High-resolution DNA Flow Cytometry Research Articles

Cooperative antitumor activities of carnosic acid and Trastuzumab in ERBB2+ breast cancer cells

BackgroundERBB2 is overexpressed in up to 20–30% of human breast cancers (BCs), and it is associated with aggressive disease. Trastuzumab (Tz), a humanized monoclonal antibody, improves the prognosis associated with ERBB2-amplified BCs. However, the development of resistance remains a significant challenge. Carnosic acid (CA) is a diterpene found in rosemary and sage, endowed with anticancer properties. In this in vitro study, we have investigated whether Tz and CA have cooperative effects on cell survival of ERBB2 overexpressing (ERBB2+) cells and whether CA might restore Tz sensitivity in Tz-resistant cells.MethodsWe have studied BC cell migration and survival upon CA and Tz treatment. In particular, migration ability was assessed by transwell assay while cell survival was assessed by MTT assay. In addition, we have performed cell cycle and apoptosis analysis by high-resolution DNA flow cytometry and annexin-V, resazurin and sytox blue staining by flow cytometry, respectively. The expression of proteins involved in cell cycle progression, ERBB2 signaling pathway, and autophagy was evaluated by immunoblot and immunofluorescence analysis. Cellular structures relevant to the endosome/lysosome and autophagy pathways have been studied by immunofluorescence and transmission electron microscopy.ResultsWe report that, in ERBB2+ BC cells, CA reversibly enhances Tz inhibition of cell survival, cooperatively inhibits cell migration and induces cell cycle arrest in G0/G1. These events are accompanied by ERBB2 down-regulation, deregulation of the PI3K/AKT/mTOR signaling pathway and up-regulation of both CDKN1A/p21WAF1 and CDKN1B/p27KIP1. Furthermore, we have demonstrated that CA impairs late autophagy and causes derangement of the lysosomal compartment as shown by up-regulation of SQSTM1/p62 and ultrastructural analysis. Accordingly, we have found that CA restores, at least in part, sensitivity to Tz in SKBR-3 Tz-resistant cell line.ConclusionsOur data demonstrate the cooperation between CA and Tz in inhibiting cell migration and survival of ERBB2+ BC cells that warrant further studies to establish if CA or CA derivatives may be useful in vivo in the treatment of ERBB2+ cancers.

DNA aneuploidy relationship with patient age and tobacco smoke in OPMDs/OSCCs.

The aim of this study was to investigate the relationship between tobacco smoke habit, patient age, DNA aneuploidy and genomic DNA copy number aberrations (CNAs) in oral potentially malignant disorder (OPMD) and oral squamous cell carcinoma (OSCC) patients. DNA aneuploidy was detected by high-resolution DNA flow cytometry (hr DNA-FCM) on DAPI stained nuclei obtained from multiple tissue samples from OPMDs/OSCCs in 220 consecutive patients. Nuclear genomic aberrations were determined in a subset of 65 patients by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. DNA aneuploidy and mean nuclear genomic aberrations were associated with patients’ age. In particular, DNA aneuploidy strongly associated with age in non-smoker OPMDs/OSCCs patients. OSCCs from smokers showed a lower prevalence of DNA aneuploidy compared to OSCCs from non-smokers. A higher occurrence of DNA aneuploidy (particularly in smokers’ OPMDs) was observed in patients characterized by involvement of a single oral subsite. Our study suggests that: 1) DNA aneuploidy in non-smokers is mainly related to aging; 2) OPMDs/OSCCs involving multiple oral subsites in smokers are less likely to develop DNA aneuploidy compared to non-smokers; 3) OSCC development is characterized by both CIN and CIN-independent mechanisms and that the latter are more relevant in smokers. This study provides evidence that DNA diploid OPMDs may be considered at lower risk of cancerization than DNA aneuploid ones in non-smokers but not in smokers.

High-resolution DNA content analysis of microbiopsy samples in oral lichen planus.

DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed.

Genomic DNA Copy Number Aberrations, Histological Diagnosis, Oral Subsite and Aneuploidy in OPMDs/OSCCs.

Oral potentially malignant disorders (OPMDs) characterized by the presence of dysplasia and DNA copy number aberrations (CNAs), may reflect chromosomal instability (CIN) and predispose to oral squamous cell carcinoma (OSCC). Early detection of OPMDs with such characteristics may play a crucial role in OSCC prevention. The aim of this study was to explore the relationship between CNAs, histological diagnosis, oral subsite and aneuploidy in OPMDs/OSCCs. Samples from OPMDs and OSCCs were processed by high-resolution DNA flow cytometry (hr DNA-FCM) to determine the relative nuclear DNA content. Additionally, CNAs were obtained for a subset of these samples by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. Our study shows that: i) aneuploidy, global genomic imbalance (measured as the total number of CNAs) and specific focal CNAs occur early in the development of oral cancer and become more frequent at later stages; ii) OPMDs limited to tongue (TNG) mucosa display a higher frequency of aneuploidy compared to OPMDs confined to buccal mucosa (BM) as measured by DNA-FCM; iii) TNG OPMDs/OSCCs show peculiar features of CIN compared to BM OPMDs/OSCCs given the preferential association with total broad and specific focal CNA gains. Follow-up studies are warranted to establish whether the presence of DNA aneuploidy and specific focal or broad CNAs may predict cancer development in non-dysplastic OPMDs.

Chromosomal Instability, DNA Index, Dysplasia, and Subsite in Oral Premalignancy as Intermediate Endpoints of Risk of Cancer

Chromosomal instability and aneuploidy may represent biomarkers of oral exposure to damaging agents and early signs of clinical disease according to the theory of "oral field cancerization." The hypothesis was tested that the DNA index (DI) values, obtained by high-resolution DNA flow cytometry (DNA-FCM), may potentially contribute to oral cancer risk prediction. For this purpose, the DI of oral fields of normal-appearing mucosa and oral potentially malignant disorders (OPMDs) in 165 consecutive patients was tested for association with dysplasia and/or the oral subsites of tongue and floor of the mouth taken as high-risk intermediate endpoints surrogate of cancer clinical endpoints. The association was evaluated by logistic regression using patient gender, age, tobacco, cigarette smoking habit, and alcohol abuse as confounding variables. Different DI models provided evidence of statistical significant associations. Subdividing the DI values in diploid, near-diploid aneuploid, and high or multiple aneuploid from both OPMDs and oral normal-appearing mucosa, ORs, respectively, of 1, 4.3 (P = 0.001), and 18.4 (P < 0.0005) were obtained. Routine DI analysis by high-resolution DNA-FCM seems potentially useful to complement dysplasia and subsite analysis for assessment of oral cancer risk prediction and for a better management of the patients with OPMDs. Work is in progress to validate the present findings in a prospective study with clinical endpoints. Identifying DNA abnormalities in oral premalignancy may lead to biomarkers of oral exposure and cancer risk and potentially to more effective prevention measures.

Chromosomal Instability, Aneuploidy and Routine High-Resolution DNA Content Analysis in Oral Cancer Risk Evaluation

Carcinogen exposure of the oral cavity is thought to create an extensive 'field cancerization'. According to this model, a very early precursor of oral cancer is a patch of normal-appearing mucosa in which stem cells share genetic/genomic aberrations. These precancerous fields then become clinically visible as white and red lesions (leuko- and erythro-plakias), which represent the vast majority of the oral potentially malignant disorders. This review focuses on aneuploidy (where it is from) and on biomarkers associated with DNA aneuploidy in oral mucosa and oral potentially malignant disorders, as detected by DNA image and flow cytometry. Data from the literature strongly support the association of DNA ploidy with dysplasia. However, work is still needed to prove the clinical value of DNA ploidy in large-scale prospective studies. Using high-resolution DNA flow cytometry with fresh/frozen material and the degree of DNA aneuploidy (DNA Index) might improve the prediction of risk of oral cancer development.

Genomic aberrations in normal appearing mucosa fields distal from oral potentially malignant lesions

ObjectivesOral fields of visually normal and non-dysplastic mucosa (ODFs) may represent the precursors of oral potentially malignant lesions (OPMLs). Aim of the study was to provide new evidence for the concept of the “field carcinogenesis” model by comparing the ODF and OPML genomic aberration profiles obtained by high resolution DNA flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). A second aim was to investigate if specific CGH aberrations were associated with DNA aneuploidy.MethodsNineteen patients with single OPMLs were recruited for the study. In parallel with obtaining samples of OPML tissue from 11 leukoplakias without dysplasia (nd-OPMLs) and 8 with dysplasia (d-OPMLs), we also obtained samples from distant ODFs. DNA aneuploid nuclei detected by hr DNA-FCM were physically separated, based on DNA content, from the DNA diploid components with a DNA-FCM-Sorter. These relatively pure subpopulations of epithelial nuclei were then submitted to DNA extraction and a-CGH for a genome-wide analysis of DNA copy number aberrations (CNAs).ResultsThe frequencies of DNA aneuploidy (DI ≠ 1) among ODFs and OPMLs were respectively 5.3% and 32%. The DI aneuploid values of ODFs and nd-OPMLs were all near-diploid (DI ≠ 1 and DI ≤ 1.4), while for d-OPMLs were high-aneuploid (DI > 1.4) in 40% of the cases. CNA averages were 1.9 in ODFs and 6.5 in OPMLs. The gain of the chromosomal region 20q13.33-qter was observed in 37% of both ODFs and corresponding OPMLs. Additional common regions included 7p22.2-pter, 11p15.5-pter and 16p13.3-pter where gains were observed. Furthermore, gains of 20q13.31–q13.33 and of 5p13.33-pter and loss of 9p21.3 were detected at high frequency (respectively, at 62.5%, 50% and 50%) only in d-OPMLs. In particular, loss at 9p21.3, gain at 5p13.33-pter and gain of 20q13.31–q13.33 were associated with DNA aneuploidy (p = 0.00004; p = 0.0005; p = 0.01).ConclusionsODFs and OPMLs showed common CNAs in specific chromosomal regions suggesting that they may represent early events of the natural history of oral carcinogenesis according to the field effect cancerization and may contribute to the ODF-OPML transition. In addition, loss at 9p21.3 and gains at 5p13.33-pter and 20q13.31–q13.33 may contribute to DNA aneuploidization.

Chromosomal aberrations and aneuploidy in oral potentially malignant lesions: distinctive features for tongue

BackgroundThe mucosae of the oral cavity are different at the histological level but appear all equally exposed to common genotoxic agents. As a result of this exposure, changes in the mucosal epithelia may develop giving rise to Oral Potentially Malignant Lesions (OPMLs), which with time may in turn progress to Oral Squamous Cell Carcinomas (OSCCs). Therefore, much effort should be devoted to identify features able to predict the likeliness of progression associated with an OPML. Such features may be helpful in assisting the clinician to establish both appropriate therapies and follow-up schedules. Here, we report a pilot study that compared the occurrence of DNA aneuploidy and chromosomal copy number aberrations (CNAs) in the OPMLs from different oral anatomical subsites.MethodsSamples from histologically diagnosed OPMLs were processed for high resolution DNA flow cytometry (hr DNA-FCM) in order to determine the relative DNA content expressed by the DNA index (DI). Additionally, array-Comparative Genomic Hybridization (a-CGH) analysis was performed on DNA obtained from diploid nuclei suspensions directly. When aneuploid nuclei were detected, these were physically separated from diploid nuclei on the base of their DI values by means of a DNA-FCM-Sorter in order to improve the a-CGH analysis.ResultsTongue OPMLs were more frequently associated with DNA aneuploidy and CNAs than OPMLs arising from all the other mucosal subsites.ConclusionsWe suggest that the follow-up and the management of the patients with tongue OPMLs should receive a distinctive special attention. Clearly, this hypothesis should be validated in a prospective clinical study.

Distinctive chromosomal instability patterns in oral verrucous and squamous cell carcinomas detected by high‐resolution DNA flow cytometry

Oral verrucous carcinomas (OVCs) are characterized by better prognosis than oral squamous cell carcinomas (OSCCs). Because chromosomal instability (CIN) in solid tumors is indicative of prognosis, this study investigated whether OVCs and OSCCs were characterized by differences in CIN biomarkers. Fresh or frozen multiple tissue samples were submitted to high-resolution DNA flow cytometry (hr DNA-FCM). DNA aneuploid sublines were detected in 6 of 9 OVCs (66.7%) and in 20 of 25 OSCCs (80.0%). Multiple DNA aneuploid sublines were observed, respectively, in 2 of 6 (33.3%) DNA aneuploid OVCs and in 14 of 20 (70%) DNA aneuploid OSCCs (P = .163). OVCs were mainly characterized by DNA Index (DI) values in the near-diploid region (DI≠1 and DI < 1.4), whereas aneuploid OSCCs carried most frequently multiple aneuploid sublines with high DI values (DI ≥ 1.4). DNA near-diploid and high aneuploid sublines were, respectively, 87.5% and 12.5% for the OVCs versus 30% and 70% for the OSCCs (P = .004). Present data suggest that OVCs are characterized by a lower degree of CIN and tumor heterogeneity than OSCCs, such that they appear as "frozen" in an early stage of DNA near-diploid aneuploidy, as previously observed for oral preneoplastic lesions. These DI characteristics, which can easily be obtained by hr DNA-FCM, appear to reflect the well-known differences in aggressiveness and prognosis of OVCs and OSCCs.

Oral Cancer Genesis and Progression: DNA Near-Diploid Aneuploidization and Endoreduplication by High Resolution Flow Cytometry

Oral potentially malignant lesions (OPMLs) with dysplasia and aneuploidy are thought to have a high risk of progression into oral squamous cell carcinomas (OSCCs). Non-dysplastic “oral distant fields” (ODFs), characterized by clinically normal appearing mucosa sited at a distance from co-existing OPMLs, and non-dysplastic OPMLs may also represent an early pre-cancerous state. ODFs, OPMLs without and with dysplasia and OSCCs were investigated by high resolution DNA content flow cytometry (FCM). ODFs and OPMLs without dysplasia were DNA aneuploid respectively in 7/82 (8.5%) and 25/109 (23%) cases. “True normal oral mucosa” and human lymphocytes from healthy donors were DNA diploid in all cases and were used as sex specific DNA diploid controls. Dysplastic OPMLs and OSCCs were DNA aneuploid in 12/26 (46%) and 12/13 (92%) cases. The DNA aneuploid sublines were characterized by the DNA Index (DI ≠ 1). Aneuploid sublines in ODFs and in non-dysplastic and dysplastic OPMLs were near-diploid (DI &lt; 1.4) respectively in all, 2/3 and 1/3 of the cases. DNA aneuploid OSCCs, instead, were characterized prevalently by multiple aneuploid sublines (67%), which were commonly (57%) high-aneuploid (DI ≥ 1.4). DNA near-diploid aneuploid sublines in ODFs and OPMLs appear as early events of the oral carcinogenesis in agreement with the concept of field effect. Near-diploid aneuploidization is likely to reflect mechanisms of loss of symmetry in the chromosome mitotic division. High DNA aneuploid and multiple sublines in OPMLs with dysplasia and OSCCs suggest, instead, mechanisms of “endoreduplication” of diploid and near-diploid aneuploid cells and chromosomal loss. High resolution DNA FCM seems to enable the separation of subsequent progression steps of the oral carcinogenesis.

Oral cancer genesis and progression: DNA near-diploid aneuploidization and endoreduplication by high resolution flow cytometry.

Oral potentially malignant lesions (OPMLs) with dysplasia and aneuploidy are thought to have a high risk of progression into oral squamous cell carcinomas (OSCCs). Non-dysplastic “oral distant fields” (ODFs), characterized by clinically normal appearing mucosa sited at a distance from co-existing OPMLs, and non-dysplastic OPMLs may also represent an early pre-cancerous state. ODFs, OPMLs without and with dysplasia and OSCCs were investigated by high resolution DNA content flow cytometry (FCM). ODFs and OPMLs without dysplasia were DNA aneuploid respectively in 7/82 (8.5%) and 25/109 (23%) cases. “True normal oral mucosa” and human lymphocytes from healthy donors were DNA diploid in all cases and were used as sex specific DNA diploid controls. Dysplastic OPMLs and OSCCs were DNA aneuploid in 12/26 (46%) and 12/13 (92%) cases. The DNA aneuploid sublines were characterized by the DNA Index (DI ≠ 1). Aneuploid sublines in ODFs and in non-dysplastic and dysplastic OPMLs were near-diploid (DI < 1.4) respectively in all, 2/3 and 1/3 of the cases. DNA aneuploid OSCCs, instead, were characterized prevalently by multiple aneuploid sublines (67%), which were commonly (57%) high-aneuploid (DI ≥ 1.4). DNA near-diploid aneuploid sublines in ODFs and OPMLs appear as early events of the oral carcinogenesis in agreement with the concept of field effect. Near-diploid aneuploidization is likely to reflect mechanisms of loss of symmetry in the chromosome mitotic division. High DNA aneuploid and multiple sublines in OPMLs with dysplasia and OSCCs suggest, instead, mechanisms of “endoreduplication” of diploid and near-diploid aneuploid cells and chromosomal loss. High resolution DNA FCM seems to enable the separation of subsequent progression steps of the oral carcinogenesis.

DNA-Ploidie und proliferative Aktivität bei Speicheldrüsentumoren

DNA ploidy and S-Phase fraction (SPF) of 279 salivary gland tumours were analysed using high-resolution DNA flow cytometry. All 229 benign neoplasms were diploid while 12 of 50 malignant tumours showed cell populations with aneuploid DNA content. The SPF values of diploid malignancies were significantly higher if compared with pleomorphic adenomas but did not differ from that of the zystadenolymphoma (Warthin tumour) group. While aneuploidy represents a distinct indicator of malignancy SPF values are of minor relevance for dignity assessment in salivary gland tumours.

High resolution DNA flow cytometry of boar sperm cells in identification of boars carrying cytogenetic aberrations

The cytogenetic quality of boars used for breeding determines the litter outcome and thus has large economical consequences. Traditionally, quality controls based on the examination of simple karyograms are time consuming and sometimes give uncertain results. As an alternative, the use of high-resolution DNA flow cytometry on DAPI-stained sperm cell nuclei (CV≤1.3%) was investigated as a screening method for the cytogenetic quality of boars. By analyzing a series of 25 animals judged normal by their fertility statistics and a series of seven animals with known reciprocal translocations, a model for identifying sperm cells from cytogenetically aberrant animals was proposed. This model was applied to a series of 50 uncharacterized animals. The model successfully identified a mosaic or chimaeric carrier of an aberrant X chromosome. However, implementation of this technique for screening purposes would necessitate essential improvements in standardization and measurement precision.

High-resolution DNA flow cytometry in papillary cystadenoma lymphomatosum (Warthin's tumour).

Twenty-eight examples of papillary cystadenoma lymphomatosum (Warthin's tumour) of the parotid gland were analysed by high-resolution DNA flow cytometry. The mean coefficient of variation was found to be 1.19% (SD: 0.41). All tumours were DNA diploid. These results did not correspond with expected deviations based on published chromosomal studies. Also, the homogeneously low S-phase fractions (mean: 4.8%; SD: 2.7) found did not support the hypothesis of etiologically distinctive subgroups in these tumours.

DNA ploidy and cell-cycle analysis in intracranial meningiomas and hemangiopericytomas: a study with high-resolution DNA flow cytometry.

Although various DNA flow-cytometric studies have been performed on meningiomas, the role of DNA ploidy and the S-phase fraction (SPF) in predicting biological tumor behavior remains unresolved. Discrepant results in earlier studies might be due to different preparing, staining and measuring techniques; different quality standards; and lack of sophisticated computer software. In this study, high-resolution DNA flow cytometry using the DNA-specific dye DAPI (4', 6'-diamidino-2-phenylindol) was performed on stored frozen tissue from 128 microsurgically resected meningiomas and 7 hemangiopericytomas, including 17 recurrent meningiomas and 4 recurrent hemangiopericytomas. The computer software Multicycle 2.5 was used to determine the ploidy level and to perform cell-cycle analysis. DNA aneuploidy and SPF were significantly higher in atypical, anaplastic and recurrent meningiomas and correlated well with histopathological features such as focal necrosis, infiltration of dura mater and mitotic activity. Among 128 meningiomas, 42 had additional DNA aneuploid stem lines. No association between hypo- and hyperploidy and either histological subtype or clinical outcome was found. In 7 hemangiopericytomas, SPF was significantly higher compared to the benign meningioma group, while only 1 tumor was aneuploid. In all 42 DNA aneuploid tumors, cell-cycle analysis was performed separately for the euploid and aneuploid stem lines. The proliferation parameters (SPF, G2/M phase) were significantly higher in the DNA aneuploid stem lines. DNA ploidy and SPF are thus useful indicators of different biological behavior within identical histological subgroups in meningiomas.

Tumor progression by dna flow cytometry in human colorectal cancer

We wished to better understand the role of aneuploidy during the progression of human colorectal cancer. Fresh or frozen multiple samples from 221 human colorectal adenomas, 93 carcinomas, and corresponding control mucosa were investigated using high-resolution DNA flow cytometry. A total number of 164 DNA abnormal clones were observed and characterized by a quantitative index of DNA aneuploidy (DI). In precancerous lesions the vast majority of DNA abnormal clones (almost 3/4 in adenomas with mild to moderate dysplasia) was hypo- and hyper-diploid with DI values from 0.8 to 1.2 (near-diploidy). In moderately to poorly differentiated carcinomas the vast majority of abnormal clones was near-triploid and hypotetraploid with DI values from 1.4 to 1.8 (near hypertriploidy) and only 12% were near-diploid. Adenomas with foci of carcinomas, a group of special interest since they represent a link in colorectal tumor progression, had median triploid DNA content. In addition to an increase in DI values, the carcinomas had a clear increase in the proportion of cells actively synthesizing DNA (S-phase fraction). These results are interpreted as evidence for a ploidy-evolution model according to which near-diploid clones in adenomas at early stages of dysplasia would derive from abnormal mitotic cells that divide their DNA unequally between two daughter cells. Tetrapolidization of these near-diploid cells and successive DNA loss would then lead in later stages of tumor progression to near-hypertriploid clones characterized by a balance of chromosomes bearing growth-promoting and growth-suppressing genes confering a selective proliferative advantage.