Abstract Background: Approximately 47-65% of estrogen receptor-positive (ER+)/HER2-negative breast cancers (BC), although classified as HER2-negative due to the absence of HER2 gene amplification, exhibit low levels of HER2 expression (referred to as HER2-low BC) with immunohistochemistry (IHC) 1+ or 2+ without amplification scores. More recently, an additional subclass—HER2-ultralow—has been proposed for tumors with HER2 IHC-0 and <10% tumor cell staining. Emerging evidence indicates that HER2-low and HER2-ultralow BC may represent biologically distinct entities compared with HER2-null BC. Here, the latest updates of the NextGIM trail, a retrospective, multicenter translational study designed to comprehensively characterize ER+/HER2-low BC, are presented. Methods: Primary tumor samples from patients enrolled in three randomized clinical trials (GIM2, GIM4, and GIM10) of the GIM (Gruppo Italiano Mammella) Investigator Group, underwent re-assessment of HER2 expression and tumor-infiltrating lymphocytes (TILs) in accordance with ASCO/CAP and TILs Working Group guidelines. In parallel, gene expression profiling was performed using the nCounter Breast Cancer 360 Panel on the NanoString platform. Clinical and survival data will be updated and integrated to evaluate potential prognostic markers and to investigate mechanisms of treatment resistance. Results: To date, from a total of 283 patients selected at participating centres, 208 samples were deemed suitable for molecular analyses after pathological and RNA quality/quantity assessment. HER2 expression has been re-evaluated in 179 samples. At diagnosis, HER2 classification was: 45.8% HER2-negative (IHC not specified), 26.8% HER2-0, 19.6% HER2-1+, and 7.8% HER2-2+. After pathological reassessment, the distribution shifted to 58.7% HER2-null, 20.7% HER2-ultralow, 15.1% HER2 1+, 5.0% HER2 2+, and 0.6% HER2 3+. Notably, HER2-null BC resulted more prevalent in older archival samples (before 2011). TILs levels ≤1% were more frequently observed in HER2-null and HER2-ultralow tumors compared with HER2-1+ and HER2-2+ (59.1% & 67.6% vs. 25.9% & 55.6%).. Genomic profiling has been completed for 208 cases and data analyses are ongoing. An integrated analysis of molecular, pathological and clinical data will be conducted upon completion of the dataset. Conclusion: The results obtained so far from HER2 re-evaluation has indicated that the interval between sample collection and reassessment can affect tissue antigenicity and immunohistochemistry results, and should be considered when re-evaluating HER2 status for therapeutic purposes. Overall, the study is expected, integrating genomic, immunological, and clinical information, to validate HER2-low and -ultralow expression as prognostic/predictive biomarkers to support personalized treatment. Citation Format: Barbara Cardinali, Alice Stella, Chiara Molinelli, Marco Bruzzone, Yanina Lizet Castillo, Francesca Pitto, Barbara Massa, Simona Pigozzi, Andrea Sciutto, Giorgia Anselmi, Maria Dono, Virna Maltoni, Benedetta Conte, Giulia Buzzatti, Davide Soldato, Gaia Griguolo, Valentina Guarneri, Mario Giuliano, Caterina Marchiò, Fabio Puglisi, Lorenzo Gerratana, Francesca Poggio, Lucia Del Mastro. Genomic, immune, and clinical characterization of estrogen receptor-positive, HER2-low breast cancer: The NextGIM trial latest updates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7907.

Abstract Introduction: While liquid biopsy shows promise as a non-invasive precision medicine tool, its ability to comprehensively represent the complexity of a tumor or only a subset of its clones remains uncertain. This issue is particularly crucial in the context of triple-negative breast cancer (TNBC), a type of cancer known for its significant heterogeneity. The goal of this study is to examine the clonal diversity within Circulating Biomaterials (CBMs): namely circulating tumor cells (CTCs), extracellular vesicles RNA (evRNA), and circulating tumor DNA (ctDNA), employing genetic barcoding and patient-derived TNBC xenografts. Methodology: Cells from TNBC patients' resected tumors were tagged with a unique barcode sequence through vector transduction. These barcoded cells were subsequently introduced into immunodeficient mice to form xenografts. When these xenografts grew to a certain size (15mm in diameter), the mice were humanely euthanized, and the tumor tissues as well as CBMs from their blood and plasma were collected. CTCs were isolated through negative enrichment and DEPArray technology; evRNA was extracted using ultracentrifugation and TRIzol™ Plus RNA Purification method; ctDNA was retrieved using the QIAamp DNA Micro Kit. Tumor tissues were processed for RNA and DNA using RNeasy micro kit and Qiamp DNA micro kit, respectively. RNA sequencing of the tumor tissues was carried out using the Lexogen - CORALL Total RNA-Seq Kit and Illumina NextSeq high output reagents. Results: Analysis of xenografts from four patients showed that ctDNA was the predominant genetic material detected through liquid biopsy. Crucially, clonal analysis using Next Generation Sequencing (NGS) demonstrated that ctDNA provides an accurate reflection of the primary tumor's clonal heterogeneity. The method efficiently identified both main tumor clones and subclones, even those constituting as little as 1% of the tumor, with a 50% detection probability. Transcriptomic analysis of the tumor tissues uncovered a distinct mesenchymal and pro-metastatic signature, more pronounced in tumors with higher ctDNA release. Clonal analysis of CTCs and evRNA is still ongoing. Conclusions: This investigation affirms the effectiveness of ctDNA analysis in liquid biopsy for identifying subclonal diversity in TNBC. Our results suggest that liquid biopsy via ctDNA is capable of managing TNBC's extensive intra-tumoral heterogeneity, including the identification of smaller subclones. Furthermore, a link was identified between the release of ctDNA and a mesenchymal, prometastatic gene expression pattern in the tumors. Citation Format: Davide Ceresa, Barbara Cardinali, Antonio Daga, Roberta Tasso, Andrea Sciutto, Irene Appolloni, Daniela Marubbi, Franca Carli, Piero Fregatti, Paolo Malatesta, Lucia Del Mastro. Harnessing liquid biopsy and genetic barcoding in evaluating diversity within triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2405.

Abstract Introduction: One of the unmet needs in Breast Cancer (BC) prevention is how to identify women at higher risk of developing this disease, who might benefit from periodic surveillance and/or from chemoprevention. The goal of this retrospective study is to assess the role miRNAs isolated from extracellular vesicles (EV-miRNAs) as possible early biomarkers for identifying patients with higher risk of developing BC, in order to personalize their screening programme. To this goal, EV-miRNAs from cyst fluid derived from women with Gross Cyst Disease of the Breast (GCDB - a benign disease of the mammary gland associated with a 2-4-fold increase in the risk of developing BC) have been analysed. Methodology: Cyst fluids have been selected among samples from a cohort of 600 patients diagnosed with GCDB between 1985 and 1993, some of whom developed BC during follow-up. EV-miRNAs have been extracted from cyst fluid using the exoRNeasy midi kit (Qiagen), qualitatively evaluated by Bioanalyzer (Agilent) and quantified by Qubit™ using the microRNA Assay Kit (Thermo Fisher Scientific). The MiRNome profiles have been assessed by microarray using an optimised protocol on Agilent platform (labelling and hybridization on SurePrint Human miR Microarrays 8 × 60 K and images acquisition by G2565CA scanner). Results: A total of 117 samples (58 cases and 59 controls, paired on the bases of clinical and pathological variables) have been analysed. Single variable logistic regression analysis on the most expressed EV-miRNAs (logFC >= 6 (n=329)), selected six EV-miRNAs (miR-6076, miR-202-3p, miR-6872-3p, miR-769-3p, miR-5195-3p, miR-4443) and one (miR-4713-3p) negatively and positively associated with BC development, respectively (raw p-value <0.05). A risk score combining the seven EV-miRNAs was derived by multivariable logistic regression modelling and reported an AUC=0.73 (95% [CI] 0.6427-0.8231). The model was refined including clinical variables and applying step-wise logistic regression analysis. The combination of 3 EV-miRNAs (miR-6076, miR-6872-3p, miR-5195-3p) with menopausal status, familiarity and type of cyst yielded an AUC of 0.8 (95% [CI] 0.7015-0.8847). Logistic regression was also performed on Type I cysts (n=78), that are correlated with a higher risk of developing BC, pointing in evidence 3 EV-miRNAs with a negative (miR-6076, miR-6872-3p) or positive (miR-4515) correlation with BC development risk (AUC = 0.74, 95% [CI] 0.6278-0.8475). In the model comprehensive of clinical variables, two of those EV-miRNAs (miR-6872-3p, miR-4515) together with familiarity and menopausal status were selected, giving an AUC of 0.8 (95% [CI] 0.6961-0.8991). Conclusion: This study allowed the identification of a EV-miRNA-based BC risk signature obtained by liquid biopsy on cyst fluid. The final purpose of this project is to transfer this signature in the clinic by testing it prospectively on plasma samples. Citation Format: Barbara Cardinali, Patrizia Piccioli, Francesco Boccardo, Alessandra Rubagotti, Linda Zinoli, Andrea Sciutto, Simona Coco, Giovanna Chiorino, Paola Ostano, Emir Sehovic, Zita Cavalieri, Cristina Bruzzo, Silvia Marconi, Roberta Tasso, Rodolfo Quarto, Davide Ceresa, Paolo Malatesta, Lucia Del Mastro. The EsomiR project: A case control study to assess the role of exosomal miRNAs in breast cancer cancerogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1072.

Breast cancer (BC) is the first cancer in terms of frequency and mortality in women. The aim of this retrospective study is to identify the role of exosome microRNAs (Exo-miRNAs) as possible early biomarkers for identifying patients with higher risk of developing the disease and to personalize their screening programmes. For this purpose, Exo-miRNAs derived from cyst fluid of women affected by Gross Cyst Disease of the Breast (GCDB - a benign disease of the mammary gland associated with a 2-4-fold increase in the risk of developing BC) have been analysed. Cyst fluids have been selected among samples from 600 patients diagnosed with GCDB between 1985 and 1993, some of whom developed BC during follow-up. Exo-miRNAs were extracted from 400 μl of cyst fluid using the exoRNeasy midi kit (Qiagen). Quality control was assessed by Bioanalyser (Agilent) and Qubit™ using the microRNA Assay Kit (Thermo Fisher Scientific). Exo-miRNome profiles were investigated by microarray on Agilent platform using an optimised protocol (labelling and hybridization on SurePrint Human miR Microarrays 8×60 K and image acquisition using the G2565CA scanner). A total of 46 samples (23 cases and 23 controls, paired on the bases of clinical and pathological variables) have been analysed. The analysis allowed to identify 10 Exo-miRNAs slightly modulated between cases and controls, and 7 Exo-miRNAs in the Type 1 cyst, that are correlated with a higher risk of developing BC. A single variable logistic regression model on the most expressed Exo-miRNAs detected, pointed out 3 Exo-miRs (miR-6076, miR-3660, miR-6879-5p), whose expression was negatively associated to the development of BC (raw p-value <0.05). A risk score combining the 3 Exo-miRNAs by multivariable logistic regression modelling showed an optimal accuracy (AUC = 0.8, 95%, [CI] 0.6703-0.9282). These data, to be validated in further 72 samples equally divided between cases and controls, show that Exo-miRNAs are promising minimally invasive biomarkers of BC risk. In order to transfer these data to the clinic, the Exo-miR signature will be tested on plasma of patients with BC enrolled in a prospective study.

The study of circulating cancer-derived components (circulome) is considered the new frontier of liquid biopsy. Despite the recognized role of circulome biomarkers, their comparative molecular profiling is not yet routine. In advanced breast cancer (BC), approximately 40% of hormone-receptor-positive, HER2-negative BC cases harbor druggable PIK3CA mutations suitable for combined alpelisib/fulvestrant treatment. This pilot study investigates PIK3CA mutations in circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (EVs) with the aim of determining which information on molecular targetable profiling could be recollected in each of them. The in-depth molecular analysis of four BC patients demonstrated, as a proof-of-concept study, that it is possible to retrieve mutational information in the three components. Patient-specific PIK3CA mutations were found in both tissue and ctDNA and in 3/4 cases, as well as in CTCs, in the classical population (large-sized CD45−/EpCAM+/− cells), and/or in the “non-conventional” sub-population (smaller-sized CD44+/EpCAM−/CD45− cells). Consistent mutational profiles of EVs with CTCs suggest that they may have been released by CTCs. This preliminary evidence on the molecular content of the different circulating biomaterials suggests their possible function as a mirror of the intrinsic heterogeneity of BC. Moreover, this study demonstrates, through mutational assessment, the tumor origin of the different CTC sub-populations sustaining the translational value of the circulome for a more comprehensive picture of the disease.

Mutations of the KRAS2 protoncogene and inactivation of the TP53 oncosuppressor gene have been suggested to contribute to chromosomal instability (CIN) and aneuploidy in colorectal cancer (CRC). Previous work has also shown that the degree of DNA ploidy [DNA index (DI)], as obtained by flow cytometry in CRC, is non‐randomly distributed and, in particular, that DI near‐diploid and near‐triploid values are well separated by a low‐probability valley region. At present, it is not known whether a relationship exists between DI and the mutational status of KRAS2 and TP53 . Multiple samples obtained from 35 human sporadic CRCs have been used to provide nuclei suspensions for flow cytometric analysis and sorting of specific DI subpopulations. Sorted nuclei were then used to analyze the high‐microsatellite‐instability (MSI‐H) phenotype and the mutation spectrum of the KRAS2 and TP53 genes. A single MSI‐H case was detected. There were 6 DNA diploid (DI = 1) and 29 aneuploid (DI ≠ 1) CRCs, with the DI aneuploid cases non‐randomly subdivided in 9 near‐diploid (DI ≠ 1 and DI ≤ 1.4), 8 near‐triploid (1.4 < DI < 1.6), and 12 high‐aneuploid (DI ≥ 1.6) cases. Proximal CRCs were more often DNA diploid and near‐diploid than distal ones, and Dukes' C cases were more commonly high‐aneuploid than Dukes' B. Moreover, the incidence of mutations of the KRAS2 and TP53 genes was lowest among the DNA near‐triploid subpopulations and highest among the near‐diploid ones. We suggest that DNA near‐diploid and near‐triploid subpopulations in human sporadic CRC reflect different genetic mechanisms of CIN and have a potentially different clinical behavior. © 2003 Wiley‐Liss, Inc.

Background & Aims: Current models of colorectal adenoma to carcinoma progression do not fully reflect the genetic heterogeneity and complexity of the disease. The aim of the present study was to identify genetic changes discriminating adenomas that have progressed to carcinoma from adenomas that have not progressed, and to refine the current genetic models of colorectal adenoma to carcinoma progression, based on a genome-wide analysis of chromosomal aberrations. Methods: Sixty-six nonprogressed colorectal adenomas, 46 progressed adenomas (malignant polyps), and 36 colorectal carcinomas were screened for chromosomal aberrations by comparative genomic hybridization, and for mutations in the adenomatous polyposis coli (APC) and K-ras gene. Data analysis focused on cancer-associated genetic changes in adenomas. Results: Accumulation of losses in 8p21-pter, 15q11-q21, 17p12-13, and 18q12-21, and gains in 8q23-qter, 13q14-31, and 20q13 were strongly associated with adenoma-to-carcinoma progression, independent of the degree of dysplasia. Hierarchic cluster analysis demonstrated the presence of 3 distinct subgroups of adenomas, characterized by unique combinations of genetic aberrations in the adenomas (17p loss and K-ras mutation, 8q and 13q gain, and 18q loss and 20q gain, respectively). Conclusions: The presence of 2 or more of the aforementioned 7 chromosomal changes was associated with progressed colorectal adenomas and colorectal cancer. In addition, evidence was found that these chromosomal abnormalities occurred in specific combinations of a few abnormalities rather than as a mere accumulation of events, indicating the existence of multiple independent chromosomal instability pathways of colorectal cancer progression.

BACKGROUND:Activation of the k-ras2 pathways and chromosomal instability leading to aneuploidy in human sporadic colorectal cancer (sCRC) is essential to the tumor cell ability to survive, grow, and metastatize.METHODS:The study included 135 patients with sCRC who were followed up for a median of 72 months. Multiple fresh-frozen fragments obtained from superficial and invasive areas of the tumors were mixed and used to detect the degree of DNA aneuploidy (DNA index [DI]) and S-phase fraction by two scatter signals and 4,6-diamidino-2-phenylindole-2-hydrocloride (DAPI) fluorescence flow cytometry (FCM). PCR amplification and k-ras2 mutation spectrum analysis were performed using enriched epithelial nuclei after sorting DNA aneuploid nuclei and DNA diploid nuclei from which tissue-infiltrating lymphocytes were absent.RESULTS:DNA aneuploidy was detected in 98 (73%) and k-ras2 mutations in 54 cases (40%). Univariate analyses of overall survival with both Dukes' A to D or B to C series of cases showed that DNA multiple aneuploidy, k-ras2 mutations, older age, and distal site, but not increased S-phase fraction, were predictive of worse outcome. Multivariate Cox models strongly indicated that k-ras2 mutations, but neither single nor multiple DNA aneuploidy, were an independent prognostic factor in both series of patients. In particular, with B and C Dukes' stage patients (n = 110), the relative risk (RR) of death was above 2.5 with k-ras2 mutations and above 3 with the G-->C/T transversions.CONCLUSION:Combined FCM and k-ras2 analysis may be used to predict long-term increased risk of death in sCRC patients.

Mutant ras genes occur frequently in human neoplasia and, in particular, in pancreatic, colorectal, and lung adenocarcinomas, Recent evidence suggests that G-->T and G-->C transversions of the Ki-ras gene in codon 12 may lead to biological effects in vitro and in vivo that may be associated with an abnormal cell cycle and increased tumour aggressiveness. The role of Ki-ras activation (a G-->C transversion in codon 12, arginine for glycine) in the cell cycle and apoptosis was investigated using control and permanently transfected NIH3T3 mouse fibroblasts. Flow cytometry was used to evaluate the G1-, S- and G2M-phase transit times, the potential doubling time, the growth fraction, and the cell loss factor during asynchronous exponential growth. Apoptosis was induced in both cell lines by absence of growth factors for an extended period of time (72 h) and quantitatively evaluated using the TUNEL method coupled with flow cytometry, It was found that codon 12 G-C Ki-ras transfected cells compared with controls, had a significant prolongation of G1 by about 50%, a reduction of the G2M transit time by 30%, and a decrease of the cell loss factor by about 90%. Apoptotic cells were about 10% in control and less than 0.5% in Ki-ras transfected cells after 72 h starvation-confluency. These data suggest that codon 12 G-->C Ki-ras activation in mouse NIH3T3 fibroblasts is associated with deregulation of checkpoint controls in the GI and G2M phases of the cell cycle and inhibition of apoptosis. It appears plausible that these cell mechanisms are related to a proliferative advantage and that they may also be important in the progression of human tumours characterized by specific Ki-ras mutations. Copyright (C) 2000 John Wiley & Sons, Ltd.

The possible role of K‐ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP) adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%), low grade dysplasia (63%), and were preferentially located in the right colon (60%). Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K‐ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO) hybridization, while flow cytometry (FCM) was used for evaluating degree of DNA ploidy and S‐phase fraction. Overall, incidences of K‐ras2 mutations, DNA aneuploidy and high S‐phase values (>7.2%) were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K‐ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K‐ras2 and size, DNA ploidy and size and K‐ras2 and S‐phase (p). In particular, among the wild type K‐ras2 adenomas, high S‐phase values were detected in 8% of the cases versus 57% among the K‐ras2 mutated adenomas (p=0.0005). The present series of FAP adenomas indicates that K‐ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

BACKGROUND The origin and evolution of somatic chromosome aberrations in colorectal cancer is still poorly understood. The data in the literature suggest that some specific chromosome aberrations are more common. It is not known, however, if there is a correlation of these with near-diploid and high aneuploidy previously proposed to be a characteristic of the adenoma-carcinoma sequence. METHODS Chromosome 1, 7, 17 and 18 numerical aberrations and 1p deletions were evaluated by fluorescence in situ hybridization analysis for 20 human sporadic colorectal adenocarcinomas in 70 distinct tumor sectors and correlated with flow cytometric DNA index (DI) values. RESULTS Aneusomy for at least one of the investigated chromosomes was observed in 60 of 70 tumor sectors corresponding to 19 of 20 adenocarcinomas (95%). Deletions at 1p, observed in 8 of 18 adenocarcinomas (44%), were intratumor homogeneous in 7 of 8 tumors. In contrast, the other aberrations were intratumor heterogeneous. Aneusomies of chromosomes 1, 7, and 17 were strongly associated with DNA high aneuploidy (DI > or = 1.4), whereas aneusomy of chromosome 18 and 1p deletions were equally common among DNA diploid and near-diploid tumors (DI < 1.4 and DI not equal to 1). CONCLUSIONS Overall, these data suggest the existence of different aneuploidization routes correlated with specific chromosome aberrations. In addition, intratumor homogeneity of 1p deletions appears to be an indication of early occurrence or strong selection. We also suggest that tumors with monosomies and in particular monosomies-trisomies for the same chromosomes support a model of aneuploidization and chromosome instability during the colorectal tumor progression based on loss of symmetry during chromosome segregation (Giaretti: Lab Invest 71:904-910, 1994).

Recent studies indicate that p21ras proteins mediate their multiple cell functions through interactions with multiple effectors and that the number of new effectors is growing. We recently reported that K-ras2 mutations in human colorectal adenomas were associated with chromosome instability and proliferation changes. In the present study, we extend these previous observations. Hereditary and multiple (n > or = 5) adenomas and adenomas with early cancer were excluded. Dysplasia was moderate in 91 cases and high in 25, and the median adenoma size was 1.5 cm. K-ras2 spectrum analysis was done by sequence-specific oligonucleotide hybridization using nuclear suspensions provided by analysis and sorting of multiparameter flow cytometry. In particular, tissue inflammatory cells were separated for DNA diploid tumors, whereas DNA aneuploid epithelial subclones were analyzed separately. K-ras2 mutations and DNA aneuploidy were both detected in 29 of 116 (25%) cases. DNA aneuploid index was in the near-diploid region in the majority of cases. DNA aneuploidy was strongly associated with G-->C/T transversions. An association was also found between low S-phase values and G-->A transitions. These findings were confirmed using multivariate logistic regression analysis to account for the effects of size, dysplasia, site, type, age, and sex. These data suggest that specific K-ras2 mutations in a subgroup of human sporadic colorectal adenomas play a role in chromosome instability and, contrary to expectations, are associated with inhibition of proliferation.

Human sporadic colorectal adenomas are characterized by a relatively high occurrence of aneuploidy. Similarly, 1p deletions have been reported to be an early event in colorectal tumorigenesis, while chromosome 7, 17 and is gain/losses were also found. The present study investigated 1p deletions, the numerical aberrations of chromosomes 1, 7, 17 and 18, and the nuclear DNA content as obtained by flow cytometry in a series of 34 human sporadic colorectal adenomas. From these adenomas, 51 intra-adenoma regions were microdissected according to 2 degrees of dysplasia and presence of foci of early cancer. Isolated epithelial nuclei were analyzed by fluorescence in situ hybridization interphase cytogenetics using centromeric probes for chromosomes 7, 17 and 18 and, in a double-target analysis, a centromeric probe for chromosome 1 simultaneously with a telomeric probe mapping to the 1p36 band. Aneuploidy incidence due to presence of numerical aberrations for at least one among the investigated chromosomes and/or abnormal flow-cytometric DNA content was 35%, while 1p deletion incidence was 38%. The correlation of 1p deletions with aneuploidy was statistically highly significant (P = 0.003), suggesting that loss of genes in this region may be implicated in chromosome instability. (C) 1998 Wiley-Liss, Inc.

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases. Five tumours (25%) were DNA diploid, while 15 tumours (75%) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60% of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p < 0.04, Fisher’s exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86%, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation. The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.