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Flow cytometry system, a device used to count and measure the physical fluorescent properties of live cells or other biological elements, is being developed to provide invaluable testing and interpretive testing to physicians for management of patients. In Roswell Park Cancer Institute in Buffalo, NY, data collected from the cytometer is transferred to a personal computer to distinguish cancer cells from normal cells. However, the lack of standardization has relegated the wide use of flow cytometry in cancer diagnosis., The development of instrumentation to measure the fluorescence of live cells in solution initiated the use of flow cytometry for oncology diagnostics. In the 1980s, enhancements such as the discovery [...]

Older individuals are more prone to sickness compared to their younger counterparts. This is explained by an age-related decline in immune function and the immunosuppressive effects of stressful events that are particularly common among elderlies, such as death of a loved one and relocation. Certain hormones such as cortisol are elevated during stress, so determining their levels using radioisotopes may be a way of detecting proneness to sickness. Flow cytometry may also be utilized to assess the number of immune cells. These laboratory procedures may be useful in the early identification of immunosuppression among elderlies., The combination of an aging immune system and a stress-inducing event can spell illness for elderly Americans. Newly emerging evidence suggests that elevated levels of selected hormones may signal a [...]

Background: Most patients receiving therapy for acute myeloid leukemia (AML) enter an interval in which leukemic blast cells cannot be detected by light microscopy (i.e., morphologic remission). However, many of these patients experience a subsequent relapse. Multidimensional flow cytometry, which allows the discrimination of antigens expressed on normal and malignant cells, can detect small numbers of cancer cells in bone marrow or peripheral blood specimens. This technique enables the detection of one leukemic blast cell among [10.sup.3] to [10.sup.2] normal regenerating hematopoietic cells. Purpose: We determined whether the presence of residual leukemic blast cells, identified in the bone marrow of pediatric patients with AML by use of multidimensional flow cytometry, would be predictive of subsequent leukemic relapse. Methods: Multidimensional flow cytometry was performed on 205 marrow specimens collected throughout the course of treatment from 39 patients who had achieved morphologic remission. The analyses employed monoclonal antibodies directed against CD45 in combination with mixed pairs of monoclonal antibodies directed against 10 other antigens. A time-varying Cox regression analysis that controlled for sample time intervals, age, sex, morphologic classification of disease, and white blood cell count at diagnosis was used to relate the multidimensional flow cytometric results to the risk of relapse after achieving remission. Reported P values are two-sided. Results: Thirty-five of the 39 patients had bone marrow specimens available from the time that first morphologic remission was achieved. Leukemic blast cells were detected in the specimens from 19 (54%) of these 35 patients. Twenty-five of the 35 patients did not receive an allogeneic (i.e., from a different genetic background) bone marrow transplant during first morphologic remission, and 13 of 14 with residual leukemic cells experienced a relapse at a median time of 153 days after diagnosis (range, 48-863 days). Nine of the 11 patients who did not receive an allogeneic bone marrow transplant and lacked evidence of leukemic blast cells at first morphologic remission relapsed at a median time of 413 days after diagnosis (range, 321-794 days). Among the 10 individuals who received an allogeneic bone marrow transplant during first morphologic remission, five were positive for leukemic blast cells and five were negative; one of these patients (positive for leukemic blast cells) experienced a relapse 265 days after diagnosis, and three others died of transplant-related complications. The estimated risk of relapse during intervals of multidimensional flow cytometric positivity (i.e., intervals of remission for which the immediately preceding cytometry measurement was positive) was 2.8 times greater than that during negative intervals (95% confidence interval = 1.1-7.0; P = .02). Conclusions and Implications: Multidimensional flow cytometry identifies residual leukemia in more than half of the patients with AML who are in morphologic remission. The detection of leukemic blast cells in these patients by multidimensional now cytometry is predictive of a more rapid relapse. [J Natl Cancer Inst 1996;88:1483-8]

Background. The survival of 52 patients with hepatocellular carcinoma (HCC) seen during the last 4 years was analyzed prospectively on the basis of disease stage and nuclear DNA content. Methods. Ploidy was measured by flow cytometry (FCM). Cells for cytologic diagnosis and FCM were collected by ultrasound-guided fine needle aspiration. Results. DNA aneuploidy, which was detected in 62% of the patients, did not correlate with clinicopathologic features, except in the sonographic aspect (P = 0.03). However, ploidy correlated significantly with survival; the survival times for patients with an aneuploid DNA index were significantly shorter than for those with a diploid index (P = 0.02). In a Cox multivariate analysis, DNA content was prognostically significant, as were the grade of cirrhosis severity and the echographic aspect. Conclusions. In addition to the clinicopathologic features observed, FCM DNA analysis of ultrasound-guided fine needle aspirates from HCC is a simple and valid method for estimating a prognosis of these patients. Cancer 1994; 74:599-605.

We analyzed the patterns of DNA ploidy in 31 colorectal carcinoid tumors from paraffin-embedded tissues by DNA flow cytometry and the relationship of the patterns of DNA ploidy to prognosis. Diploid DNA was found in 78% (24 of 31) of carcinoids and tetraploid in 6% (2 of 31). Five (16%) carcinoids were DNA aneuploid, and four of the patients with ancuploidy showing a near-hypertriploid pattern died during the first 5 years of follow-up. The association of aneuploidy with stage, size, and invasion of tumor was significant. However, our data indicated that DNA aneuploidy of a near-hypertriploid pattern was the most precise and reliable parameter for predicting the prognosis of colorectal carcinoid tumors.

Background. Flow cytometry (FCM)-measured DNA content may be a predictor in the prognosis of ovarian cancer. Multiple specimens taken from the same ovarian tumor may show a variation in DNA content (i.e., intratumoral DNA heterogeneity). We measured the FCM DNA content of multiple specimens from the same tumor in ovarian cancer, and the relationship among DNA ploidy, intratumoral DNA heterogeneity, retroperitoneal lymph node involvement, and surgical staging was evaluated. Methods. Forty-one patients with primary epithelial ovarian cancer were included in the study. The FCM-measured DNA content of multiple fresh tumor specimens taken from different parts of the same ovarian tumor from each patient was measured. When aneuploidy was observed in at least one specimen from the same tumor, the tumor was defined as an aneuploid tumor. If there were two or more different aneuploid stem lines with a variation of DNA indices (differences of the DNA indices >0.15) from the same tumor, the presence of intratumoral DNA heterogeneity was defined. Results. Diploid tumor was found in 8 (19.5%) of the 41 patients, and aneuploid tumor with intratumoral DNA heterogeneity was found in 20 (48.8/o). None of the eight patients with diploid tumors demonstrated lymph node involvement. In contrast, lymph node involvement was found in 14 (70.0%) of 20 patients with intratumoral DNA heterogeneity. There was a significant different incidence of lymph node involvement between the groups with and without intratumoral DNA heterogeneity (P < 0.01). The incidence of intratumoral DNA heterogeneity significantly correlated with the International Federation of Gynecology and Obstetrics staging (P < 0.01), while that of aneuploid tumor did not. Conclusions. Intratumoral DNA heterogeneity reflected a malignant potential for lymph node involvement and its progression in epithelial ovarian cancer. Cancer 1994; 73:3011-4. Key words: flow cytometry, DNA ploidy, intratumoral DNA heterogeneity, lymph node involvement, ovarian cancer.

Background. DNA ploidy is a significant prognostic factor in patients with prostate cancer. Using DNA/nuclear protein flow cytometry, a subpopulation of tumors with near-diploid DNA is identifiable. The prognostic significance of near-diploidy was examined. Methods. Paraffin-embedded formalin fixed prostate tumor tissue from patients treated at M. D. Anderson Cancer Center with external beam radiation therapy was processed for DNA/nuclear protein flow cytometry. All patients had pretreatment and follow-up serum prostate specific antigen (PSA) levels. Seventy-six specimens were suitable for flow cytometric analysis. Tumors were classified as either diploid (n = 30), near-diploid (n = 24), or nondiploid (n = 22, tetraploid and aneuploid). Median follow-up time was 36 months. Results. Diploid tumors were associated with a significantly better actuarial outcome at 4 years, compared with near-diploid tumors, using either biochemical relapse (rising PSA) or a composite end point of a rising PSA or clinical relapse (16% versus 52% relapse, P < 0.05, log-rank). Moreover, patients who had nondiploid tumors had the worst prognosis (77% relapse, composite end point). No significant difference was observed between diploid and near-diploid neoplasms regarding actuarial local control, freedom from metastasis, freedom from clinical relapse, or overall survival time. A Cox proportional hazards model, using the composite end point of a rising PSA or relapse, was performed with ploidy categorized as diploid, near-diploid, and nondiploid; pretreatment PSA, DNA ploidy, and tumor grade were found to be independent prognostic factors. When ploidy was categorized as diploid or near-diploid (nondiploid tumors excluded), pretreatment serum PSA and DNA ploidy were independent predictors of outcome. Ploidy remained an independent prognostic factor even when nondiploid tumors were excluded. Conclusions. These data show that patients who have near-diploid tumors have an intermediate prognosis between the more favorable diploid tumors and the less favorable nondiploid tumors. Cancer 1994; 73: 1895-903. Key words: prostate cancer, radiation therapy, flow cytometry, ploidy, DNA, nuclear protein.

Background. The prognostic significance of flow cytometric DNA ploidy and S-phase fraction (SPF) in ovarian cancer has been controversial. In the current study, the authors analyzed tumor heterogeneity in respect to DNA index DI and SPF. Methods. Flow cytometric variation in DI and SPF among representative fresh tumor material from the primary tumor, metastasis, and malignant effusions from the same patient was analyzed. Results. One hundred thirty-two samples from 47 patients were analyzed, and 119 samples from 42 patients were evaluable. Stable DI between different samples was found in 34 patients, whereas heterogeneity was found in 8 patients (19%). The metastases showed stable DNA content. The malignant effusion samples often lacked tumor cells. The representative ones were often DNA diploid. In 21% of the aneuploid samples, the SPF could not be analyzed. In 38% of the aneuploid samples, the stem line constituted less than 15% of measured nuclei. In these samples, a negative correlation between SPF and percentage of aneuploid cells was found, making SPF unreliable. Correct SPF measurement was thus possible in only 41% of the aneuploid samples, and in these tumors, SPF values varied considerably among different samples from the same patient, illustrated by a median SPF difference of 11% (range, 0-28%). Conclusions. Tumor DI heterogeneity existed in 19% of tumors. SPF depended on the amount of aneuploid cells in case of small stem lines and varied considerably, making its use as a prognostic factor doubtful. To ensure that all tumor stem lines are represented, at least two biopsy specimens from any solid tumor should be analyzed. Cancer 1994; 73:1870-7. Key words: ovarian carcinoma, tumor heterogeneity, DNA ploidy, S-phase fraction.

Background. Aneuploidy in DNA flow cytometry (FCM) of musculoskeletal tumors is generally considered to be a sign of malignancy. Previously, giant cell tumor of the bone has been reported to contain aneuploid (near0diploid) DNA stemlines. Otherwise, only sporadic cases have been reported. The authors wanted to study the relationships among DNA FCM, histology, and clinical course of nonmalignant musculoskeletal lesions. Methods. Twenty-eight histologically benign tumors and seven nonnepolastic lesions were subjected to DNA FCM. After tissue preparation mechanically and with ribonuclease and trypsin, the isolated nuclei were stained with propidium iodine using chicken and rainbow trout erythrocytes as controls. In the DNA FCM histograms, ploidy and cell cycle fractions were determined using a computerized mathematical model. The histologic diagnoses were made without knowledge of the DNA FCM results. Results. Aneuploidy was found in eight lesions. A shoulder in the diploid peak, suggesting a diploid and a near-diploid population, was found in DNA histograms of a condensing osteitis of the clavicle (a benign inflammatory process) and of a giant cell tumor of bone. The latter lesion also had a tetraploid population. Six benign tumors--two enchondromas, one osteochondroma, one subcutaneous and one intramuscular lipoma, and a calcifying aponeurotic fibroma--showed clear aneuploidy with separate peaks. The S-phase fraction was less than 10% in all cases. The highest aneuploid population, DNA index = 1.70, in a subcutaneous lipoma, was small, with an undetectable S phase. Despite nonradical operations in seven lesions, no recurrences were observed during a median follow-up of 49 months (range, 28--73 months). Conclusions. Small aneuploid populations with low DNA synthetic activity may be compatible with a benign histologic picture and uneventful clinical course of the musculoskeletal lesion. Cancer 1994; 73:1200--5.

The deoxyribonucleic acid (DNA) index appears to be the strongest predictor of health outcomes in patients with endometrial cancer. The DNA index is a comparison of the tumor DNA content to a normal DNA standard. Researchers performed flow cytometry on fresh tumor samples from 74 patients with endometrial cancer. Fifty-three tumor samples had normal numbers of chromosomes and 21 samples had abnormal chromosome numbers according to the DNA index. Fourteen patients had recurrences of the disease and 10 patients died from endometrial cancer. The recurrence rate was about 8% for tumors with normal chromosome numbers and about 48% for tumors with abnormal numbers. The mortality rate was about 4% and the disease-free survival rate was about 89% among patients who had tumors with normal DNA content, compared to 38% and 36% among those with abnormal tumor DNA. The DNA index and the stage and grade of disease were significant predictors of survival.

DNA flow cytometry of the lymph node metastases from 56 patients was used to retrospectively evaluate the prognostic significance of DNA ploidy in patients with stage III melanoma. The findings were correlated with traditional prognostic factors and patient survival. Multivariate regression analysis revealed that, in addition to the number of positive lymph nodes and patient gender, the DNA index was a significant predictor of patient survival (all p

Background. Although flow cytometry (FCM) has become a widely used technique for the measurement of DNA content in solid tumors, the correlation of ploidy analysis by FCM with cytogenetic analysis (CGA) is not well described. The sensitivities of G-banded CGA and FCM were compared to determine the accuracy of the DNA index value (DI) as a measurement of chromosome number. Methods. Tumor specimens from 56 pediatric cases were analyzed for DNA content by both FCM and CGA. Nuclei for FCM were prepared from archival tissue in 53 specimens using a modification of the Hedley technique and from fresh tissue in 3 specimens. Metaphase chromosomes for CGA were prepared from standard solid tumor harvests and Giemsa-trypsin banding procedures. Ploidy status for this study was defined as (1) diploid--DI between 0.97 and 1.03 by FCM or chromosome number [+ or -] 2 from normal by CGA (44-48); and (2) aneuploid--DI < 0.97 or > 1.03 by FCM or total chromosomes < 44 or > 48 by CGA. Results. Forty-nine of the 56 pediatric specimens were evaluable by both techniques. Concordance was observed in 34 cases (69%) between the two techniques in assigning similar ploidy status to a tumor (22 diploid and 12 aneuploid). It also was observed that among the aneuploid concordant cases, the actual DI obtained from archival material could predict total chromosome number with 95% accuracy. The 15 discordant cases showed a distinct aneuploid population by FCM, but were diploid by CGA. Conclusions. A correlation of 69% was obtained between both techniques to assign a similar pleidy status (diploid versus aneuploid) in 56 pediatric solid tumors. These results support the combined use of CGA and FCM to obtain the most complete analysis of DNA content and chromosome abnormalities in pediatric solid tumors. FCM on formalin-fixed, paraffin-embedded tissue can be used to measure total DNA content. Cancer 1993; 72:2033-41.

Background The recurrence or mortality rate of axillary lymph node-negative invasive breast cancer has been associated with the tumor S-phase fraction, which is measured by DNA flow cytometry. Because many of the studies that established this association were performed using frozen, pulverized tumor specimens, this association could not be tested for independence from the established prognostic factors of histologic and nuclear grading. Methods. Histologic, nuclear, and mitotic grades, DNA ploidy, and S-phase fraction (SPF) were determined from paraffin-embedded tumors obtained from 280 women with node-negative invasive ductal carcinomas using standard grading schemes and flow cytometric techniques. These variables were compared with disease-free and cancer-specific survival (CSS) in univariate and multivariate analyses of these patients. Results. Tumor diameter, SPF, histologic grade, and nuclear grade were significant predictors of disease-free survival (DFS); diameter and SPF had significant associations with CSS. Cox analysis showed histologic grade to be the only independent predictor of relapse, whereas diameter and SPF were independent predictors of mortality. The patients with low nuclear or histologic grade tumors had only a 5% risk of recurrence at 5 years. In contrast, 36% of patients in this series with medium-grade or high-grade high SPF tumors had a 30% risk of recurrence over the same interval. Conclusions. Histopathologic grading and flow cytometric determination of SPF appear to provide additive prognostic information for patients with early invasive ductal carcinomas of the breast. Cancer 1993; 72: 1926-32.

Background. The prognostic value of flow cytometric DNA analysis on paraffin-embedded tumor samples has been controversial in esophageal cancer. To clarify its true significance, the authors developed an improved method that excludes the possibility of contamination by lymphocytes in tumor sample. Methods. Single nuclear suspension was prepared from paraffin-embedded samples on 103 patients with squamous cell carcinoma of the esophagus. Both DNA content and nuclear size were simultaneously measured by flow cytometry on 30,000 nuclei, and contaminated lymphocyte nuclei were eliminated from the data by optimal gating. Correlation between DNA ploidy and postoperative survival was examined. Results. Analysis using a flow cytometric cell sorter showed that the frequency of tumor cells in the lymphocyte-reducing gating fraction (LGF) was significantly higher than that in the conventional nongating fraction (NGF). LGF analysis showed aneuploid peaks in 58 patients (56.3%), but NGF analysis showed aneuploid peaks in only 38 patients. LGF analysis revealed that the aneuploid tumors had higher histologic grading (P < 0.05) and worse survival rate (P < 0.01) compared with diploid tumors. However, conventional methods could not detect this difference. Conclusions. Flow cytometric analysis gating by nuclear size may be helpful to detect aneuploid peaks, and for predicting prognosis of patients with squamous cell carcinoma of esophagus. Cancer 1993; 72:1813-8.

Background. DNA ploidy and S-phase fraction measured by DNA flow cytometry were shown to correlate with several clinicopathologic characteristics in several types of tumors. Methods. DNA flow cytometry was performed on 329 samples (164 normal mucosa, 165 tumors) obtained from 165 patients (112 men) with stomach cancer, and the findings were correlated with various clinicopathologic characteristics of the patients in a prospective manner. Results. Seventy-nine of 165 samples (48%) from the tumors gave an aneuploid histogram. None of 164 samples from the normal mucosa showed aneuploidy. There was no significant difference in the frequency of DNA aneuploidy in terms of age, sex, symptom duration, bleeding history, gastric outlet obstruction, weight loss, performance status, serum hemoglobin level, albumin level, creatinine level, tumor size, tumor location in the stomach, and TNM stage. Moderately well differentiated tumors had a significantly higher frequency of aneuploidy compared to well differentiated or undifferentiated tumors. S-phase fraction was obtained in 162 of 164 samples from the normal mucosa, and 123 of 165 samples from the tumors. The overall mean of the S-phase fraction was 4.01% (range, 0.5-23.6%) for the normal mucosa and 13.8% (range, 0-51.9%) for the tumors. Higher S-phase fraction of tumors was correlated with history of weight loss, poorer performance status, and histologically less differentiated tumors. Conclusions. The overall frequency of aneuploidy was 48% in stomach cancer. DNA aneuploidy showed significant correlation with histologic differentiation of tumors. S-phase fraction of the tumors showed significant correlartion with history of weight loss, performance status of the patients, and histologic differentiation of tumors. Cancer 1993; 72:1819-26.

Background. The prognostic value of flow cytometric analysis of DNA content is contradictory in renal cell carcinoma (RCC). Methods. Flow cytometric analysis was performed on 72 patients with RCC, and the relationship between the DNA content and pathologic or clinical features was investigated. Results. Aneuploidy was observed in 36 (50%) of these patients. Two tissue samples from primary tumors of 12 patients were analyzed, and heterogeneity was found in 7 (58%) of these patients. The incidence of aneuploidy was significantly higher in high-grade than in low-grade tumors (P = 0.0328). All five tumors with a maximum diameter of 2.5 cm or smaller (T1N0M0) were diploid, and a trend for more aneuploid tumors among high-stage diseases was observed. The survival rate of patients with diploid tumors was not significantly different from that of those with aneuploid tumors in the low-or high-stage group. Conclusions. DNA ploidy assessed with one sample is not a prognostic factor, and heterogeneity between different samples of a given tumor indicates that one sample is not enough for DNA flow cytometry in RCC. Cancer 1993; 72:1319-23.

Background. Although it is important to determine any relationship between tumor DNA ploidy and its biologic behavior, the correlation between DNA-ploidy and the prognosis of patients with ovarian cancer is not conclusive. Accordingly, the authors evaluated the clinical application of DNA ploidy in ovarian cancer. Methods. Flow cytometric measurements were performed in 45 selected patients with well-differentiated serous cystadenocarcinoma of the ovary, Stages Ic-IV. All of them had the same surgical procedure, with retroperitoneal lymphadenectomy including paraaortic nodes, followed by the same postoperative chemotherapeutic regimen. Results. Of the 45 ovarian cancers, 28 were diploid and 17 were aneuploid. The 2-year survival rate and the estimated 5-year survival rate for patients with diploid tumors were significantly greater than those for patients with aneuploid tumors (73.2% versus 46.7% and 29.1% versus 22.4%, respectively). The 2-year survival rate in patients with advanced disease (Stage III or IV) was also significantly higher for those with diploid tumors (53.3% versus 37.4%, respectively), but the estimated 5-year survival rate was similar in both groups (8.9% versus 9.1%, respectively). Patients with advanced disease had aneuploid tumors more frequently than those with early-stage disease. A significantly higher incidence of retroperitoneal lymph node metastasis was observed in aneuploid tumors than in diploid tumors (43.8% in diploid tumors versus 86.7% in aneuploid tumors). The authors found no difference in the response to chemotherapy between diploid and aneuploid tumors. Conclusions. Although tumor DNA ploidy was not as reliable as conventional parameters such as surgical stage in establishing prognosis, it may provide an indicator of lymph node involvement.

Background. The incidence of upper urinary tract tumors is relatively high in southern Taiwan. DNA analysis by means of flow cytometry is not well investigated with regard to tumors of the upper urinary tract and the differences in DNA ploidy between transitional cell carcinomas in endemic and nonendemic areas. Methods. A retrospective nuclear DNA ploidy analysis by flow cytometry comprised 41 formaldehyde solution-fixed, paraffin-embedded tissue specimens of transitional cell carcinoma of the renal pelvis and ureter. The preparation of nuclear suspensions from paraffin-embedded tissue blocks and staining were modified by means of the techniques of Hedley and Vindelov. Results. There was no statistical correlation between DNA ploidy, histologic grade, and pathologic stage; however, 82% of the DNA nondiploid tumors showed tumor progression. In contrast, only 46% of the DNA diploid tumors revealed tumor progression. Among Grade 2 tumors, 85% of the DNA nondiploid tumors showed postoperative tumor progression, whereas only 31% of the DNA diploid tumors showed tumor progression. Seventy-nine percent of the nondiploid patterns were present in patients native to the Pa Chang Valley, where the so-called 'blackfoot disease' and urothelium tumor are endemic, whereas only 22% appeared in patients living. in other areas. Conclusions, DNA flow cytometry can identify a group of patients with poor outcome unpredictable by pathologic examination, and is an important tool in research into the pathogenesis of cancer.

A patient is described with chronic myelogenous leukemia in blastic crisis, in whom numerous circulating platelet fragments and megakaryocytic nuclei were present, with 50% blasts and 50% micromegakaryocytes in the marrow. The blasts expressed myeloid-associated antigens CD34, CD33, and CD13, whereas the micromegakaryocytes were positive for CD41, CD42b, and CD61. These findings suggested a myeloblastic transformation with a possible megakaryoblastic component. Cytogenetic analysis showed rearrangement of 3q26 in the form of t(2;3) (p13; q26), in addition to t(9; 22) (q34; q11). Dual-color flow cytometric analysis of DNA content of CD42b-positive cells showed that the micromegakaryocytes were predominantly 2N, indicating a maturation block before nuclear endoreplication and polyploidization. These findings confirmed a combined myeloblastic and megakaryoblastic transformation. It is concluded that dual-color flow cytometric DNA analysis is a useful method for the investigation of abnormal megakaryocytopoiesis in hematologic malignancies.