Aberrations In Tumor Cells Research Articles

Alternative Non-Homologous End-Joining: Error-Prone DNA Repair as Cancer's Achilles' Heel.

Simple SummaryCancer onset and progression lead to a high rate of DNA damage, due to replicative and metabolic stress. To survive in this dangerous condition, cancer cells switch the DNA repair machinery from faithful systems to error-prone pathways, strongly increasing the mutational rate that, in turn, supports the disease progression and drug resistance. Although DNA repair de-regulation boosts genomic instability, it represents, at the same time, a critical cancer vulnerability that can be exploited for synthetic lethality-based therapeutic intervention. We here discuss the role of the error-prone DNA repair, named Alternative Non-Homologous End Joining (Alt-NHEJ), as inducer of genomic instability and as a potential therapeutic target. We portray different strategies to drug Alt-NHEJ and discuss future challenges for selecting patients who could benefit from Alt-NHEJ inhibition, with the aim of precision oncology.Error-prone DNA repair pathways promote genomic instability which leads to the onset of cancer hallmarks by progressive genetic aberrations in tumor cells. The molecular mechanisms which foster this process remain mostly undefined, and breakthrough advancements are eagerly awaited. In this context, the alternative non-homologous end joining (Alt-NHEJ) pathway is considered a leading actor. Indeed, there is experimental evidence that up-regulation of major Alt-NHEJ components, such as LIG3, PolQ, and PARP1, occurs in different tumors, where they are often associated with disease progression and drug resistance. Moreover, the Alt-NHEJ addiction of cancer cells provides a promising target to be exploited by synthetic lethality approaches for the use of DNA damage response (DDR) inhibitors and even as a sensitizer to checkpoint-inhibitors immunotherapy by increasing the mutational load. In this review, we discuss recent findings highlighting the role of Alt-NHEJ as a promoter of genomic instability and, therefore, as new cancer’s Achilles’ heel to be therapeutically exploited in precision oncology.

Therapeutic Potential of PARP Inhibitors in the Treatment of Metastatic Castration-Resistant Prostate Cancer.

Simple SummaryIn recent years, the development of sequencing techniques to reveal the genomic information of prostate cancer tumors has allowed for the emergence of targeted therapies. Genomic aberrations in tumor cells have become popular due to the successful development of PARP inhibitors, which are particularly active in those tumors harboring DNA repair genomic defects. This review focuses on PARP inhibitors, two of which were approved for use by the US Food and Drug Administration in 2020 in metastatic castration-resistant prostate cancer. The article highlights the development of PARP inhibitors in the preclinical setting, summarizes the impactful clinical trials in the field, and discusses the need for continued research for further success in treating men with advanced prostate cancer.Metastatic castration-resistant prostate cancer (mCRPC) is an incurable malignancy with a poor prognosis. Up to 30% of patients with mCRPC have mutations in homologous recombination repair (HRR) genes. Poly (ADP-ribose) polymerase (PARP) inhibitors take advantage of HRR deficiency to kill tumor cells based on the concept of synthetic lethality. Several PARP inhibitors (PARPis) have been successful in various malignancies with HRR gene mutations including BRCA1/2, especially in breast cancer and ovarian cancer. More recently, olaparib and rucaparib were approved for mCRPC refractory to novel hormonal therapies, and other PARPis will likely follow. This article highlights the mechanism of action of PARPis at the cellular level, the preclinical data regarding a proposed mechanism of action and the effectiveness of PARPis in cancer cell lines and animal models. The article expands on the clinical development of PARPis in mCRPC, discusses potential biomarkers that may predict successful tumor control, and summarizes present and future clinical research on PARPis in the metastatic disease landscape.

Quantification of Multiple Tumor Clones Using Gene Array and Sequencing Data.

Cancer development is driven by genomic alterations, including copy number aberrations. The detection of copy number aberrations in tumor cells is often complicated by possible contamination of normal stromal cells in tumor samples and intratumor heterogeneity, namely the presence of multiple clones of tumor cells. In order to correctly quantify copy number aberrations, it is critical to successfully de-convolute the complex structure of the genetic information from tumor samples. In this article, we propose a general Bayesian method for estimating copy number aberrations when there are normal cells and potentially more than one tumor clones. Our method provides posterior probabilities for the proportions of tumor clones and normal cells. We incorporate prior information on the distribution of the copy numbers to prioritize biologically more plausible solutions and alleviate possible identifiability issues that have been observed by many researchers. Our model is flexible and can work for both SNP array and next-generation sequencing data. We compare our method to existing ones and illustrate the advantage of our approach in multiple datasets.

Quantification of intratumoral heterogeneity of HER2 status in breast cancer.

1036 Background: Intratumoral heterogeneity (ITH) occurs as a consequence of epigenetic aberrations in tumor cells with genetic diversity. HER2 ITH can be classified into genetic (a mixture of tumor cells with and without HER2 gene amplification) and epigenetic ITH (a mixture of HER2 gene-amplified tumor cells with and without HER2 protein overexpression). However, the both effects of genetic and epigenetic ITH on HER2-targeted therapy have not been clearly demonstrated. In order to implement ITH as a referenced factor for treatment selection, the ITH quantification is necessary. Gene-protein assay (GPA), in which immunohistochemistry and dual in situ hybridization are simultaneously performed on a single slide, allows bright-field analyses of both gene and protein status. We aimed to quantify HER2 ITH by the combination of gene and protein status and clarify its clinical significance. Methods: Fifty three patients with HER2-positive breast cancer, who underwent neoadjuvant trastuzumab with chemotherapy, were examined. Five representative microscopic images were captured from a GPA slide of a pre-therapeutic biopsy material. All evaluable tumor cells in the images were scored according to the HER2 status determined by the combination of gene copy number and protein expression (Table). We investigated the relationship between the HER2 scores and pathological complete response (pCR) to the neoadjuvant treatment by the logistic analysis. Results: The average of HER2 scores, indicating the degree of the HER2 status, varied from 2.21 to 5.98. It was significantly related to pCR (Estimate: 1.21, Std. error: 0.46, RR: 3.34, P=0.009, 95%CI: 1.35-8.25). The standard deviation of HER2 scores, indicating the degree of the HER2 ITH, varied from 0.13 to 1.37. It was significantly related to pCR (Estimate: -2.09, Std. error: 0.83, RR: 0.12, P=0.012, 95%CI: 0.02-0.63). Conclusions: HER2 ITH, quantified by GPA, is a predictive factor for the therapeutic effect to trastuzumab-based treatment in HER2-positive breast cancer. [Table: see text]

Abstract 3061: A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of circulating tumor cells

Abstract Background: Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis as “liquid biopsy”; however, attempts to isolate rare CTCs for further molecular analysis remain technically challenging by the currently available enrichment methods. In this study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology. Methods: We have developed a rare cell capture platform with On-Chip Sort (On-Chip Biotechnologies), a novel benchtop cell sorter equipped with a disposable microfluidic chip. Spike-in experiments using lung cancer cell lines with varying EpCAM expression levels (H1975, A549 and H1755) were conducted to assess capture and purification efficiency of our platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by fixation and labeling with cytokeratin (CK)-FITC, vimentin-PE, CD45-Alexa Fluor 700 antibodies, and/or Hoechst 33342 for nuclei staining. The enriched and labeled samples were then sorted on On-Chip Sort based on CK, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification (WGA) followed by mutation analysis using deep targeted sequencing. Amplicon libraries were generated using Illumina TruSeq Amplicon Cancer Panel and sequenced on a MiSeq sequencer. Copy number analysis of the WGA samples were performed by quantitative PCR (qPCR). Results: Spike-in experiments revealed excellent overall mean detection, recovery and purity rates of 85.0 ± 9.9%, 70.9% ± 10.6 and 72.9% ± 15.1, respectively (n=27). These results were obtained from samples containing as few as 5 tumor cells per 4 mL of blood, independent of EpCAM expression. A 100% success rate of mutation detection in EGFR, KRAS and BRAF genes from captured cells (n=27) was achieved by pyrosequencing as well as by deep targeted sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR gene n captured tumor cells. Conclusions: Using a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients. Further evaluation using clinical samples is warranted. Citation Format: Yasuhiro Koh, Masaru Watanabe, Takeshi Sawada, Hirotsugu Kenmotsu, Masakuni Serizawa, Masayuki Ishige, Kazuo Takeda, Nobuyuki Yamamoto, Toshiaki Takahashi, Fumiaki Koizumi. A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3061. doi:10.1158/1538-7445.AM2014-3061

A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood.

BackgroundPersonalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis; however, the technical difficulties in isolating and analyzing rare CTCs have limited progress to date. In this preclinical study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology.MethodsWe developed a cell capture platform using On-chip Sort (On-Chip Biotechnologies), a novel bench-top cell sorter equipped with a disposable microfluidic chip. Spike-in experiments comprising a series of lung cancer cell lines with varying epithelial cell adhesion molecule (EpCAM) expression levels were conducted to assess the capture and purification efficiency of the platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by sample fixation and labeling. The enriched and labeled samples were then sorted by On-chip Sort based on cytokeratin, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification followed by mutation analysis using deep targeted sequencing, and copy number analysis using quantitative polymerase chain reaction (qPCR).ResultsSpike-in experiments revealed an excellent overall mean capture rate of 70.9%. A 100% success rate in the detection of EGFR, KRAS and BRAF mutations from captured cells was achieved using pyrosequencing and deep sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR in captured tumor cells.ConclusionsUsing a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Abstract LB-227: Targeted next-generation sequencing (NGS) of circulating tumor cells (CTCs) and matched primary tumors.

Abstract Introduction: Personalizing cancer care relies on accurate detection of actionable genomic aberrations in tumor cells. Conventionally, this strategy relies on analysis of primary tumor samples, which are often temporally and biologically distinct from recurrent, metastatic or treatment-resistant disease. As an alternative, CTCs offer real-time cancer tissue for analysis that may more accurately represent the current state of a patient's disease. In this pilot, we used CTCs as source material for targeted NGS across a range of malignancies. Methods: Under IRB approval, blood samples from patients with advanced cancer were labeled with EpCAM ferrofluid and placed into the LiquidBiopsy® platform (Cynvenio Biosystems, Inc.), an immunoaffinity-based microfluidic device tailored to query genomic events. CTCs were identified by CK, CD45 and DAPI expression. A matched WBC pellet served as a control representing germline sequence. Amplicon libraries were generated using Life Technologies AmpliSeq 2.0 and sequenced with an Ion Torrent sequencer. When available, matched formalin fixed paraffin embedded (FFPE) primary tumor tissue from the same patient was analyzed in parallel. Somatic single nucleotide variants (SNV) present in CTCs or FFPE samples but not in WBC were identified. Results: CTCs were detected in 18 of 19 patients with advanced prostate (8), breast (6), renal cell (2), bladder (1), lung (1) and rectal (1) cancer (CTC median 54, range 15-421). Germline SNPs were consistently detected across WBC, CTC and FFPE samples. Significant SNVs (occurring in > 1% of DNA in a sample) were found in 6 of 18 patient CTC samples (range 1-6 SNVs/sample, frequency 1.1%-11.9% with 620X-14,422X sequence coverage depth). Numerous SNVs were identified in all 9 matched primary tumor FFPE specimens but did not correlate with the SNVs identified in the CTCs. Conclusion: This pilot demonstrates the feasibility of using CTCs as a real-time disease relevant substrate for NGS to identify personalized genomic targets. A high number of CTCs were detectable across malignancies, and CTC germline variants correlated with matched WBC controls. Cancer relevant SNVs were detected in a third of patients even using the relatively narrow primary tumor derived AmpliSeq platform. The FFPE specimens generated a high number of SNVs but did not correlate with CTC profiles, likely reflecting biological disparity between early localized tumors and advanced metastatic disease, as well as genomic artifacts introduced into primary tumor specimens by FFPE preservation. These data demonstrate the feasibility and potential biological and technical advantages of CTCs over traditional FFPE samples for genomic analysis in the pursuit of personalized cancer medicine. Citation Format: Stephen V. Liu, Paul W. Dempsey, William Strauss, Yucheng Xu, Tong Xu, Jessamine Winer-Jones, Tim J. Triche, David I. Quinn, Janine McMurdie, Andre Defusco, Amir Goldkorn. Targeted next-generation sequencing (NGS) of circulating tumor cells (CTCs) and matched primary tumors. [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 LB-227. doi:10.1158/1538-7445.AM2013-LB-227

330 Fluorescence in situ hybridization (FISH) for determination of chromosomal aberrations in tumor cells in patients with bladder cancer and in urothelial cells of healthy people

330 Fluorescence in situ hybridization (FISH) for determination of chromosomal aberrations in tumor cells in patients with bladder cancer and in urothelial cells of healthy people

Identification of Single Nucleotide Polymorphism Interactions Associated with Survival and Risk In Multiple Myeloma Using Novel Data Mining Methods

AbstractAbstract 2973Disease risk and therapeutic outcomes are impacted by both tumor heterogeneity as well as germline variations found in the population. Multiple myeloma (MM) shows significant heterogeneity in genetic aberrations in tumor cells, that together with inherited polymorphisms, affects disease risk and therapeutic response. In order to identify the impact of genetic variations (SNPs) on MM we have developed a Bank On A Cure platform for examining 3404 SNPs, selected in 983 genes associated with pathways affecting cellular functions important in cancer. Using SNP data sets we sought to identify genetic interactions, beyond single univariate association analysis. The challenge was to use data mining methods that take into account relatively small cohorts of patients, in which false discovery rates typically exceed the power of the study. We report results from using novel computational approaches that efficiently identify higher order SNP interactions associated with disease risk as well as survival outcomes, while minimizing the false discovery rate. The BOAC SNP panel was used to develop a data base on 143 patients selected for short (<1yr) versus long (>3yr) survival in ECOG 9486 and SWOG 9321; as well as 247 newly diagnosed patients and equal number of controls for disease risk analysis. One algorithm developed employs a discriminative pattern mining approach in which defined pathway sets of SNPs are used in combination testing. A second algorithm used identified SNPs that had some association with outcome (survival or disease status); but demonstrated a significant increase in associations when examined in combinations – we refer to this as a p-value jump association. Variations in genes associated with cell cycle, apoptosis, drug metabolism, stress response and immunity reached very low p-values, and survived multiple comparison testing when analyzed in combinations associated with both survival (PFS) predictions as well as analysis of case-control disease risk. Some of the key genetic variations identified in various combinations, included: PTRB, PTEN, CDK5, XRCC4, GSTA4, GPX, DYPD, PCNA, CYP4F2, VEGF, PON1, ALK, and BAG3. The data mining methods and algorithms used, and specific combinations associated with risk and survival, will be presented. These results are being further validated in new cohorts, and functional implications of identified genetic variants are being investigated in HapMap cell lines. Disclosures:No relevant conflicts of interest to declare.

FOXP1 and BCL2 Show Similar Immunoenzymatic Pattern in Bone Marrow Trephines of Chronic Lymphocytic Leukemia Patients

Indolent B lymphoproliferative disorder, chronic lymphocytic leukemia (CLL) represents one of the most common hematologic diseases in the Western world. Although there are many disease development markers known so far, for example, B-cell lymphoma/leukemia (BCL) 2, new ones are needed for better understanding course of the disease. FOXP1 is known to be strongly expressed after B-cell activation. Its essential role in B-cell development suggested that it could also have a role in a various tumor B-cells. We have analyzed 74 bone marrow samples from B-CLL patients for presence of FOXP1 and its gene aberrations in tumor cells. Our results showed presence of FOXP1 protein mostly in the same tumor cells as BCL2 protein, and their specific immunostaining pattern. Diffuse immunostaining pattern of both proteins is present in patients with higher clinical stages of B-CLL and with some other markers that indicate worse outcome of the disease. Thus, FOXP1 and/or BCL2 immunostaining of bone marrow trephine sections could serve as an immunohistochemical marker in B-CLL.

Quantification of Normal Cell Fraction and Copy Number Neutral LOH in Clinical Lung Cancer Samples Using SNP Array Data

BackgroundTechnologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.Principal FindingUsing SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.ConclusionGenome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.

Absence of Ras mutations in rat DMBA‐induced mammary tumors

Animal cancer models reduce genetic background heterogeneity and thus, may facilitate identification and analysis of specific genetic aberrations in tumor cells. Rat and human mammary glands have high similarity in physiology and show comparable hormone responsiveness. Thus, spontaneous and carcinogen (e.g., NMU and DMBA)-induced rat mammary models are valuable tools for genetic studies of breast cancer. In NMU-induced rat mammary tumors, activating mutations in Hras codon 12 have frequently been reported and are supposed to contribute to the mammary carcinogenic process. Involvement of Ras mutations in DMBA-induced tumors is less clear. In the present study we investigated the mutation status of the three Ras genes, Hras, Kras, and Nras, in DMBA-induced rat mammary tumors. We examined codons 12, 13, and 61 of all three genes for mutations in 71 tumors using direct sequencing method that in experimental conditions is sensitive enough to detect single nucleotide mutations even when present in only 25% of the test sample. No activating Ras gene mutation was found. Thus, in contrast to NMU-induced rat mammary tumor, tumorigenesis in DMBA-induced rat mammary tumors seems to be independent on activating mutations in the Ras genes. Our finding suggests that the genetic pathways selected in mammary tumor development are influenced by and perhaps dependent on the identity of the inducing agent, again emphasizing the importance of tumor etiology on the genetic changes in the tumor cells.

Stability analysis of mixtures of mutagenetic trees

BackgroundMixture models of mutagenetic trees are evolutionary models that capture several pathways of ordered accumulation of genetic events observed in different subsets of patients. They were used to model HIV progression by accumulation of resistance mutations in the viral genome under drug pressure and cancer progression by accumulation of chromosomal aberrations in tumor cells. From the mixture models a genetic progression score (GPS) can be derived that estimates the genetic status of single patients according to the corresponding progression along the tree models. GPS values were shown to have predictive power for estimating drug resistance in HIV or the survival time in cancer. Still, the reliability of the exact values of such complex markers derived from graphical models can be questioned.ResultsIn a simulation study, we analyzed various aspects of the stability of estimated mutagenetic trees mixture models. It turned out that the induced probabilistic distributions and the tree topologies are recovered with high precision by an EM-like learning algorithm. However, only for models with just one major model component, also GPS values of single patients can be reliably estimated.ConclusionIt is encouraging that the estimation process of mutagenetic trees mixture models can be performed with high confidence regarding induced probability distributions and the general shape of the tree topologies. For a model with only one major disease progression process, even genetic progression scores for single patients can be reliably estimated. However, for models with more than one relevant component, alternative measures should be introduced for estimating the stage of disease progression.

Fluorescence in situ hybridization: a multitarget approach in diagnosis and management of urothelial cancer

Noninvasive tests for detecting genetic or molecular alterations in urine indicative of urothelial cancer are increasingly becoming the focus of urological cancer research. Since its approval by the US FDA in 2001, the fluorescence in situ hybridization test (Vysis®UroVysion™) has been widely evaluated. In general, published data demonstrate better sensitivity and equal or better specificity compared with routine cytology, which is still considered the ‘gold standard’ in diagnosing and monitoring bladder tumors. However, the fluorescence in situ hybridization test seems to provide not only a useful tool in bladder cancer detection, but also in the diagnosis of upper urinary tract tumors, surveillance and determining therapy effectiveness. This multitarget assay that detects four different chromosomal aberrations in tumor cells is a kind of objective molecular cytology and has proven advantages over routinely used cytology.

Enhancement of chromosomal aberrations in tumor cells with a non-labeled Cu–PTSM and irradiation with Cu K-shell monochromatic X rays

This study aims to clarify the enhancement effect on chromosomal aberrations via selective energy absorption by Cu atoms in cultured bone cancer cells with non-labelled Cu-pyruvaldehyde-bis(N(4)-methylsemicarbazone) (Cu-PTSM) and monochromatic X rays. The X rays having the energy of the Cu K-shell absorption edge arising from synchrotron radiation (KEK-PF) was used as radiation source. Cu-PTSM labelled with copper radionuclides has been developed for medical imaging using positron emission tomography (PET). Evidence clearly showed that the absorption of X rays in the Cu K-shell caused this enhancement. After cells were treated with 1000 nM Cu-PTSM, the enhancement ratio of the initial yield of isochromatid breaks caused by CuK-H X-ray irradiation to that by 200 kV(p) X rays was approximately 2.8. About 5.6 times of the remaining isochromatid breaks were observed at 4 h in Cu-PTSM treated samples irradiated with CuK-H X rays comparing to that with 200 kV(p) X rays. In this study, uniqueness in physical property such as Cu atom K-shell ionisation was applied for the enhancement of biological effects in cancer cells.

Constitutional genomic instability, chromosome aberrations in tumor cells and retinoblastoma

Although retinoblastoma (Rb) is initiated as a result of biallelic inactivation of the RB1 gene, additional genetic events (M3) in tumor cells are indicative of their role in the full transformation of retinal cells. We investigated the constitutional genetic instability by fragile site (FS) expression studies and checked its relationship with loci of tumor cytogenetics in a series of 36 retinoblastoma patients (34 nonfamilial and 2 familial cases). Tumor cytogenetics revealed −13/+13, del/t(13)(q14) (50%), +1/del/t(1p/q) (65%), +6/i(6p) (60%), and del(16)(q13)/(q22∼q23) (60%). Conventional cytogenetics in leukocytes revealed constitutional del(13q14) in five unilateral Rb (URB) and one trilateral Rb (TRB). Constitutional del(16)(q22) and t(6;12) were also identified in two cases. Constitutional FS analysis showed a significant increase in the cellular fragility, with high prevalence at 13q14, 3p14, 6p23, 16q22∼q23, and 13q22 loci in retinoblastoma patients ( P<0.05). Patients with constitutional del(13)(q14) demonstrated higher fragility than those with normal constitution. A strong correlation between loci of constitutional FSs and loci of recurrent chromosomal abnormalities in tumors strengthen and support the proposal that FS loci present as inherent genomic instability in retinoblastoma. The chromosomal changes and resultant genetic mutations, along with RB1 mutation events, probably contribute synergistically to the development and progression of Rb malignancy. Implementation of fluorescence in situ hybridization to nonfamilial Rb on a large scale (113 cases) could detect constitutional RB1 deletion in 12.3% of cases, with equally higher incidence in URB (14.7%) and bilateral Rb (13.6%), demonstrating that the true prevalence of patients with predisposition to RB1 mutation in sporadic URB is definitely higher in our populations. Also, higher incidence of constitutional RB1 deletion mosaicism in unilateral than in bilateral Rb indicates that the constitutional genetic mosaicism in URB should be given serious consideration during genetic counseling.

Chromosome aberrations in B-cell chronic lymphocytic leukemia: reassessment based on molecular cytogenetic analysis.

In B-cell chronic lymphocytic leukemia (B-CLL) clonal chromosome aberrations are detected in approximately 40-50% of tumors when using conventional chromosome banding analysis. Most studies find trisomy 12 to be the most frequent chromosome aberration, followed by structural aberrations of the long arm of chromosomes 13 and 14. Trisomy 12 and the "14q+" marker are associated with shorter survival times, while the patients with 13q abnormalities have a favorable outcome, similar to those with a normal karyotype. The development of molecular cytogenetic techniques has greatly improved our ability to detect chromosome aberrations in tumor cells. Using fluorescence in situ hybridization, chromosome aberrations can be detected not only in dividing cells but also in interphase nuclei, an approach referred to as interphase cytogenetics. The prevalence of specific aberrations in B-CLL is currently being reassessed by interphase cytogenetics. By far the most frequent abnormality are deletions involving chromosome band 13q14, followed by deletions of the genomic region 11q22.3-q23.1, trisomy 12, deletions of 6q21-q23, and deletions/mutations of the TP53 tumor suppressor gene at 17p13. The evaluation of the true incidence of these aberrations now provides the basis for more accurate correlations with clinical characteristics and outcome. Deletions/mutations of the TP53 gene have been shown to be associated with resistance to treatment and to be an independent marker for poor survival. 11q deletions have been associated with extensive nodal involvement, rapid disease progression, and short survival times. Whether trisomy 12, 13q14, and 6q deletions have a prognostic impact awaits further study. The application of these molecular cytogenetic techniques will also contribute to the identification of the pathogenetically relevant genes that are affected by the chromosome aberrations in B-CLL.

Tumor Invasion: Role of Growth Factor-Induced Cell Motility

Cancer progression to the invasive and metastatic stage represents the most formidable barrier to successful treatment. To develop rational therapies, we must determine the molecular bases of these transitions. Cell motility is one of the defining characteristics of invasive tumors, enabling tumors to migrate into adjacent tissues or transmigrate limiting basement membranes and extracellular matrices. Invasive tumor cells have been demonstrated to present dysregulated cell motility in response to extracellular signals from growth factors and cytokines. Recent findings suggest that this growth factor receptor-mediated motility is one of the most common aberrations in tumor cells leading to invasiveness and represents a cellular behavior distinct from-adhesion-related haptokinetic and haptotactic migration. This review focuses on the emerging understanding of the biochemical and biophysical foundations of growth factor-induced cell motility and tumor cell invasiveness, and the implications for development of targeted agents, with particular emphasis on signaling from the epidermal growth factor (EGF) and hepatocyte growth factor (HGF) receptors, as these have most often been associated with tumor invasion. The nascent models highlight the roles of various intracellular signaling pathways including phospholipase C-gamma (PLC gamma), phosphatidylinositol (PI)3'-kinase, mitogen-activated protein (MAP) kinase, and actin cytoskeleton-related events. Development of novel agents against tumor invasion will require not only a detailed appreciation of the biochemical regulatory elements of motility but also a paradigm shift in our approach to and assessment of cancer therapy.

Detection of Chromosomal Aberrations in Tumor Cells and Tumor Infiltrating Lymphocytes by Molecular Cytogenetics in Patients with Gynecological Cancer

Conventional cytogenetic studies of tumor cells from patients with breast or ovarian cancer have shown multiple chromosomal abnormalities including chromosomes 7, 12, and 17. This study was designed to analyze the cytogenetic features of tumor cells and tumor infiltrating lymphocytes (TILs) by using a combination of magnetic activated cell sorting (MACS) and fluorescence in situ hybridization (FISH). Tumor cell, peripheral blood (PB), and TIL samples from 37 patients (20 ovarian tumors, 13 breast cancers, 3 uterine sarcoma, 1 carcinoma of the filamentary tube) were analyzed for the presence of numerical aberrations of chromosomes 7, 12, and 17. All of the tumor cells showed a high frequency of numerical aberrations of chromosomes 7, 12, and 17, especially trisomies or tetrasomies. There was no statistically significant difference in the incidence of chromosomal abnormalities in tumor tissue and effusions, or between primary and relapsed disease in patients with breast or ovarian tumors. However, tumor cells from patients with solid metastatic disease had significantly higher numbers of aberrations of chromosome 7 in the primary tumor than in tumors from patients without metastases (P = 0.049), suggesting that chromosome 7 is frequently involved in the progression of disease. Monosomies and trisomies of chromosomes 7 and 12 also occurred at a low percentage of TILs without any statistically significant difference between primary and relapsed tumors. The presence of these aneuploidies might be responsible for treatment failures in the immunotherapy of gynecological cancer.

Use of FISH for analysis of structural and numerical chromosome aberrations in tumor cells from paraffin blocks: W. Lee, K. Han, C. Harris and L. Meisner University of Wisconsin, State Lab of Hygiene. Madison, WI U.S.A.

Use of FISH for analysis of structural and numerical chromosome aberrations in tumor cells from paraffin blocks: W. Lee, K. Han, C. Harris and L. Meisner University of Wisconsin, State Lab of Hygiene. Madison, WI U.S.A.