Comparison of DNA content parameters in paired, fresh tissue pretreatment biopsies and surgical resections from squamous cell carcinoma of the head and neck.

OBJECTIVE: Cellular DNA characteristics derived from pretreatment biopsy (PTB) may become important for predicting treatment outcomes in patients with head and neck squamous cell cancer (HNSCC). Whether the PTB adequately represents the whole specimen is of critical importance. STUDY DESIGN: In a series of >700 HNSCCs, we identified 59 cases in which the PTB and the surgical resection (SR) met the following criteria: PTB and SR were from the same site, and SR was obtained within 5 weeks of PTB with no intervening treatments. RESULTS: Twenty-nine percent of the PTB specimens were DNA diploid. Only 1 of the 11 subsequent DNA diploid SR was associated with a DNA aneuploid PTB (91% concordance). Of the 48 DNA aneuploid tumors, 3 were associated with DNA diploid PTB (94% concordance). Three other DNA aneuploid SRs were associated with PTB of poor quality. CONCLUSION: With respect to DNA ploidy, PTB are representative of SR specimens.

Comparative image and flow cytometric TUNEL analysis of fine needle samples of breast carcinoma.

The assessment of apoptosis in solid tumors is of interest because of its biological role in tumor evolution and response to therapy. A commonly used method for apoptosis measurement is the TUNEL 3' end-labeling technique, which has shown wide variations in results when applied to solid tumors. Thirty-one fine needle breast carcinoma samples were analyzed by fluorescent TUNEL assay and DNA content using image analysis and flow cytometry. TUNEL positivity, seen both in cells with apoptotic morphology and in a subset of morphologically normal cells, was categorized into five staining patterns and quantitated. Values for patterns of TUNEL-positive cells were compared with TUNEL positivity measured by flow cytometry. Flow cytometric quantitation showed a mean of 24.3% positive cells, which correlated (P < 0.02) with total positive cells (all patterns) measured by image (22.4%). Image analysis quantitation of morphologically apoptotic cells (4.2%) did not correlate with flow cytometric TUNEL positivity and the majority of TUNEL-stained cells were morphologically normal (17%). Image analysis allows discrimination of TUNEL-positive morphologically apoptotic and nonapoptotic cells, which are included in the total number of TUNEL-positive events measured by flow cytometry.

Differential expression of Bax and Bcl-2 in the assessment of cellular dynamics in fine-needle samples of primary breast carcinomas.

The rates of cell proliferation and programmed cell death (apoptosis) reflect tumor cell dynamics and are considered to directly influence biological progression and tumor response to therapy. Bax and Bcl-2 are members of a gene family that influence apoptosis and have been used as surrogate markers in the evaluation of this process. Sixty-three fine-needle tumor samples from an equal number of patients with breast carcinomas were analyzed for Bax, Bcl-2, and DNA content by flow cytometry. The results were correlated with classical clinicopathological parameters. Bax values varied widely among tumors and showed no significant correlation with any of the clinicopathological parameters analyzed. Bcl-2 levels ranged from 4% to 91%, correlated positively with estrogen (P = 0.0004) and progesterone (P = 0.0045) receptor positivity, and were more associated with low S-phase tumor values. In contrast, high S-phase values correlated with estrogen receptor negativity, high grade, and DNA aneuploidy. The study results indicate that Bcl-2 and S-phase analysis of fine-needle samples of breast carcinomas provide a convenient tool for the assessment of these tumors.

Ubiquitin-dependent protein processing controls radiation-induced apoptosis through the N-end rule pathway.

The ubiquitination of nuclear proteins activated in human lymphocytes undergoing radiation-induced apoptosis and the subsequent downstream proteasomal protein processing, shown to be involved in apoptotic death control, may be dependent on an amino-terminal sequence identity of ubiquitin target proteins, the "N-end rule" pathway. Here we report that this selective pathway controls radiation-induced apoptosis and that it is involved in the initiation of this type of cell death. Dipeptide competitors of protein ubiquitination/processing dependent solely on the basic amino-terminal residues (type I) efficiently inhibited the radiation-induced apoptotic death phenotype, indicating that only the substrates of ubiquitination with basic NH2-terminal amino acids are involved in apoptotic death control. This selective inhibition was followed by an early, overall but also target-specific inhibition of ubiquitination and by an activation and stabilization of poly(ADP-ribose) polymerase (PARP) that occurs through inhibition of ubiquitination of its cleaved form (85 kDa). Interestingly, caspases-3 and -7 were not activated following irradiation, further suggesting that PARP cleavage may be regulated by an N-end rule pathway in a caspase-independent manner. These results highly suggest involvement of this subset of the ubiquitin system in the apoptotic death control and in the specific regulation of PARP activity.

Detection and quantitation by fluorescence in situ hybridization (FISH) and image analysis of HER-2/neu gene amplification in breast cancer fine-needle samples.

BACKGROUND: Fine-needle sampling, although a practical and noninvasive method of tissue acquisition, has rarely been used for HER-2/neu fluorescent in situ hybridization (FISH). To assess HER-2/neu gene amplification in mammary carcinoma, FISH signals on cytology and corresponding tissue biopsies were detected visually and measured by image analysis. The results were correlated with patient and tumor characteristics. METHODS: In situ HER-2/neu DNA probe hybridization was performed on 61 cytology specimens and on 47 corresponding frozen sections of breast carcinomas. Tumors were classified by visual evaluation as unamplified, moderately amplified, or highly amplified. Multiparametric image analysis was performed using the Discovery automated image analyzer (Becton Dickinson, Leiden, Netherlands). The integrated fluorescence ratio (IFR) was calculated for each sample as the integrated FISH fluorescence of the tumor cells divided by the integrated FISH fluorescence of internal control cells containing two spots. The percentage positive nuclear area (PPN), calculated as the area of FISH fluorescence divided by the area of nuclear DNA fluorescence, and the PPR, ratio of the PPN of the tumor cells divided by the control cells, were also calculated for each sample. RESULTS: Visual analysis yielded 46 unamplified and 15 (24.6%) amplified (seven moderately amplified and eight highly-amplified) tumors. Strong (P < 0.001) correlation between results on cytological and histological materials was obtained. The FISH spots on the cytological preparations were more easily visualized and scored than those on the corresponding tissue sections. Visual HER-2/neu signal scoring was strongly correlated with IFR (P = 0.0001) and PPR (P = 0.0001). Within the tumors classified as highly amplified by visual examination, quantitation of the degree of amplification fluorescence signal was possible using image analysis. CONCLUSIONS: Cytologic specimens were a suitable and representative source of materials for detection and quantitation of HER-2/neu gene amplification by FISH and image analysis. Cancer (Cancer Cytopathol)

The value of pretreatment cell kinetic parameters as predictors for radiotherapy outcome in head and neck cancer: a multicenter analysis.

PURPOSE: The aim of this study was to assess the potential of pre-treatment cell kinetic parameters to predict outcome in head and neck cancer patients treated by conventional radiotherapy. MATERIALS AND METHODS: Data from 11 different centers were pooled. Inclusion criteria were such that the patients received radiotherapy alone, and that the radiotherapy was given in an overall time of at least 6 weeks with a dose of at least 60 Gy. All patients received a tracer dose of either iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) intravenously prior to treatment and a tumor biopsy was taken several hours later. The cell kinetic parameters labeling index (LI), DNA synthesis time (Ts) and potential doubling time (Tpot) were subsequently calculated from flow cytometry data, obtained on the biopsies using antibodies against I/BrdUrd incorporated into DNA. Each center carried out their own flow cytometry analysis. RESULTS: From the 11 centers, a total of 476 patients conforming to the inclusion criteria were analyzed. Median values for overall time and total dose were 49 days and 69 Gy, respectively. Fifty one percent of patients had local recurrences and 53% patients had died, the majority from their disease. Median follow-up was 20 months; being 30 months for surviving patients. Multivariate analysis revealed that T-stage, maximum tumor diameter, differentiation grade, N-stage, tumor localization and overall time correlated with locoregional control, in decreasing order of significance. For the cell kinetic parameters, univariate analysis showed that only LI was significantly associated with local control (P=0.02), with higher values correlating with a worse outcome. Ts showed some evidence that patients with longer values did worse, but this was not significant (P=0.06). Tpot showed no trend (P=0.8). When assessing survival in a univariate analysis, neither LI nor Tpot associated with outcome (P=0.4, 0.4, respectively). Surprisingly, Ts did correlate with survival, with longer values being worse (P=0.02). In the multivariate analysis of local control, LI lost its significance (P=0.16). CONCLUSIONS: The only pretreatment kinetic parameter for which some evidence was found for an association with local control (the best end-point for testing the present hypothesis) was LI, not Tpot, and this evidence disappeared in a multivariate analysis. It therefore appears that pretreatment cell kinetic measurements carried out using flow cytometry, only provide a relatively weak predictor of outcome after radiotherapy in head and neck cancer.

Comparative analysis of apoptosis measured by Hoechst and flow cytometry in non-Hodgkin's lymphomas.

Fine-needle samples of 75 non-Hodgkin's lymphomas were investigated for apoptosis immediately and after 24 h of culture after in vitro irradiation (2 Gy, 10 Gy, and nonirradiated controls). Apoptotic cells were simultaneously quantified by fluorescence microscopic enumeration of apoptotic cells using Hoechst 33342 staining, and by flow cytometric detection of sub-G1 peak cells. The nonirradiated controls showed a similar mean percent apoptotic cells using both methods, analyzed immediately (9% by morphology vs. 10% by flow) or after 24 h of culture (40% by morphology vs. 41% by flow). In the irradiated samples, the mean percent apoptotic cells quantified by morphology was higher than by flow cytometry (64% by morphology vs. 55% by flow after 2 Gy irradiation, and 71% vs. 58% after 10 Gy). The results of the two methods were correlated, although large differences were seen between the techniques in individual tumors. In our system, flow cytometric sub-G1 peak analysis appears to underestimate apoptosis. Of these two methods, we find the Hoechst morphology method to be more reliable for quantitation of apoptosis utilizing fresh fine-needle sample material, in that discrimination of apoptotic cells from debris is easier and that both early and late apoptotic cells are detectable.

In vitro radiation-induced apoptosis and early response to low-dose radiotherapy in non-Hodgkin's lymphomas.

PURPOSE: Prospective investigation of spontaneous and in vitro radiation-induced apoptosis to predict early response to palliative radiotherapy in patients with non-Hodgkin's lymphomas. PATIENTS AND METHODS: Fine-needle sampling was performed in 28 tumor sites (26 patients) and yielded adequate cell numbers in 27 cases. Apoptotic cells were counted by fluorescence microscopy immediately after sampling and after 24-h culture (spontaneous apoptosis) and 24 h after 2- and 10-Gy in vitro irradiation (radiation-induced apoptosis). Early response to low-dose in vivo radiotherapy (mostly 4 Gy in two fractions over 3 days) was evaluated 15 days after treatment. RESULTS: The tumor response rates at 15 days were 11 (39%) complete responses, nine (32%) responses of greater than 50% reduction in volume, six (21%) responses of less than 50% reduction in volume and two (7%) cases of no response. Tumors achieving complete or major response after in vivo irradiation had higher percentages of apoptotic cells after in vitro irradiation, while no significant differences in terms of spontaneous apoptosis were observed between responders and non-responders. CONCLUSION: Spontaneous and in vitro radiation-induced apoptosis can be easily and quickly assessed on cells obtained by fine-needle sampling of non-Hodgkin's lymphoma lesions. The present results suggest that in vitro radiation-induced apoptosis could be used as a predictive assay of early response to low-dose in vivo irradiation in patients with non-Hodgkin's lymphomas.

Comparison of fixation procedures for fluorescent quantitation of DNA content using image cytometry.

DNA quantitation using fluorescent dyes is of interest in image cytometry in that it is nondestructive in its mode of staining and is compatible with techniques such as FISH and immunofluorescence, allowing multicolor analysis of a wide range of cellular markers of interest. Optimal preparation techniques were sought using human peripheral blood lymphocytes and fine needle samples of breast carcinomas. Unfixed (air-dried only), ethanol, ethanol/acetic acid, and paraformaldehyde/ethanol fixations were tested. Unfixed or fixed cells were placed on slides as a drop or by cytocentrifugation, stained with 4',6-diamidino-2-phenylindole dihydrochloride and DNA content was measured using image analysis. Histogram quality was evaluated using G0G1 peak coefficient of variation, and compared to those generated by flow cytometry. Drop preparations of ethanol/acetic acid fixed and cytospin preparations of paraformaldehyde/ethanol fixed cells appeared to give the best histograms for image analysis, which were inferior in quality to those generated by flow cytometry. Comparison of breast carcinoma histograms generated by flow and image showed the same DNA aneuploid populations but with slightly higher DNA indices measured by image analysis.

Development and optimization of tissue preparative methodology for DNA content analysis of soft tissue neoplasms.

Data regarding DNA content parameters in soft tissue sarcoma is limited. Development and optimization of tissue specific preparative techniques for DNA flow cytometry was undertaken prior to routine DNA content analysis of soft tissue neoplasms; 154 soft tissue tumors were studied. Dissociation dependent differences in cellular yields, viabilities, maintenance of DNA aneuploid populations, coefficients of variation, and DNA index supported the need for these developmental studies. Fifty-six of eighty-nine patients had DNA aneuploid soft tissue sarcomas. A relationship between DNA aneuploidy and grade was seen in this series with 38% with low grade, 59% with moderate grade, and 69% with high grade tumors demonstrating DNA aneuploid populations (P < 0.005). The mean S-phase fraction for DNA diploid and aneuploid sarcomas was 7.2% and 13.3%, respectively (P < 0.001). When classified by histologic grade of the primary tumor, a greater percentage of metastatic lesions were DNA aneuploid (4 of 7 grade 2 lesions, and 15 of 16 grade 3 lesions). Decreases in cellular yields and rate of DNA aneuploidy were observed in a subgroup of patients with localized high grade sarcoma treated preoperatively, as compared to patients treated with initial surgery. Prospective correlation of DNA content parameters to prognosis and response to cytotoxic therapy are now possible and are ongoing.

Flow cytometric DNA analysis of fresh prostatic resections. Correlation with conventional prognostic parameters in patients with prostate cancer.

BACKGROUND: DNA ploidy analysis has been investigated as a prognostic indicator in prostate cancer. Most of the data is derived from retrospective studies using paraffin-embedded tissue. This method has drawbacks related to the quality of DNA histograms and uncontrolled data collection. METHODS: DNA ploidy analysis of freshly resected prostatic tissue was prospectively compared with conventional prognostic variables in 97 men treated with radical prostatectomy for localized prostate cancer. RESULTS: Regarding the patients, 31.9% were African American and 66% had pathologic Stages C or D1 disease. Only 9.6% of patients with Stages A2 and B had a prostate-specific antigen (PSA) value greater than 10 ng/ml, whereas 97% of patients with PSA values greater than 20 ng/ml had pathologic Stages C and D1. PSA levels correlated with Gleason score (P = < 0.05); 51% and 100% of patients with Gleason score 5-7 and 8-10, respectively, had PSA values greater than 10 ng/ml. Twenty-two patients (23%) had DNA aneuploid tumors. Comparisons of mechanical to enzymatic cell suspensions indicated that DNA aneuploidy was better preserved in mechanical cell preparations. DNA ploidy correlated with pathologic stage (P = < 0.05) and Gleason score (P = < 0.05). Fifteen of 79 patients (18.9%) with Gleason score 5-7 had DNA aneuploid tumors versus 71.4% of patients with Gleason score 8-10. PSA groups correlated with ploidy status (P = 0.01). Although the majority of patients (19 of 22) with DNA aneuploid tumors had elevated preoperative PSA levels, none had a PSA value greater than 50 ng/ml. CONCLUSIONS: DNA ploidy analysis correlated with established prognostic indicators in prostate cancer; however, its independent correlation with natural history and treatment outcome must be established for it to have an effect on therapeutic decisions.

DNA content parameters of paraffin-embedded soft tissue sarcomas: optimization of retrieval technique and comparison to fresh tissue.

DNA content analysis of formalin fixed paraffin embedded (FFPE) tissue permits determination of the influence of DNA content on the prognosis in cohorts of patients for whom the clinical outcome is known. Of key importance in such an analysis is the accuracy of DNA content determination. Variations in the quality of DNA histograms from FFPE tissues of different types prompted a comparative evaluation of the preparative methodology of FFPE soft tissue sarcomas for DNA flow cytometry. Following deparaffination and rehydration of fixed tissue, and prior to fluorochrome staining, tissue blocks of 15 DNA aneuploid soft tissue sarcomas were subjected to repeated experimental (time x concentration) enzyme exposures. The goal of these studies was to define the optimal tissue specific retrieval technique with the coefficient of variation, maintenance of DNA aneuploidy, and DNA index as endpoints. After optimizing the technique, the DNA content of 50 soft tissue neoplasms derived from FFPE specimens was compared to the corresponding fresh surgical tissue. The observed 14 percent error rate in the determination of DNA ploidy status suggest limited utility for FFPE tissue in prospective therapeutic trials of soft tissue sarcoma.

Variations in DNA aneuploid cell content during tumor dissociation in human colon and head and neck cancers analyzed by flow cytometry.

Experimental research involving human solid tumors often requires single cell suspensions of high yield that are representative of the tissue of origin and in which the cellular property of interest is preserved. This is particularly necessary for the determination of DNA ploidy by flow cytometry. Mechanical dissaggregation and proteolytic enzyme digestion are the most commonly employed dissociation techniques for solid tumors. Comparative testing of techniques is often not performed. Mechanical and proteolytic enzyme dissociation techniques were comparatively tested in 77 human squamous cell cancers of the head and neck (SCCHN) and 25 human colon cancers for cellular yield, dye exclusion viability, quality, and morphology of DNA histograms, and the presence and proportion of DNA aneuploid subpopulations. Significant and consistent DNA aneuploid subpopulation losses were noted in mechanical preparations of SCCHN and enzymatic preparations of colon cancers. The frequency of SCCHN specimens with DNA aneuploid subpopulations was underestimated by 52% in mechanical cell suspensions, and the proportion of DNA aneuploid cells was diminished in an additional 30% of the specimens. Conversely, the frequency of specimens with DNA aneuploid subpopulations was underestimated by 38% in cell suspensions from enzymatically dissociated human colon cancer and their proportion diminished in an additional 50% of the specimens. Incubations of human colon cancers with three commonly employed proteolytic enzymes demonstrated a progressive loss of DNA aneuploid subpopulations as a function of enzyme concentration and incubation time. This is a serious potential source of error in the flow cytometric determination of DNA ploidy in human solid tumors, and may contribute to the diversity of results obtained and occasional contradictory conclusions reached in such studies.

DNA ploidy of primary and metastatic squamous cell head and neck cancers.

Regional metastases are a major determinant in the treatment outcome of patients with squamous cell carcinoma of the head and neck. Metastases do not respond as well to cytotoxic therapy as do primary tumors. DNA diploid tumors or tumor components also respond poorly to intermittent cytotoxic therapy. In our series of 497 patients with squamous cell carcinoma of the head and neck, the percentage of pure DNA diploid tumors and the mean DNA indexes in 497 primary tumors and 82 regional metastases were 34% and 1.54 and 50% and 1.34, respectively. Paired comparisons were performed in 61 patients and revealed a statistically significant increase in the frequency of DNA diploid tumors (27.4% to 41.2%) in associated lymph node metastases. The clinical observation that patients with squamous cell carcinoma of the head and neck and regional lymph node metastases have a poorer prognosis and a poorer response to cytotoxic therapy may in part be explained by the increased incidence of DNA diploid tumors in their regional lymph nodes, and the poorer response of such tumors to cytotoxic therapy.

Cellular DNA content parameters in untreated and recurrent squamous cell cancers of the head and neck.

The presence and degree of DNA aneuploidy as measured by the DNA index (DI) and the S phase fraction (SPF) were determined by flow cytometry in 294 specimens from 237 patients with untreated and recurrent squamous cell carcinomas of the head and neck (SCCHN). A descriptive analysis was performed in which the specimen DNA parameters were correlated with stage, size of primary, degree of lymph node involvement, morphological grade, and treatment status of the corresponding patients. Approximately 70% of the previously untreated specimens contained DNA aneuploid populations (DI greater than 1.10) and three quarters had SPF that were above 15%. There was a strong, direct association between DI and SPF (P less than 0.001). There was no correlation of the presence or degree of DNA aneuploidy with the stage of the tumor or the size of the primary or conventional morphological grade of the tumor. Specimens from patients with recurrent tumors and untreated patients with N3 lymph nodes had significantly lower rates of DNA aneuploidy and mean DI. Serial determinations of DNA aneuploidy in patients with SCCHN undergoing cytotoxic therapy are ongoing and may prove useful in the identification and understanding of resistance and response in this tumor.

Solid tumor preparation for flow cytometry using a standard murine model.

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well-differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3-methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol-water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.

Solid tumor preparation for clinical application of flow cytometry.

Intense interest in advanced squamous cell cancers of the head and neck (SCC of H&N) has resulted from the recent progress made in tumor responses with chemotherapy and radiotherapy. Unfortunately, the response patterns and clinical outcome of such patients are not adequately predicted on an individual patient basis using clinical parameters or conventional morphology. The study of flow cytometrically determined cellular parameters in such tumors is therefore of interest, but is hindered by inadequate tumor preparative technology. The previous article (10) in this journal describes the use of a murine SCC tumor, LC12, which was employed for comparative testing and determination of optimum techniques of preparation for this tumor. This report describes the application of these techniques to 144 specimens of human SCC of H&N. The mean total yield for these specimens is 7.4 X 10(7) cells/g of tissue. The mean viable enzymatic yield (3.3 X 10(7) cells/g) was higher than the mean viable mechanical yield (2.0 X 10(7) cells/g) except when lymph nodes were the source of the specimen (5.4 X 10(7) cells/g). The mean dye exclusion viability from enzymatically dissociated specimens were above 90%. Significant aneuploidal subpopulation losses were evident in mechanically dissociated and enucleated specimens. 65% of the enzymatically dissociated specimens were successfully cultured with a mean cloning efficiency of 2.1 X 10(-3). Preparative techniques derived from comparative testing with a murine standard tumor have been successfully applied to 144 specimens of SCC of H&N with resultant high yields and excellent viability. Technical problems detected during the preliminary testing with LC12 were confirmed in the human tumors.