11 mutation. We also identified RAS mutations in ten patients and FLT3 internal tandem duplication (FLT3/ITD) in one patient. None of the patients had simultaneous mutations in PTPN11 and RAS, while one patient had both PTPN11 and FLT3 mutations. These data suggest that PTPN11 mutation may play an important role for leukemogenesis in a proportion of children with ALL, particularly B precursor ALL.     

Overexpressed Skp2 within 5p amplification detected by array-based comparative genomic hybridization is associated with poor prognosis of glioblastomas

To better understand the pathogenesis of glioblastoma multiforme (GBM) and to increase the accuracy of predicting outcomes for patients with this disease, we performed genome-wide screening for DNA copy-number aberrations in 22 glioma-derived cell lines using a custom-made comparative genomic hybridization-array. Copy-number gains were frequently detected at 3q, 7p, 7q, 20q, Xp and Xq, and losses at 4q, 9p, 10p, 10q, 11q, 13q, 14q, 18q, and 22q. Among several non-random chromosomal aberrations, the gain/amplification of DNA at 5p, which has never been reported before in GBM, was detected with a relatively high ratio (log2 ratio = 0.41-1.19) in four cell lines. Amplification and subsequent overexpression of SKP2, a possible target of amplification within 5p, were detected in four of the 22 cell lines. We also investigated the expression of the gene product in primary GBM by immunohistochemistry, which revealed increased levels of Skp2 in 11 of the 35 tumors examined (31.4%). Heightened expression of Skp2 was associated with shorter overall survival (P = 0.001, logrank test), especially in patients younger than 65 years. These results suggest that overexpression of Skp2 through gene amplification may contribute to the pathogenesis of GBM, and that overabundance of the protein might be a useful prognostic tool in patients with this disease.     

BRAF and RAS mutations in human lung cancer and melanoma

BRAF encodes a RAS-regulated kinase that mediates cell growth and malignant transformation kinase pathway activation. Recently, we have identified activating BRAF mutations in 66% of melanomas and a smaller percentage of many other human cancers. To determine whether BRAF mutations account for the MAP kinase pathway activation common in non-small cell lung carcinomas (NSCLCs) and to extend the initial findings in melanoma, we screened DNA from 179 NSCLCs and 35 melanomas for BRAF mutations (exons 11 and 15). We identified BRAF mutations in 5 NSCLCs (3%; one V599 and four non-V599) and 22 melanomas (63%; 21 V599 and 1 non-V599). Three BRAF mutations identified in this study are novel, altering residues important in AKT-mediated BRAF phosphorylation and suggesting that disruption of AKT-induced BRAF inhibition can play a role in malignant transformation. To our knowledge, this is the first report of mutations documenting this interaction in human cancers. Although >90% of BRAF mutations in melanoma involve codon 599 (57 of 60), 8 of 9 BRAF mutations reported to date in NSCLC are non-V599 (89%; P < 10(-7)), strongly suggesting that BRAF mutations in NSCLC are qualitatively different from those in melanoma; thus, there may be therapeutic differences between lung cancer and melanoma in response to RAF inhibitors. Although uncommon, BRAF mutations in human lung cancers may identify a subset of tumors sensitive to targeted therapy.     

Numeric chromosome aberrations in prostate cancer detected by in situ hybridization

Background This retrospective study was designed to examine the applicability of in situ hybridization to the study of numeric chromosome aberrations in conventionally fixed, embedded tissue sections of human prostate cancers. Methods By use of in situ hybridization with chromosome-specific DNA probes, the copy number of pericentromeric sequences on chromosomes 7, 11, 17 X, and Y was detected within interphase nuclei in formalin-fixed and paraffin-embedded tissue specimens from 21 patients with adenocarcinoma of the prostate. The percentage of hyperdiploid cells (3 or more spots) was estimated by using light microscopy. Results The percentage of hyperdiploid cells for chromosomes 7 and 17 was highly correlated with increasing tumor Mostofi grade (P<0.05, Spearman rank correlation) or increasing Gleason score (P<0.05). The percentage of hyperdiploid cells for chromosome 11 was not correlated with either tumor Mostofi grade or Gleason score (P>0.05). Conclusion Since high tumor grade is indicative of more aggressive tumor behavior and a worse prognosis, these findings suggest that the percentage of hyperdiploid cells may be highly predictive of prostate tumor aggressiveness. Our preliminary results suggest that measurement of numeric chromosome aberrations using in situ hybridization in prostate cancer may serve as a predictive factor for the prognosis of prostate cancer.     

p53 and RAS gene mutations in multiple myeloma.

We analysed genomic DNA from 30 patients with multiple myeloma (MM), searching for alterations in the p53 and RAS genes by a combination of polymerase chain reaction and single-strand conformation polymorphism techniques. Mutations in the p53 gene were observed in 20% (6 out of 30) of the patients, and were located in conserved sequence blocks within exons 5 and 7. These were single-nucleotide substitutions and consisted predominantly (4/6) of G:C to A:T transitions. Of the six patients with a mutated p53 gene, four were in the terminal phase of the disease. RAS gene mutations were found more frequently since they occurred in 47% (14 out of 30) of the patients. Mutations consisted of single-nucleotide substitutions, located in codons 12, 13 and 61 of either K- or N-RAS, to the exclusion of H-RAS. Moreover, one patient bore two simultaneous mutations, affecting simultaneously the K- and the N-RAS genes. RAS gene mutations were more frequently observed in patients with fulminating disease (10/15, 67%) than in patients with less aggressive forms of the disease (4/15, 26%). We also analysed genomic DNAs from 10 human myeloma cell lines, of which two bore mutations affecting codon 12 of the K-RAS gene, and one codon 12 of the N-RAS gene. The first two cell lines were obtained from freshly explanted tumor cells in which we observed identical mutations. Results presented here show that activating mutations in the RAS genes are, in MM, more frequent than those affecting the p53 gene and suggest that both events are related to terminal phases of the disease.     

Numerical chromosome 1, 7, 9, and 11 aberrations in bladder cancer detected by in situ hybridization

Forty transitional cell carcinomas of the human urinary bladder (TCCs) were examined for numerical aberrations of chromosomes 1, 7, 9, and 11 by in situ hybridization using chromosome-specific probes. Our interphase cytogenetic study of 24 low-grade, noninvasive TCCs, which were near-diploid by flow cytometry, showed a numerical aberration for at least 1 of these chromosomes in 14 of these cases. Most strikingly, a monosomy for chromosome 9 was found in 9 of 24 low-grade TCCs. A trisomy for chromosomes 1, 7, and 11 was detected in 5, 2, and 1 case(s), respectively. In 1 case a monosomy for chromosome 1 was detected by in situ hybridization. Monosomy for chromosome 9 was the only detected numerical change in 5 low-grade TCC cases. Examination of 16 invasive TCCs showed extra copies for chromosomes 1 and 7 in 7 flow cytometrically diploid cases with numerical chromosome aberrations; also, loss of chromosome 9 was detected. In 5 invasive and 2 noninvasive aneuploid/tetraploid TCCs a profound imbalance between the different chromosomes was found. In 5 of these cases an evident underrepresentation of chromosome 9 in comparison to chromosomes 1, 7, and 11 was detected. This underrepresentation of chromosome 9 in diploid, as well as aneuploid, TCCs, and in some cases the constant ratio between this chromosome and the other chromosomes, may be explained by a process of tetraploidization. Therefore, loss of chromosome 9 may be one of the primary genetic events in TCC oncogenesis, with secondary events, such as tetraploidization, correlated to tumor progression. Our results show that in situ hybridization can be routinely used to study important cytogenetic changes which occur during the development of a malignant disease.     

Targeted therapy in melanoma – the role of BRAF,RAS and KIT mutations

Melanoma today is considered as a spectrum of melanocytic malignancies characterised by clinical and molecular features, including targetable mutations in several kinases such as BRAF or c-KIT. The successful development of therapies targeting these mutations has resulted in new specific treatment options. These include vemurafenib, dabrafenib, trametinib, imatinib and other kinase inhibitors that are selected when the respective mutation is present.The BRAF inhibitor vemurafenib has resulted in improved survival in patients with BRAF-mutated advanced melanoma. Dabrafenib has shown similar efficacy. The MEK inhibitor trametinib also improved overall survival. In addition, the MEK inhibitor MEK 162 was investigated in a phase II clinical trial and showed promising efficacy in terms of response rate and progression-free survival (PFS) in NRAS-mutated melanomas. After this first success in the treatment of advanced melanoma, there is expectation that combinations of kinase inhibitors will additionally improve overall survival rates and PFS in advanced melanoma.     

Ploidy, as detected by fluorescence in situ hybridization, defines different subgroups in multiple myeloma.

Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma. However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients) or a DNA index calculation by flow cytometry (not routinely performed in myeloma). We validated a novel method based on interphase fluorescence in situ hybridization that can be utilitized to analyze almost all the patients. The method was very specific and sensitive for the detection of hyperdiploidy. Extended studies showed that most recurrent 14q32 translocations occur in nonhyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48 vs 66%). Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma.     

RAS mutations in patients following cytotoxic therapy for lymphoma

The occurrence of Myelodysplastic Syndrome (MDS) and Acute Myeloblastic Leukaemia (AML) following cytotoxic therapy for neoplastic disease is well recognised. RAS mutations are common in patients with MDS and AML. To determine whether these lesions are found as early markers of secondary disease, we have studied the incidence of RAS mutations in the peripheral blood of 70 patients in complete remission from lymphoma. Patients were treated by standard chemotherapy regimes and/or localised radiotherapy. Treatment had been given 6 months to 14 1/2 years previously and no patient showed any sign of residual disease. Genomic DNA from peripheral blood leukocytes was amplified in vitro at target codons of N, K and H RAS genes, and mutations detected by hybridisation with oligonucleotide probes. RAS mutations were detected in 9 subjects. One patient with an N12 valine (Val) substitution had been in complete remission from Hodgkin's disease (HD) for 9 years. DNA from this patient registered in a nude mouse tumorigenicity assay (NMT). The N12 Val mutation was not detected in the original tumour tissue from the same patient. A second patient in remission from HD showed evidence of co-existent N12 cysteine (Cys) and N13 valine (Val) substitutions which were not detected in presentation material or unaffected tissues. All patients are currently haematologically normal, indicating that clones of mutant RAS bearing cells may be detected prior to any overt sign of disease.     

Interglandular cytogenetic heterogeneity detected by comparative genomic hybridization in pancreatic cancer

The aim of this study is to explore the mechanisms of intratumoral cytogenetic heterogeneity (ICH) in pancreatic cancer. Using comparative genomic hybridization (CGH) analysis, we investigated interglandular variation in 20 primary invasive ductal adenocarcinomas of the pancreas. Three or four adjacent neoplastic glands were individually microdissected from a tumor specimen. Extracted DNA from each gland was amplified by degenerate oligonucleotide primed-PCR, followed by CGH. In addition, DNA index (DI) was measured by laser scanning cytometry in each case. CGH profiles displayed a wide variety of differences between glands within the same tumor in all cases, i.e., interglandular cytogenetic heterogeneity was distinct in pancreatic cancers. In this study, genetic changes detected in all regions of a tumor were classified as "region-independent" alterations, whereas changes seen in at least one, but not all regions were designated as "region-dependent" alterations, which resulted in ICH. The degree of ICH, which was manifested as the ratio of these two types of alterations, correlated closely with DI (Spearman rho = 0.842; P = 0.0002). Therefore, DI might be a surrogate marker for ICH. These results suggest that with tumor progression, ICH and DNA aneuploidy result from the successive appearance of region-dependent alterations attributable to chromosomal instability in tumor cells. Our data support a concept of individual cell heterogeneity in pancreatic cancer.