Aneuploid Carcinomas Research Articles

Abstract A007: Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F)

Abstract Background: Although a range of therapeutic options are available in the management of invasive breast carcinomas, spatial segregation of tumour subclones has hampered biomarker identification in single-region samples. Representative sampling (RS) overcomes spatial bias by sampling from a homogenized and well-mixed cancer specimen. The Homogenization of Leftover Surgical Tissue Feasibility Study (HoLST-F - NCT03832062) is a prospective trial aiming to assess the feasibility of RS in tumor tissue leftover after pathology sampling. An interim analysis of the breast cancer cohort is presented. Methods: In the context of the HoLST-F study and pre-specified end-points, representative samples derived from 75 leftover invasive breast carcinoma specimens underwent flow cytometric analysis of CK8/18, CD3, Ki67 and DAPI. Tumor cell enrichment by CK8/18 positivity and ploidy (in aneuploid carcinomas) was performed for downstream DNA extraction and whole exome sequencing. Somatic variants were determined using a bespoke pipeline to remove artefacts associated with fixation. Variant oncogenicity and therapeutic evidence levels were assigned by OncoKB variant annotation. Quantitative image analysis was applied to tissue sections from diagnostic FFPE blocks stained with Ki67 immunohistochemistry (n=78) and H&E for lymphocytic infiltration scores (n=175) to correlate with flow cytometry. Results: In enriched representative samples, oncogenic mutations were commonly identified at high variant allele frequency (VAF) in known breast carcinoma driver genes including PIK3CA, CDH1, TP53, KMT2C and GATA3. Other clinically relevant oncogenic mutations were identified in ESR1, PTEN, ERBB2, RB1, AKT1, BRCA1, NF1 and FOXA1 which have been associated with resistance to various anti-cancer therapies. These mutations occurred at low and high VAFs indicative of both of clonal and sub-clonal resistance mechanisms. Recurrent variants in ESR1 (e.g. D538G) and PIK3CA (e.g. H1047R/L) were associated with Level 1 evidence for use as predictive biomarkers, while variants in twelve other genes across the cohort were associated with Level 2-4 evidence. Lymphocyte infiltration scores and Ki67 expression varied by tumor region, however RS by flow cytometry showed strong correlation with a weighted average across multiregional quantification of Ki67 expression (R=0.81, p=2.8 × 10-8) and lymphocyte infiltration (R=0.61, p<2.2 × 10-16). Conclusions: RS of invasive breast carcinoma in the HoLST-F trial has recapitulated the expected genomic driver landscape in breast cancer, in addition to identifying both clonal and subclonal genomic mechanisms of therapy resistance. Many of these mutations are either current or emerging therapeutic targets. Flow quantification of cells expressing phenotypic biomarkers (Ki67 and CD3) is feasible through RS and early analysis indicates that RS correlates with a weighted average across multiple regions. Leftover surgical tissue is an underutilized resource for biomarker assessment in breast carcinomas and can be examined by RS. Citation Format: Brian Hanley, Lisa Gallegos, Lavinia Spain, Husayn Pallikonda, Zayd Tippu, Samantha Hill, Aoune Barhoumi, Fiona Byrne, Yulia Dogva, Ashley Gilchrist, Glenn Noel-Storr, Hannah Veloz, Stacey Stanislaw, Harold Sansano, Kim Edmonds, Eleanor Carlyle, Nicholas Turner, James Larkin, Nelson Alexander, Samra Turajlic. Representative sampling of invasive breast carcinomas: Interim report from a prospective study (HOLST-F) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A007.

Abstract 5381: Elevated PD-L1 and HER2 expression on aneuploid carcinoma cells in malignant ascites accelerate stumor cell immune evasion in gastrointestinal cancer patients

Abstract Background Peritoneal dissemination (PD) is a type of metastasis frequently found in gastrointestinal (GI) cancer patients, resulting in the production of massive malignant ascites which associate with poor prognosis. However, the question of how the expression of tumor biomarkers on cancer cells in malignant ascites correlates with PD is yet to be answered. This study aims to investigate the clinical significance of PD-L1 and HER2 expression on malignant ascites disseminated tumor cells (maDTCs) in GI carcinoma patients. Patients and Methods Paired blood and malignant ascites specimens were respectively collected from 24 of metastatic GI subjects, including 15 gastric, 2 pancreatic, 1 esophageal, and 6 colorectal cancer patients. Non-hematologic CTCs and maDTCs were enriched by subtraction enrichment (SE), followed by immunostaining-FISH (iFISH) to co-examine PD-L1 and HER2 expression on aneuploid maDTCs and CTCs. In addition, CD4+, CD8+, Treg and naive T cells in the same cohort of samples were quantified by flow cytometry. Results In situ karyotypic and phenotypic characterization performed by iFISH showed that the average number of maDTCs in the 24 patients was 32755/ml ascites, which was significantly higher than the average number of CTCs detected (3.5/ml blood) (P<0.0001). The number of maDTCs and CTCs exhibited no correlation between each other (Pearson's product-moment correlation coefficient, R=-0.03, P=0.89). Further examination indicated that PD-L1 was expressed on 50% of maDTCs compared to only 11% for CTCs (P=0.0002). No correlation of PD-L1 expression between maDTCs and CTCs was observed (Pearson's product-moment correlation coefficient, R=0.25, P=0.24). Co-expression of PD-L1 and HER2 on CTCs and maDTCs was respectively examined in the recruited 15 gastric cancer (GA) patients. All 15 patients were histopathological HER2 negative (hHER2-). One subject (1/15=6.7%) showed 1 double HER2+/PD-L1+ CTC (1/63 CTCs in 15 GA patients=1.6%). In comparison, 6 patients (6/15=40%) had a total of 290377 HER2+/PD-L1+ maDTCs detected (290377/1306783 overall maDTCs=22%). In addition, flow cytometry analyses revealed that 5% of lymphocytes in malignant ascites were immunosuppression relevant regulatory T cells (Tregs), compared to only 2% of Tregs identified in the paired peripheral blood sample of the same patient. Conclusions Our study suggested that a higher proportion of Tregs in malignant ascites may contribute towards fostering an immunosuppressive microenvironment.PD-L1+/HER2+ maDTCs, in such an immunosuppressive microenvironment, might be able to evade immune system attack. Moreover, PD-L1 and HER2 identified on maDTCs could serve as therapy targets with respect to inhibition of peritoneal metastasis, potentially leading to an additional treatment strategy for GI cancer patients with malignant ascites. Citation Format: Yang Chen, Changsong Qi, JiaJia Yuan, Dan Liu, Zhi Peng, Xiaotian Zhang, Jian Li, Pin Peter Lin, Yilin Li, Lin Shen. Elevated PD-L1 and HER2 expression on aneuploid carcinoma cells in malignant ascites accelerate stumor cell immune evasion in gastrointestinal cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5381.

Abstract 4735: Single-cell sequencing from formalin-fixed paraffin-embedded breast cancers: a powerful tool to address intratumor genetic heterogeneity

Abstract Background: At diagnosis, tumors often consist of multiple, genotypically distinct cell populations. Genome sequencing of single cells has opened new avenues of investigation, yet the application of this technology remains limited to fresh/frozen tissue samples. Here, we describe and validate a method to perform single-cell massively parallel sequencing using DNA extracted from individual nuclei from FFPE tumor samples. Methods: Tissue sections are deparaffinized and the area of interest microdissected, DNA is reverse-crosslinked and the extracellular matrix is digested. Extracted single nuclei are then FACS-sorted, lysed and the whole genome repaired using a broad-spectrum DNA-repair enzyme cocktail. Repaired DNA is whole genome amplified (WGA) using GenomePlex WGA4 (Sigma-Aldrich) with modifications for heavily damaged genomic templates. Illumina sequencing libraries are generated using standard approaches followed by multiplex sequencing on an Illumina HiSeq2000. To obtain copy number (CN) profiles, single-cell sequencing data are mapped to the reference genome with PCR duplicates removed. Uniquely mapped reads are allocated and counted in genomic intervals of variable length (bins) with CN states roughly proportional to the number of allocated sequencing reads. Bin counts are then normalized on the basis of GC-content, segmented and transformed to CN values to identify long contiguous regions of equivalent CN. Results: As a proof-of-principle we performed single-cell CN profiling on two aneuploid synchronous ductal carcinomas in situ (DCIS)/invasive ductal carcinomas (IDCs), where both FFPE and frozen material was available. Using our novel methodology, 24 single nuclei were multiplexed/lesion. An average of 3 million reads/cell (average coverage of 0.1X/cell) was obtained, providing sufficient data to infer CN profiles of single cells accurately. These data were successfully employed to identify non-neoplastic cells and distinct clonal lineages of neoplastic cells within each lesion. The data obtained from the analysis of FFPE samples were concordant with those obtained from the analysis of matched frozen samples. Conclusions: We developed a robust procedure to perform single-cell massively parallel sequencing of individual nuclei isolated from FFPE samples, providing the opportunity to unlock pathology archives for studies aiming to catalog and dissect the biological and clinical relevance of intra-tumor genetic heterogeneity. Citation Format: Luciano G. Martelotto, Rita A. Sakr, Timour Baslan, Linda Rodgers, Hilary Cox, Jude Kendall, Tari A. King, Britta Weigelt, James Hicks, Jorge S. Reis-Filho. Single-cell sequencing from formalin-fixed paraffin-embedded breast cancers: a powerful tool to address intratumor genetic heterogeneity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4735. doi:10.1158/1538-7445.AM2015-4735

Aurora B expression and histone variant H1.4S27 phosphorylation are no longer coordinated during metaphase in aneuploid colorectal carcinomas.

Experimental model systems identified phosphorylation of linker histone variant H1.4 at Ser 27 (H1.4S27p) as a novel mitotic mark set by Aurora B kinase. Here, we examined expression of Aurora B and H1.4S27p in colorectal carcinoma (CRC) cell lines (HCT116, DLD1, Caco-2, HT29) and tissue specimens (n = 36), in relation to microsatellite instability (MSI) status and ploidy. In vitro, Aurora B (pro-/meta-/anaphase) and H1.4S27p (pro-/metaphase) were localized in mitotic figures. The proportion of labeled mitoses was significantly different between cell lines for Aurora B (p = 0.019) but not for H1.4S27p (p = 0.879). For Aurora B, these differences were not associated with an altered Aurora B gene copy number (FISH) or messenger RNA (mRNA) expression level (qRT-PCR). Moreover, Aurora B expression and H1.4S27 phosphorylation were no longer coordinated during metaphase in aneuploid HT29 cells (p = 0.039). In CRCs, immunoreactivity for Aurora B or H1.4S27p did not correlate with T- or N-stage, grade, or MSI status. However, metaphase labeling of H1.4S27p was significantly higher in diploid than in aneuploid CRCs (p = 0.011). Aurora B was significantly correlated with H1.4S27p-positive metaphases in MSI (p = 0.010) or diploid (p = 0.003) CRCs. Finally, combined classification of MSI status and ploidy revealed a significant positive correlation of Aurora B with H1.4S27p in metaphases of diploid/MSI (p = 0.010) and diploid/microsatellite-stable (MSS; p = 0.031) but not of aneuploid/MSS (p = 0.458) CRCs. The present study underlines the functional link of Aurora B expression and H1.4S27p during specific phases of mitosis in diploid and/or MSI-positive CRCs in vitro and in situ. Importantly, the study shows that the coordination between Aurora B expression and phosphorylation of H1.4 at Ser 27 is lost in cycling aneuploid CRC cells.

Immunoreactivity of the 14F7 Mab (Raised against N-Glycolyl GM3 Ganglioside) as a Positive Prognostic Factor in Non-Small-Cell Lung Cancer

Lung carcinoma is the leading cause of cancer-related mortality worldwide. Therefore, numerous studies are focusing on the assessment of other biological and molecular prognostic factors in these tumors. We evaluated the relationship between 14F7 Mab reactivity, pathological features, DNA-content and S-phase fraction (SPF), and their impact in the survival of NSCLC patients. Hematoxylin and eosin staining and immunohistochemistry optical microscopy assays as well as DNA content and SPF measuring using flow cytometry were performed. The 14F7 reactivity was widely observed in NSCLC sections, no depending of the clinicopathological characteristics. We also obtained differences in the intensity of reaction with 14F7 as well as in the SPF between diploid and aneuploid carcinomas. Patients with diploid tumors showing higher SPF and 14F7 reaction joint to a low mitotic index displayed higher survival rates. Our results are in agreement with the assumption of the possible positive prognostic value of 14F7 staining in NSCLC.

HDAC2 and TXNL1 distinguish aneuploid from diploid colorectal cancers

DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Further understanding of the translation of DNA aneuploidy into protein expression will help to define novel biomarkers to improve therapies and prognosis. DNA ploidy was assessed by image cytometry. Comparison of gel-electrophoresis-based protein expression patterns of three diploid and four aneuploid colorectal cancer cell lines detected 64 ploidy-associated proteins. Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in two overlapping high-ranked networks maintaining Cellular Assembly and Organization, Cell Cycle, and Cellular Growth and Proliferation. CAPZA1, TXNL1, and HDAC2 were significantly validated by Western blotting in cell lines and the latter two showed expression differences also in clinical samples using a tissue microarray of normal mucosa (n=19), diploid (n=31), and aneuploid (n=47) carcinomas. The results suggest that distinct protein expression patterns, affecting TXNL1 and HDAC2, distinguish aneuploid with poor prognosis from diploid colorectal cancers.

CA15.3 sérico y proliferación celular en carcinomas ductales infiltrantes de mama

Objective To study the possible correlations between the preoperative CA15.3 serum levels and the cellular proliferation, measured by S-phase (SP), in patients having infiltrating ductal carcinomas (IDC) of the breast Material and methods The study group included 79 patients with an age ranged between 39 and 86 yrs (64,8 ± 11,8).Ploidy and S-phase were measured by cytometry (Fascam. Beckton Dikinson. USA) in fresh samples Results Using as cut-off for SP the value of 7 %, which represents the median obtained previously in 321 patients with IDC (r: 0,8-51,2; 9,3 ± 7,9; percentiles 25 y 75; 4,3 y 11,8 %), we can observed that the antigenic levels were higher (p:0,015) in the tumors with low SP. These same behavior was noted when 30U/ml was used as cut-off for CA15.3. Likewise, the levels of the tumor marker increased significantly (p:0,007) when the SP moved from < 4,3 % to 7,1 %, to decrease later (p:0,010) when the SP value was comprised between 7,11% and 11,8 %. The same behavior of this tumor marker in relation to the SP was noted in tumors without axillary involvement tumors, as well as in aneuploid carcinomas. Conclusion a) Release of CA15.3 happens when SP increases to rise the 7,1 % value, to decrease later although that goes on increasing, and b) The same behaviour of this marker with the S-phase was observed in tumors without axillary involvement, as well as in aneuploid carcinomas.

Preoperative CA15.3 serum levels and cellular proliferation in patients with infiltrating ductal carcinomas of the breast

Objective To study the possible correlations between the preoperative CA15.3 serum levels and the cellular proliferation, measured by S-phase (SP), in patients having infiltrating ductal carcinomas (IDC) of the breast Material and methods The study group included 79 patients with an age ranged between 39 and 86 yrs (64.8 ± 11.8). Ploidy and S-phase were measured by cytometry (Fascam. Beckton Dikinson. USA) in fresh simples Results Using as cut-off for SP the value of 7 %, which represents the median obtained previously in 321 patients with IDC (r: 0.8-51.2; 9.3 ± 7.9; percentiles 25 y 75; 4.3 y 11.8 %), we can observed that the antigenic levels were higher (p: 0.015) in the tumors with low SP. These same behavior was noted when 30U/ml was used as cut-off for CA15.3. Likewise, the levels of the tumor marker increased significantly (p: 0.007) when the SP moved from < 4.3 % to 7.1 %, to decrease later (p: 0.010) when the SP value was comprised between 7.11% and 11.8 %. The same behavior of this tumor marker in relation to the SP was noted in tumors without axillary involvement tumors, as well as in aneuploid carcinomas. Conclusion a) Release of CA15.3 happens when SP increases to rise the 7.1 % value, to decrease later although that goes on increasing, and b) The same behaviour of this marker with the S-phase was observed in tumors without axillary involvement, as well as in aneuploid carcinomas.

Prospective study on prognostic significance of DNA ploidy and Ki-67 expression in colorectal cancer

21184 Background: The prognostic value of flow cytometry DNA ploidy and Ki-67 expression in colorectal carcinomas has not been defined yet. The study tries to correlate tumoral DNA ploidy and proliferative activity (Ki-67) with therapy response, Overall Survival (OS), Disesase Specific Survival (DSS) and Disease Free Survival (DFS). Methods: From 01/09/02 to 30/06/05, 3 samples of cancer tissue and 1 sample of normal mucosa has been collected from all operating pieces of colorectal cancer. These samples were frosen and later disgregated, treated and coloured with Propidium Iodide. DNA ploidy was evaluated by FACSCalibur cytofluorimetry. Normal mucosa tissue was our internal control. Ki-67 was evaluated by immuno-histochemistry in all tumoral samples. All the patients were cured with chemo- and/or radiotherapy in our divisions. Results: 67 patients (M/F 35/32); median age was 70; staging: 19% I, 33% II, 30% III, 18% IV. We found aneuploidy in 65,7% of carcinoma and Ki-67 median expression was 55%. DNA tumoral heterogeneity was present in 27% of patients. DNA aneuploidy correlates with advanced disease stage at diagnosis (p&lt;0,01), with high number of metastatic lymphnodes (p&lt;0,005) and with serological markers positivity (p&lt;0,04). After surgery and chemotherapy 35% of the patients with aneuploid carcinoma and high proliferative activity (Ki-67&gt;55%) do not show evident disease versus 100% of patients with DNA diploidy and lower Ki-67. Tumoral aneuploidy correlates in a significative way with lower OS (48% vs 89% of diploid patients), lower DSS (tumor death happened just in patients with aneuploid DNA in every disease stages), with lower DFS (18% vs 86% of diploid patients). Univariated analysis stated that aneuploidy determinates an Odd Ratio=5,7 to develop disease progression (p=0,033). At the moment Ki-67 expression with 55% cut-off does not seem to correlate with OS, DSS and DFS. Conclusions: Preliminar results (the study is still in progress) seem to suggest that cytofluorimetric DNA-ploidy has a prognostic and predictive significance in colo-rectal carcinomas. Ki-67 expression (immuno-histochemistry) has an uncertain significance. The small number of patients and the short follow-up do not allow us to reach any definitive conclusions, but the study is worth to go on. No significant financial relationships to disclose.

Correlation between DNA ploidy by flow cytometry and chromosome 3 aberration in oral squamous cell carcinoma

Although flow cytometric DNA ploidy has turned out as a significant predictor of survival in oral squamous cell carcinoma, little is known about the underlying karyotypic structure of gross aneuploidy. We therefore analysed one diploid and 9 aneuploid carcinomas with relative DNA contents between 1.1 and 2.8 by fluorescence in situ hybridization with topologic markers for the centromere (3cen) and the terminal regions (3p, 3q) of chromosome 3. Progressing deviation of aberrant DNA contents from the normal diploid value correlated with increasing 3cen copy numbers per cell. A pronounced marker heterogeneity suggested that DNA-aneuploid cell populations consisted of karyotypically different clones. Monosomy of 3p was the only chromosomal alteration in the DNA diploid tumour. A significant 3p underrepresentation was a recurrent finding also in 7 of 9 aneuploid carcinomas while a subset of cells in each of 2 other cases showed a complete loss of one sister chromosome 3. In contrast, 7 of 9 aneuploid tumours exposed corresponding 3q and 3cen copy numbers, 2 showed a substantial 3q overrepresentation. It appears that amplification of chromosome 3 plays a role in the development of aneuploidy and the concurrent overexpression of 3q target genes. Acquired loss of the short arm of chromosome 3 in DNA-diploid tumour cells may contribute to the manifestation of recurrent 3q deletions in aneuploid cell populations.

Genomic alterations detected by comparative genomic hybridization in primary lung adenocarcinomas with special reference to the relationship with DNA ploidy

To clarify the clinicopathological and biological significance of genomic alterations in pulmonary adenocarcinomas, we examined chromosomal DNA sequence copy number aberrations (DSCNAs) and DNA ploidy in 42 surgically resected specimens by comparative genomic hybridization (CGH) and laser scanning cytometry (LSC), respectively. The number of DSCNA did not affect the size of carcinoma or number of nodal metastasis. More than 60% of carcinomas showed a 1q21-23 gain or 13q21 loss. Gains of 9q22-33 and 10q26-qter and a loss of 15q14-qter were significantly associated with nodal metastasis (p<0.05). Of 42 pulmonary adenocarcinomas, 32 (76%) showed DNA aneuploidy. The number of DSCNAs in aneuploid carcinomas was larger than that in diploid carcinomas (p<0.01). Our results suggest that diploid and aneuploid types are cytogenetically different in pulmonary adenocarcinomas and aneuploid carcinomas are genetically more unstable and aggressive than diploid carcinomas.

Investigation of the relationship between cell proliferative activity and invasion, metastasis and prognosis of gastric cancer

Objective: To evaluated Bromodeoxyuridine (BrdUrd)/DNA doubleparametric method in detection of gastric carcinoma and to analyze the relationships of cellular BrdUrd labeling indices(LI), G2/M-phase fraction(G2/MPE) and DNA content to the depth of invasion, lymphatic vessel invasion, lymphatic node metastasis, peritoneal dissemination and blood vessel invasion and prognosis. Methods: Fresh tumor samples from 60 cases were examined by BrdUrd/DNA doubleparametric flowcytometry. Propidium iodide(PI) was used as fluorescent probe for total cellular DNA and a monoclonal antibody against BrdUrd incorporated into DNA. Fluorescein-labeled goat anti-mouse antibody was used as second antibody. Results: The values of BrdUrd LI in patients with serosa invasion was significantly higher than those without serosa invasion (P<0.01); there was statistical significance in 5-year survival rate between the two groups (P<0.01). Both BrdUrd LI and G2/MPF values were significantly higher in patients with lymphatic vessel invasion than those without invasion (P<0.01); the patients with lymphatic vascular invasion carried a significantly poor prognosis (P<0.01). Both BrdUrd LI and G2/MPF values were significantly higher in patients with lymphatic node metastasis than those without metastasis (P<0.01), there was a statistical significance in 5 years survival between these 2 groups. The incidence of lymphatic node metastasis was significantly higher in aneuploid carcinoma (P<0.05), and the patients with aneuploid carried a significantly poor prognosis (P<0.05). Patients with peritoneal dissemination had a significantly worse prognosis (P<0.01). G2/MPF values were significantly higher in patients with blood vessel invasion than those without invasion (P<0.01). Conclusion: Cellular BrdUrd LI, G2/MPF and DNA content are related to depth of invasion, lymphatic vessel invasion, peritoneal dissemination, blood vessel invasion and prognosis of gastric carcinoma.

Improved grading of breast adenocarcinomas based on genomic instability.

Numerous investigations have shown that in primary breast adenocarcinomas DNA aneuploidy in contrast to DNA diploidy indicates high malignancy potential. On the basis of the study of 104 breast carcinomas, we describe a subtype of aneuploidy, which demonstrates a low degree of malignancy. In image cytometric DNA histograms, this subtype possessed a low percentage (< or = 8.8%) of nonmodal DNA values as measured by the stemline scatter index (SSI), which is defined as sum of the percentage of cells in the S-phase region, the G(2) exceeding rate and the coefficient of variation of the tumor stemline. The cut point of SSI = 8.8% (P = 0.03) enabled us to also subdivide diploid and tetraploid tumors into clinically low and high malignant variants. One possible reason for aneuploidy is impaired distribution of chromosomes at mitosis caused by numerical or structural centrosome aberrations. Cyclins A and E seem to be involved in centrosome duplication. Real-time quantitative PCR measurements of cyclin A and E transcript levels and immunohistochemical determination of cyclin A protein expression showed statistically significantly increased values in the tumors with a high SSI (>8.8%), compared with those with a low SSI. A pilot study demonstrated centrosomal aberrations in an average of 9.6% of the measured cells in four aneuploid carcinomas with high SSI values and in an average of 2.5% of the cells in three aneuploid and three diploid tumors with low SSI. Our data indicate that the SSI, most likely reflecting the degree of genomic instability, allows additional classifying of the known aneuploid, diploid, and tetraploid categories of primary breast adenocarcinomas into low and high malignant subtypes.

Evolution of DNA ploidy state and DNA index in colorectal adenomas and carcinomas using the crypt isolation technique: new hypothesis in colorectal tumorigenesis.

The evolution of DNA diploid, aneuploid and multiploid (diploid and aneuploid) states that represent DNA types that are independent of genetic alterations in colorectal tumors were examined. Changes in the DNA index (DI) accompanying tumor development from adenoma to carcinoma were assessed. In colorectal adenomas and early cancers, the DNA was diploid or multiploid. A pure aneuploid state was observed in advanced carcinomas only, whereas the aneuploid DI values of adenomas were characterized by two distinct peaks. The DI values for the carcinomas were randomly distributed. However, in advanced carcinomas, aneuploid carcinomas tended to have lower DI whereas aneuploid populations within multiploid carcinomas tended to have higher DI. Early cancers were subdivided into two groups: a cancer region associated with an adenomatous region (group A tumors) and a cancer region that exhibited an absence of or a very limited adenomatous region (group B tumors). Group A tumor DI were lower than group B. It is suggested that low DI adenomas might transform into group A tumors, which consequently progress to advanced aneuploid carcinomas. In addition, group B tumors might derive predominantly from high DI adenomas or from group A tumors by high DI evolution, and might progress into advanced multiploid carcinomas. Therefore, the evolution of the DNA index might play an important role in the development of colorectal tumors.

Cell proliferation, nuclear ploidy, and EGFr and HER2/neu tyrosine kinase oncoproteins in infiltrating ductal breast carcinoma

The purpose of this work was to study if the variations in the nuclear DNA content and the expression of EGFr and HER2/neu transmembrane oncoproteins were related and if this influences the rate of cell proliferation modifying, in each case, the potential aggressivity of the neoplasia. Thirty-four ductal breast carcinoma tissue samples of tumors of up to 2 cm in diameter were analyzed by flow cytometry. HER2/neu and EGFr were measured by immunohistochemical methods. Twenty cases were diploid (DNA index of 1) and 14 cases were aneuploid (DNA index other than 1). The expression of EGFr and HER2/neu was significantly higher in aneuploid tumors compared with diploid tumors. The cell proliferation rate was significantly higher in tumors with an aneuploid pattern. The expression of EGFr was associated with a higher rate of cell proliferation. The higher expression of EGFr and HER 2/neu oncoproteins in aneuploid tumors suggests that the increased proliferative activity of aneuploid carcinomas is influenced by the activity of such oncoproteins, which favors a more aggressive biological behavior.

Frequent allelic imbalance at the ATM locus in DNA multiploid colorectal carcinomas.

DNA multiploidy may involve specific DNA ploidy states with respect to genetic alterations such as oncogenes, tumor suppressor gene mutation and microsatellite instability. To clarify the role of DNA multiploidy in colorectal cancer, we analysed allelic imbalance involving the ATM gene, localized to chromosome 11q22-23 and thought to be involved in genetic stability, in a series of multiploid colorectal carcinomas. In addition, p53 gene mutation (exons 5-8) and allelic imbalance at 11q24 loci distal to the ATM locus were also examined. The crypt isolation technique coupled with DNA cytometric sorting and polymerase chain reaction assay using 10 microsatellite markers tightly linked to the ATM gene were used to study ATM allelic imbalance in 55 colorectal carcinomas (15 diploid, 13 aneuploid, 27 multiploid). While allelic imbalance at the ATM locus was rarely observed in diploid and aneuploid carcinomas, multiploid carcinomas exhibited a high frequency of ATM allelic imbalance. In multiploid carcinoma samples, diploid subpopulations showed a smaller range of allelic imbalance at the loci tested compared to aneuploid subpopulations that demonstrated allelic imbalance over a relatively large region. Also, the frequency of AI at 11q24 showed a similar tendency to that at the ATM locus for each DNA ploidy state. An association between p53 gene mutation and ATM allelic imbalance in multiploid carcinoma was also observed. Our results suggest that ATM allelic imbalance and p53 gene mutations occur during the progression from diploid to aneuploid cell populations in multiploid colorectal carcinomas.

DNA proliferative index as a marker in Iraqi aneuploid mammary carcinoma

This study estimated nuclear DNA ploidy and DNA proliferative indices (PI) in mammary ductal carcinomas from 120 Iraqi female patients. Of the examined specimens, 82.7% were aneuploid. DNA ploidy correlated significantly with histological grade and estrogen receptor content of the primary neoplasm. In aneuploid carcinomas, high PI showed a clearer association than aneuploidy with menopausal status and progesterone receptor content of the tumour. PI and percentage aneuploidy were higher in larger tumours; nodal status showed no association with these cytometric findings. Using PI, patients classified as having Auer aneuploid carcinomas can be divided into subsets with different tumour characteristics, thus improving the selection of those whose high risk, node-negative presentation makes them candidates for adjuvant systemic therapy.

Multiparameter flow cytometry for simultaneous assessment of p53 protein expression and cellular DNA content in oral squamous cell carcinomas: evidence for the development of aneuploid clones from p53-deficient diploid progenitor cells.

Diploid tumour cells regularly continue to progress after the development of aneuploid cell populations in head and neck squamous cell carcinomas. The coexistence of aneuploid clones with their diploid progenitor cells provides a unique opportunity to study the order of appearance of p53 mutation and aneuploidy in the same tumour. Multiparameter flow cytometry was therefore applied to 22 oral squamous cell carcinomas to simultaneously assess cellular DNA content and p53 protein expression on a single-cell basis. Concurrent measurements of cytokeratin expression served to identify tumour cells of epithelial origin. One of 5 diploid and 2 of 17 aneuploid carcinomas were p53-negative. For 15 p53-positive aneuploid tumours, overexpression of p53 protein was identified for the aneuploid clones as well as for coexisting diploid tumour cell populations in 14 cases. On the understanding that coexisting diploid and aneuploid tumour cell populations have a common clonal origin, these results provide evidence that aneuploid tumour clones typically develop from p53-deficient diploid progenitor cells. Loss of wild-type p53 function may therefore contribute to the development of aneuploidy in head and neck cancer.

Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations

Measurement of the nuclear DNA content allows classification of human cancers as either diploid or aneuploid. To gain further insight into mechanisms of aneuploidy, we compared the cytogenetic profile of mismatch-repair-deficient diploid versus mismatch-repair-proficient aneuploid colorectal carcinoma cell lines using comparative genomic hybridization and spectral karyotyping. Aneuploid carcinomas revealed an average of 19 chromosomal imbalances per cell line. Such numerical aberrations were exceedingly scarce in the diploid tumors. This pattern of chromosomal aberrations is consistent with a mechanism involving the impairment of chromosome segregation fidelity during mitotic cell division. In support of this idea, we demonstrate the exclusive occurrence of centrosome amplification and instability in all of the aneuploid tumor cell lines analyzed. All diploid tumors contained centrosomes that were functionally and structurally indistinguishable from those in normal human fibroblasts. Due to the observed differences in centrosomes between these two classes of tumors, we incubated the cells with the microtubule depolymerizing drugs nocodazole and griseofulvin. Our results indicate that the aneuploid tumor cell lines have an increased sensitivity to these reagents and a delay in aster formation and microtubule regrowth. However, microtubule nucleation was initiated from one or two centers in both the diploid and aneuploid cells. These observations support the notion that the integrity of the centrosome plays a central role in the development of aneuploidy. Genes Chromosomes Cancer 27:183-190, 2000. Published 2000 Wiley-Liss, Inc.

Genetic alterations in DNA diploid, aneuploid and multiploid colorectal carcinomas identified by the crypt isolation technique.

Loss of heterozygosity (LOH) and microsatellite instability (MSI) commonly occur in colorectal carcinomas. However, the role of these genetic alterations in determining DNA ploidy status of tumors (diploid, aneuploid and multiploid) remains unclear. In the present study, we attempted to clarify the relationship between genetic alterations and DNA ploidy status. Crypt isolation coupled with DNA cytometric sorting and polymerase chain reaction assay (17 microsatellite markers) were used to study allelic losses and MSI in 59 colorectal carcinomas (diploid, 15; aneuploid, 10 and multiploid, 34). Of the 15 diploid carcinomas, 6 exhibited MSI in which allelic losses were rarely found. The other 9 diploid tumors mostly exhibited allelic losses, but none displayed MSI status. Whereas allelic losses frequently occurred in the aneuploid carcinomas and the aneuploid populations of multiploid carcinomas, they were rarely detected in the diploid populations of multiploid carcinomas. MSI status was not observed in aneuploid carcinomas nor in either population of multiploid carcinomas. Although multiploid carcinomas genetically resemble aneuploid carcinomas in the expression of the severe LOH phenotype, the genetic alterations seen in the diploid populations of multiploid carcinomas may differ from those of diploid carcinomas. Furthermore, all diploid, aneuploid and both the diploid and aneuploid fractions of the multiploid tumors that were non-MSI exhibited a high rate of LOH, suggesting that LOH is independent of the tumor's ploidy status.