Prognostic significance of ploidy determination in rectal cancer.

Between 1965 and 1981, 154 patients with potentially curable rectal adenocarcinoma underwent surgical treatment at the University of Chicago Medical Center. In 134 cases, enough histological material was available to perform determinations of DNA content by the cytophotometric method (n = 108), or by the flow cytometric technique (n = 109). In 83 cases, DNA content was analyzed in the same specimen with both techniques, and in 77 of these cases the sections obtained from the paraffin blocks were contiguous. When using flow cytometry, 62% of stage B and 74% of stage C lesions were classified as aneuploid on the basis of a DNA index greater than 1. This correlation was statistically significant (p = 0.002). Patients with diploid tumors had an actuarial five-year survival equal to 62% in comparison with a 46-51% five-year survival for patients with aneuploid tumors. This difference was not statistically significant and it was explained by the tendency for aneuploid tumors to be in an advanced histopathological stage.     

The importance of blast cell DNA content for prognosis of childhood acute lymphoblastic leukemia

The prognostic value of cellular DNA content measured by static cytophotometry was evaluated in 69 children with acute lymphoblastic leukemia (ALL) using the pretreatment distribution of the DNA content in blast cells of bone marrow and peripheral blood. The median follow-up of the whole group of patients was 45 months. Aneuploidy was detected in 71% of children, most of them showing a hyperdiploid content (DNA index greater than 1.05). The duration of complete remission was significantly longer in patients with distinct hyperdiploid DNA content (DNA index greater than 1.16) than in those with less hyperdiploid and diploid DNA content (DNA index less than 1.16). The results achieved by static cytophotometry were compared with flow cytometry analysis and with cytogenetic investigations of chromosomal abnormalities in leukemic cells. Higher correlation was found between flow cytometry and cytogenetics. Flow cytometry proved to be a more convenient method for detection of the DNA content in leukemic cells than static cytophotometry.     

Detection of clonal excess in lymphoproliferative disease by kappa/lambda analysis: correlation with immunoglobulin gene DNA rearrangement

Previous studies have suggested that analysis of the distribution of surface immunoglobulin light chain isotypes by flow cytometry provides evidence for monoclonality of B cell tumors and may detect populations of circulating tumor cells in patients with lymphoproliferative disease. We have used simultaneous flow cytometry and DNA restriction enzyme analysis on 58 samples of tissue and blood to determine whether lymphocyte populations detected by "kappa/lambda" analysis are indeed monoclonal. In greater than 90% of cases, abnormalities detected by flow cytometry correlated with monoclonal rearrangements of immunoglobulin genes as detected by Southern blot analysis. By analyzing tissue and blood from the same patients, we have also demonstrated that monoclonal circulating cells detected by flow cytometry reflect peripheral circulating tumor cells, since DNA from these cells shows the same immunoglobulin rearrangement as DNA from the original tumors in these patients. Although mixing studies suggested that DNA rearrangement studies were more sensitive than was flow cytometry in detecting minor populations of monoclonal lymphocytes, we found only one case in which this affected the diagnostic accuracy of the kappa/lambda analysis, with one notable exception, that of detection of a monoclonal proliferation of B cells that did not express surface immunoglobulin. The kappa/lambda test thus offers a powerful diagnostic tool in the evaluation of lymphoproliferative disease.     

Prognostic implications of DNA ploidy in squamous cell carcinomas of the tongue assessed by flow cytometry

A total of 47 primary squamous cell carcinomas of the tongue were analysed by DNA flow cytometry. With respect to their clonal DNA content two distinct tumor groups could be distinguished. In 14 cases the tumors (29.8%) were dipoid, whereas in 33 cases (70.2%) additional cell lines characterized by abnormal DNA content could be detected. A significant increase of aneuploid cases with tumor size as well as with decreasing histological differentiation could be detected. Aneuploid cell lines are lacking in T1 as well as in most G1 carcinomas but predominate in T3 and G3. Furthermore, cervical lymph node involvement was recognized in the majority of aneuploid primary carcinomas (81.8%) but was largely lacking in diploid tumors (21.4%). Hence, the presence of aneuploid cell lines is clearly connected with clinical and histopathological parameters, each of which have turned out to worsen the prognosis in tongue carcinomas.     

Ploidy in colorectal cancer

For several authors, DNA tumoral cell content represents an important prognostic factor in colorectal cancer. Samples obtained from 65 human colorectal cancers operated on between 1983 and 1986 were studied. Of 52 cases studied by flow cytometry 60 p. 100 were aneuploid tumors. The proliferative index was calculated in slightly over 50 p. 100 of the cases by DNA histogram analysis. During the same period 30 tumoral karyotypes were established by cytogenetic analysis. In 17 cases a comparison was possible between flow cytometry and cytogenetic results. In all cases a significant correlation was seen between the DNA histogram modal value and the mean number of chromosomes counted by cytogenic analysis. In this study, there was no statistical correlation between flow cytometry results and Dukes classification. Because of the short follow-up in our series, no prognostic value may be attributed to the DNA index.     

Numerical Aberrations of Chromosomes 16, 17, and 18 in Hepatocellular Carcinoma (A FISH and FCM Analysis of 20 Cases)

Conventional cytogenetic studies havedemonstrated frequent abnormalities of specificchromosomes in hepatocellular carcinoma, although thereare few reports examining the relationship betweenchromosomal aberrations and clinicopathologic features. Inthis study, numerical aberrations of chromosomes 16, 17,and 18 were examined by fluorescence in situhybridization using pericentromeric DNA probes in 20 cases of surgically removed hepatocellularcarcinoma. DNA ploidy analysis was also performed byflow cytometry. Numerical abnormalities of chromosomes16, 17, and 18 were found in 7 of 19 cases, 15 of 20 cases, and 12 of 20 cases, respectively. Gainand/or loss of more than one chromosome was detected in16 of 19 cases. However, aneuploidy was seen in only 9of 20 tumors by flow cytometry. The incidence of aneusomy 17 and 18 increased with tumor sizeand stage progression. Fluorescence in situhybridization analysis demonstrated that numericalchromosomal aberrations accumulated with tumorprogression in hepatocellular carcinoma.     

Cytophotometric and flow cytometric DNA content of isolated glands in gastric neoplasia.

The gland isolation method was applied to various gastric lesions to measure DNA content by cytophotometry and flow cytometry for the first time. By incubating and agitating fresh specimens from surgically resected stomachs in calcium-magnesium free Hanks's balanced salt solution (CMFH) containing EDTA, many neoplastic glandular epithelial cells were successfully isolated from the stroma, and their characteristic three dimensional features were seen morphologically. The DNA content of pure nuclear suspensions of isolated glands was obtained by cytophotometry and flow cytometry staining with 4',6-diamino-2-phenylindole dihydrochloride (DAPI) and propidium iodide, respectively. Compared with histological grading, the frequency of the DNA aneuploidy of cancer with moderate or poor differentiation by cytophotometry (75%) was significantly higher than that of well differentiated cancer (25%), but the histological typing of gastric cancer DNA frequency were not correlated. This method allowed us to detect small aneuploid peaks by flow cytometry, which were previously masked by contaminating interstitial cells. The frequency of DNA aneuploidy detected by flow cytometry (87.5%) was higher than detected by cytophotometry (58.3%). The results of these studies shows the feasibility of this technique for analysing the DNA content of various lesions of the stomach.     

DNA ploidy and c-Kit mutation in gastrointestinal stromal tumors

AIM: To investigate the prognostic significance of c-Kit gene mutation and DNA ploidy in gastrointestinal stromal tumors (GISTs). METHODS: A total of 55 cases of GISTs were studied for the expression of c-Kit by immunohistochemistry, and the c-Kit gene mutations in exons 9, 11, 13, and 17 were detected by polymerase chain reaction-single strand confirmation polymarphism (PCR-SSCP) and denaturing high performance liquid chromatography (D-HPLC) techniques. DNA ploidy was determined by flow cytometry. RESULTS: Of the 55 cases of GISTs, 53 cases (96.4%) expressed c-Kit protein. The c-Kit gene mutations of exons 11 and 9 were found in 30 (54.5%) and 7 cases (12.7%), respectively. No mutations were found in exons 13 and 17. DNA aneuploidy was seen in 10 cases (18.2%). The c-Kit mutation positive GISTs were larger in size than the negative GISTs. The aneuploidy tumors were statistically associated with large size, high mitotic counts, high risk groups, high cellularity and severe nuclear atypia, and epithelioid type. There was a tendency that c-Kit mutations were more frequently found in aneuploidy GISTs. CONCLUSION: DNA aneuploidy and c-Kit mutations can be considered as prognostic factors in GISTs.     

Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome

The myelodysplastic syndromes (MDSs) are characterized by bilineage or trilineage dysplasia. Although diagnostic criteria are well established for MDS, a significant number of patients have blood and bone marrow findings that make diagnosis and classification difficult. Flow cytometric immunophenotyping is an accurate and highly sensitive method for detection of quantitative and qualitative abnormalities in hematopoietic cells. Flow cytometry was used to study hematopoietic cell populations in the bone marrow of 45 patients with straightforward MDS. The results were compared with those obtained in a series of patients with aplastic anemia, healthy donors, and patients with a history of nonmyeloid neoplasia in complete remission. The immunophenotypic abnormalities associated with MDS were defined, and the diagnostic utility of flow cytometry was compared, with morphologic and cytogenetic evaluations in 20 difficult cases. Although morphology and cytogenetics were adequate for diagnosis in most cases, flow cytometry could detect immunophenotypic abnormalities in cases when combined morphology and cytogenetics were nondiagnostic. It is concluded that flow cytometric immunophenotyping may help establish the diagnosis of MDS, especially when morphology and cytogenetics are indeterminate. (Blood. 2001;98:979-987)     

Prognostic relevance of ploidy and proliferative activity evaluation by flow cytometry in poor prognosis non-Hodgkin's lymphomas.

The DNA content and S-phase fraction (SPF) of pathological paraffin-embedded lymph node tissue from 47 patients with poor prognosis non-Hodgkin's lymphoma (NHL) were analyzed by flow cytometry (FCM). Fifteen of 42 evaluable cases proved to be aneuploid. The DNA aneuploidy was not correlated with age, histology, stage, presence of systemic symptoms, clinical response or overall survival (OS) of patients. Mean SPF evaluated in 35 cases was 22.9%. After 78 months of follow-up, nine patients with diploid low SPF tumors had a significantly longer disease-free survival (DSF) with respect to the group of patients (33 cases) with aneuploid or diploid high SPF tumors (100% vs 59%; log rank test, p = 0.04).     

A prognostic factor in childhood acute lymphoblastic leukemia: implication of cellular DNA contents. Children's Cancer and Leukemia Study Group

The cellular DNA content of marrow blasts were measured by flow cytometry in 250 children with acute lymphoblastic leukemia (ALL). Abnormal DNA stemlines were detected in 51 cases (26%) of 196 children with untreated ALL and in 10 cases (18.5%) of 54 children with relapsed ALL. In untreated cases, the distribution of cellular DNA content for DNA aneuploidy showed that all except one hypodiploid case, had hyperdiploid DNA contents (DI greater than 1.0). Children with hyperdiploid DNA stemline had significantly better prognosis than did those with diploid DNA stemline (DI = 1.0) in both low-and high risk groups; risk assignment was based on an initial WBC count and age at diagnosis. The relative risk of failure to treatment for the hyperdiploid group was one third that of the diploid group in low-risk ALL and one fifth that of the diploid group in high-risk ALL. In relapsed ALL, there was no significant correlation between the cellular DNA contents and their prognosis after relapse. DNA aneuploidy was detected more frequently in late relapsed cases (40%) than in early relapsed cases (6.2%). These results show that cellular DNA content before treatment is an important prognostic factor in childhood ALL.     

Cytostatic effects in osteosarcomas as detected by flow cytometric DNA analysis after preoperative chemotherapy according to the COSS 80/82 protocol

Summary A total of 20 highly malignant osteosarcomas were studied by DNA flow cytometry after preoperative chemotherapy according to the COSS 80/82 protocol to assess their nuclear DNA content and the impact of chemotherapy on DNA ploidy and proliferation. Of the cases studied, 70% revealed aneuploid DNA stem lines with a median value of 1.67, and a median S-phase proportion of 16.1%. These data differed substantially from the results of a preceding study on untreated osteosarcomas revealing a higher frequency of DNA aneuploidy, higher DNA indices, and higher proportions of cells in S-phase. The pretreated tumors also showed a distinct relation between DNA content and the grade of tumor regression. No aneuploid DNA stem line was found in the responder group-I, whereas group-V (osteosarcomas with more than 50% viable tumor tissue) consisted almost completely of aneuploid DNA populations; all tumors with more than 2 DNA aneuploids were found in the latter group. All populations with a DNA index (DI) over 2.0 were found in the nonresponder groups IV and V, where all DNA aneuploids had an S-phase above 12%. No differences in DNA ploidy or proliferation were found in the various histological subtypes of pretreated osteosarcomas. These data indicate that flow cytometric DNA measurement in osteosarcoma may not only serve as a tumor marker, but also support the evaluation of morphologically established regression grades, thereby verifying the patient's prognosis: a high DI (over 2.0), a high number of DNA aneuploids (more than 2), and a high proliferation (S-phase above 12%) seemed to indicate a poor regression of the primary lesion, which may offer a pretherapeutic way of prognostic evaluation.Supported by Landesministerium für Forschung Nordrhein-Westfalen     

Comparison of Quantitative Real-Time PCR and Digital PCR to Detect the Polyomavirus in Merkel Cell Carcinoma

Merkel cell polyomavirus (MCPyV) prevalence in Merkel cell carcinoma (MCC) cases is controversial. The detection and quantification of MCPyV DNA is mainly performed by PCR techniques using formalin-fixed, paraffin-embedded (FFPE) tissues. The aim of this study is to compare the performance of two different molecular techniques, specifically the quantitative Real-Time PCR (qPCR) and digital PCR (dPCR). Samples from 31 cases of MCC excisional surgical biopsies were analyzed. DNA extraction and purification from clinical samples were performed using the QIAcube Qiagen automated nucleic acid extractor. After the extraction, MCPyV was detected by qPCR and dPCR using specially designed primers and probes. Of the 31 MCC samples under study, the MCPyV genome was detected in 11 samples (35%) by qPCR compared with 20 samples (65%) detected by dPCR. Notably, 65% of primary tumors were positive for MCPyV (15/23). The viral genome was detected in 75% of tumors located at UV-exposed sites (6/8), 55% of tumors at partially UV-protected sites (5/9), and 67% of tumors at UV-protected sites (4/6). Our results showed a better sensitivity of dPCR in detecting the MCPyV genome in MCC samples compared with traditional qPCR techniques.     

Prolymphocytic leukemia: Flow microfluorometric,immunologic, and cytogenetic observations

Cells isolated from four patients with prolymphocytic leukemia were evaluated by surface markers, cytogenetics, and flow microfluorometric analysis of cell size and DNA content. All four patients had B-cell markers with a high density of IgM, kappa type, and Ia-like antigen. Less intense staining for surface IgD was also observed. In each patient studied, chromosomal modes were in the hypodiploid or near-diploid range. Despite the karyotypic abnormalities, the cellular DNA content, as determined by flow microfluorometry, was within normal limits in all cases. This suggests that the variability in chromosome numbers seen in these patients may reflect an abnormality in DNA packaging rather than differences in total DNA content. The modal electronic cell size of the prolymphocytes, determined by light scatter, was readily distinguishable from that of normal peripheral blood lymphocytes and the lymphocytes of chronic lymphocytic leukemia. Fewer than 4% of the peripheral prolymphocytes had S-phase DNA content, a finding consistent with the chronic nature of this leukemia.     

Serial assessment of suspected myelodysplastic syndromes: significance of flow cytometric findings validated by cytomorphology,cytogenetics, and molecular genetics

The significance of flow cytometry indicating myelodysplasia without proof of myelodysplasia by cytomorphology remains to be clarified. We evaluated follow-up analyses in 142 patients analyzed in parallel by flow cytometry, cytomorphology and cytogenetics for suspected myelodysplasia without proof of myelodysplasia by cytomorphology. At initial assessment, flow cytometry indicated myelodysplasia in 64 of 142 (45.1%) patients. In 9 of 142 (6.3%) patients, cytogenetics revealed aberrant karyotypes at first evaluation that were found in 5 of 64 (7.8%) patients rated with myelodysplasia by flow cytometry. The remaining 133 patients without proof of myelodysplasia by cytomorphology and with normal karyotype underwent follow-up analyses that confirmed myelodysplasia by cytomorphology, cytogenetics or molecular genetics in 47 (35.3%) after a median interval of nine months (range 1-53 months). As far as initial flow cytometry results are concerned, this applied to 30 of 59 (50.1%) with myelodysplasia, 10 of 42 (23.8%) with “possible myelodysplasia” (minor antigen aberrancies only) and 7 of 32 (21.9%) without myelodysplasia (P=0.004). Notably, in these latter 7 patients, flow cytometry results changed at follow up to “possible myelodysplasia” (n=4) and “myelodysplasia” (n=2). These data argue in favor of including flow cytometry along with cytomorphology, cytogenetics and molecular genetics to diagnose myelodysplasia, and suggest a closer monitoring of patients with myelodysplasia-typical aberrant antigen expression found by flow cytometry.     

DNA densitometry of colorectal cancer.

DNA analysis was assessed by densitometry for 281 cases of colorectal adenocarcinoma. Detection of aneuploidy in a single case rose from 65% if one, to 92.5% when three or more sections, were analysed. Although aneuploid tumours had significantly larger nuclear areas than near diploid tumours (p = 0.009), densitometric measurements showed no association with clinicopathological variables. DNA content determined by densitometry was compared with that from flow cytometry on 465 tissue sections from 241 cases. Aneuploidy assessed by flow cytometry was significantly associated with that determined by densitometry (p < 0.01 for all comparisons), ploidy state being similar in 381 sections (82%, kappa = 0.63, p < 0.001), and 187 cases (77.6%, kappa = 0.57, p < 0.001). Univariate survival analysis showed that DNA densitometric variables had no significant association with survival in (a) all cases, (b) cases without lymph node metastases, or (c) cases without distant metastases. Multivariate regression analysis of densitometric and clinicopathological variables identified Dukes's stage, patient age, and tumour differentiation as the combination of variables most closely related to survival. Densitometric measurement of DNA content could not significantly improve on the prognostic model containing these three variables. It is concluded that, although the assessment of DNA content by densitometry is comparable with that of flow cytometry, conventional histological variables remain the best predictors of prognosis in colorectal cancer.     

Analysis of the cellular DNA and RNA content in acute leukemias by flow cytometry

Summary In the present study bone marrow samples from 573 patients with newly diagnosed acute myeloid (AML) and lymphoblastic or undifferentiated leukemias (ALL/AUL), were analysed for their cellular DNA und DNA/RNA content, respectively, by means of flow cytometry. From 237 patients with AML 35.4% revealed aneuploid DNA stemlines. While no relation of DNA aneuploidy with other pretherapeutic parameters, including FAB subtype, white blood cell count, lactate dehydrogenase, S-phase index and percentage of blasts in the bone marrow, was observed, cases with aneuploid DNA stemlines revealed a tendency towards longer remission duration. In ALL/AUL 21.8% of 280 patients expressed DNA aneuploidies, which were less frequently found in T-cell ALL (11.1%) as compared to common(C)-ALL (21.4%) or null-cell(null)-ALL (23.5%). DNA aneuploidy was not related with other clinically defined risk factors such as age, white blood cell count, and rapid achievement of remission. Patients with DNA indices <1.0, however, tended to have shorter remissions. A significant difference in RNA indices was observed between AML and ALL/AUL with median values of 14.4 and 10.1, respectively (P<0.05). These data indicate the usefulness of flow cytometric analyses of cellular DNA and RNA content for the characterization and classification of acute leukemias, complementing the identification of clinical risk factors, immuno-phenotyping and cytogenetics.Abbreviations AML acute myeloid leukemia - ALL acute lymphoblastic leukemia - AUL acute undifferentiated leukemia - T-ALL, C-ALL and Null-ALL T-cell, common and Null-cell ALL     

Flow cytometry in brain tumors. I. Ploidy abnormalities

Flow cytometry (FCM) was introduced for estimation of DNA content in 75 brain tumors. Among these astrocytomas (38 cases) and meningiomas (16 cases) predominated. Astrocytomas were histologically subdivided into 3 groups of malignancy grade. Aneuploid cell populations were found in 1 out of 4 cases of astrocytoma grade I, in 11 out of 20 astrocytomas grade II and in 10 out of 14 astrocytomas grade III of malignancy. DNA index (DI) in most aneuploid astrocytomas is in the range between 1 and 2. More than one cell population with aneuploid value of DNA was found in 36% of all aneuploid tumors.     

Relationship between DNA ploidy and survival in patients with exocrine pancreatic cancer

The DNA ploidy of pancreatic cancer tissue from paraffin blocks was measured by flow cytometry in 46 patients whose disease had been detected and treated with surgery. Lymph node involvement was observed at the time of diagnosis in 36% of patients with diploid tumors and in 79% of patients with aneuploid tumors (p = 0.017), but no clear relation to metastasis could be observed (p = 0.201). The S-phase fraction (SPF) was significantly higher in aneuploid than in diploid tumors (p = 0.007). All patients who underwent radical surgery had diploid DNA content and SPF below the median (11.5%). Seven patients with a diploid tumor (32%) and none of the aneuploid cases survived 1 year. Over the 1-year period, in order of importance, the type of treatment (p less than 0.001), DNA ploidy (p = 0.004), tumor size (p = 0.0046), and lymph node status (p = 0.027) predicted survival. Aneuploidy showed a significant association with decreased cumulative survival (p = 0.015), and a suggestive relationship with SPF was found. The results suggest that DNA ploidy of pancreatic cancer can be used in dividing the patients into different prognostic groups. The value of the detection of aneuploidy, however, is limited, because diploid pancreatic cancers are also generally rapidly fatal.     

Design and validation of DNA probe sets for a comprehensive interphase cytogenetic analysis of acute myeloid leukemia

The objective of this study was to design DNA probe sets that enable the detection of chromosome aberrations in acute myeloid leukemia (AML) by interphase cytogenetics using fluorescence in situ hybridization (FISH) and to compare the results of interphase cytogenetics with those of conventional chromosome banding analysis. One hundred five consecutive patients with adult AML entered on a multicenter treatment trial were studied with a comprehensive set of DNA probes recognizing the most relevant AML-associated structural and numerical chromosome aberrations: translocations t(8;21), t(15;17), and t(11q23); inversion inv(16);chromosomal deletions (5q-, 7q-, 9q-, 12p-, 13q-, 17p-, and 20q- ); and chromosomal aneuploidies. Interphase cytogenetics was particularly sensitive for detecting the AML-specific gene fusions: 3 additional cases of inv(16) and 1 additional case of t(8;21) were identified by FISH that were missed by banding analysis, whereas equal numbers of t(11q23) and t(15;17) were detected. Five additional cases of trisomy 8q, 3 more cases of trisomy 11q, and 2 more cases of trisomies 21q and 22q were shown by FISH. These aberrations were either masked in complex karyo-types or identified in cases in which conventional banding analysis failed. On the other hand, the DNA probes selected were not informative to detect 1 case of 5q-, 9q-, and 20q-. In 5 cases, clonal aberrations were detected on banding analysis for which no FISH probes were selected. In conclusion, interphase cytogenetics proved to be more sensitive for detecting AML-specific chimeric gene fusions and some partial trisomies. Interphase cytogenetics provides a powerful technique complementary and, with further development of diagnostic DNA probes, even an alternative to chromosome banding studies for the cytogenetic analysis of AML.