Median DNA Index Research Articles

Prognostic Value of DNA Index By Flow Cytometry for Minimal Residual Disease in Childhood B-Cell Acute Lymphoblastic Leukemia

Background: Prognostic factors in B-ALL represent a critical role for treatment stratification. High hyperdiploids (>50 chromosomes) have been related to better outcomes, and hypodiploids to worse prognosis. DNA Index (DI) quantifies the DNA from leukemic blast by flow cytometry and has been correlated to the number of chromosomes. Our aim was to demonstrate the prognostic value of DI for minimal residual disease in childhood B-ALL.Methods: 26 blood samples from newly diagnosed childhood B-ALL cases were analyzed between November 2017 and February 2018. DI was evaluated by flow cytometry in samples with Propidium Iodide and leukemic blasts were identified by CD19/CD22/CD10/CD20 antibodies. DI was calculated as the ratio of mean fluorescence of pathologic B blasts and normal cells (T, NK lymphocytes, neutrophils and monocytes) in G0/G1 phase. We stablished three categories: diploid (0.95 - 1.05, 46 chromosomes), hypodiploid (<0.95) and hyperdiploid (>1.05). 8-colors FacsCanto II BD flow cytometer was also used to evaluate minimal residual disease with 0.0025% threshold of detection.Results: 26 cases were evaluated and 2 died during induction treatment. Median age was 8 years (4mo - 16years) and 54% were males. At diagnosis, 62% showed DI diploid and 38% DI hyperdiploid, no hypodiploids cases were detected. Clinical characteristics were similar between both groups. Median DI in the hyperdiploids cases was 1.25 (R: 1.12 - 1.64). We only detected two hyperdiploids cases by conventional karyotype. All patients received the same BFM-based protocol. After induction, all cases achieved complete remission and 46% had MRD negative at the 28th day. DI diploid and hyperdiploid cases achieved 47% and 44% MRD negative, respectively. At the end of consolidation (R: 6-8 Mo), 77% cases achieved MRD negative, and between categories, 62% of DI diploid cases had MRD negative and 100% of DI hyperdiploid cases were negative for MRD detection by flow (p=0.02).Conclusions: The hyperdiploid DNA index by flow cytometry is associated with minimal residual disease negative at the end of consolidation.Flow cytometry offers an alternative over the conventional karyotype to detect good prognosis groups among B-ALL cases. DisclosuresNo relevant conflicts of interest to declare.

The DNA index as a prognostic tool in hilar cholangiocarcinoma.

Due to the devastating prognosis of patients suffering from hilar cholangiocarcinoma (HCCA) valid prognostic factors are urgently needed to guide treatment decisions in a personalized concept. The aim of this study was to analyze the predictive value of the DNA index in a large single-center cohort of patients undergoing resection of HCCA. A total of 154 patients who underwent resection of HCCA were included in this prospective study. The DNA index was assessed by image cytometry of fresh tumor samples and correlated, as well as standard histopathological parameters, with patient survival. The median DNA index was 1.61 ± 0.32. Univariate survival analysis identified eight parameters including DNA index, but not DNA ploidy as prognostic markers. In the Cox proportional hazard model DNA index (P = 0.021), tumor size (P = 0.029) and lymph nodes status (P = 0.039) could be shown to be independent predictors of patient survival. The DNA index represents an independent prognostic marker in HCCA which is superior to most standard histopathological factors. Since the DNA index can be assessed not only post- but also preoperatively, it might be a potential tool in the preoperative decision-making process.

MLL Rearrangements in Childhood Acute Lymphoblastic Leukemia: Clinical and Genetic Features of a Distinct Disease Entity

MLL rearrangements in childhood ALL and specifically that of the t(4;11)(q21;q23) translocation, have been associated with unfavorable prognosis; however, MLL rearrangements in children represent an extremely heterogeneous group, whose prognostic diversity needs further refinement. Aim of this study is to present the epidemiological, clinical, immunophenotypic and genetic features as well as the outcome of our cohort patients with ALL and MLLrearrangements.167 ALL pts with a median age of 5.1 yrs (range 0.5-17.5), have been retrospectively recorded and analysed. Pts were consecutively diagnosed and treated on BFM based protocols in a single Center during the years 2000-2013. Evaluation upon diagnosis included conventional cytogenetics, i-FISH and RT-PCR for the detection of MLLrearrangements in 11q23 chromosomal region and FCM for the detection of MRD. Αdditionally, this study was designed to assess the predictive value of an MTT (3-[4,5-dimethylthiazol-2,5-diphenyl] tetrazolium bromide) in vitro assay for the evaluation of leukemic cell resistance/sensitivity to cytotoxic drugs and to compare these results with clinical and laboratory parameters in cases of MLL-positive childhood ALL. Thus, the chemoresistance of leukemic cells was ascertained by means of an MTT assay in all 11 pts positive for MLL rearrangements and in vitro drug resistance to prednisolone (PDN), asparaginase (ASP), vincristine (VCR), daunorubicin (DNR), etoposide (VP-16) and cytosine arabinoside (ARA-C) was evaluated.Eleven (6/11 boys) out of the 167 pts (6.6%) were positive for 11q23 rearrangements, with a median age of 2.3 yrs (0.49-14.72). 2/11 (18.2%) presented with mediastinal, 4/11(36.4%) with CNS and 1/11(9.1%) with testicular infiltration. The median WBC was 109210/μl (4300-700000/μl), while 8/11 pts presented with WBC> 50000/μl. Immunophenotype revealed 5/11 pts with pre pre B-ALL (including 2 infant-ALL), 2/11 pre B-ALL, 2/11 B-common-ALL and 2/11 pts with T-III cortical ALL while 1/11 patient presented with mature type ALL of B-cell origin. The median DNA index was 1.0 (0.96-1.26). Cytogenetics revealed complex karyotype and t(2;9)(p13;p13-q21) in 1/11 patient, t(4;11)(q21;q23) in 2/11, atypic t(11;19)(q23;p13.3) in 2/11 and t(10;11)(p12;q23) in 1/11 patient, while no cytogenetic abnormalities were detected in 4/11 children (failure in 1/11). I-FISH analysis demostrated MLL rearrangement in all pts, with additional concomitant aberrations del9p21(1/11), 3x9q34, 3x11q23 and del 3΄MLL (1/11), 5-10x21q22(+AML1) and del5’MLL (1/11).8/11 pts were treated according to ALL BFM protocols (BFM 95/2000/ALLIC-2009), 2/11 according to Interfant protocols (Pilot 98/06) and 1/11 according to the NHL-BFM 2004 (R4 therapy group). 10/11 pts were stratified and treated according to the High Risk Arm. The results of the MTT assay revealed that all patients bearing MLL rearrangements were resistant to asparaginase, while 6/11 pts were also resistant to PDN. Infant ALLs and t(4;11)(q21;q23) cases that were associated with the most unfavourable prognosis, were all bearing extremely chemoresistant leukemic cells to all the cytotoxic drugs administered and evaluated by the MTT assay.Among the pts treated with ALL protocols, 4/10 were poor prednisone responders on day 8 of remission-induction therapy, 2/10 had M2/M3 bone marrow on day 15, while all achieved CR on day 33. 7/10 pts had detectable FC-MRD(>0.01%) on day 15 (2 with MRD>10%), while 5/10 pts had detectable MRD on day 33. 5/11 pts underwent HSCT (4 in CR1).Within a median follow up period of 26.7 months (1.8-152.5), overall survival (OS) and event-free survival (EFS) was 72.7% and 63.6%, respectively.In our study, MLL rearrangements are associated with unfavorable clinical, hematological and immunophenotypic features and have a dismal impact on outcome even in cases missing the classic t(4;11)(q21;q23) translocation. Longer follow-up and a larger number of patients is needed, in order to clarify whether the presence of the rearrangement itself constitutes a distinct entity with adverse prognostic significance or it moderates survival due to concomitant lesions and their biological features, such as 1.resistance to glucocorticoids and asparaginase as revealed in our cohort, or 2. mutations and/or overexpression of the FLT3 gene and 9p21 deletions, which are involved in the biology of the disease and adversely modify prognosis. DisclosuresNo relevant conflicts of interest to declare.

DNA Index as a Strong Prognostic Factor in Patients With Adenocarcinoma of the Pancreatic Head

To improve the devastating prognosis of pancreatic cancer; the identification of reliable predictive factors is crucial. The aim of the present study was to prospectively assess the prognostic value of DNA index determined by image cytometry as an predictive factor in pancreatic head cancer. The DNA ploidy and the DNA index of 61 patients were evaluated by DNA image cytometry and were found to be correlated, as well as standard histopathologic parameters, with patient survival. Through the DNA image cytometry, 15 tumors (24.6%) were identified as diploid and 46 (75.6%) as nondiploid. The median DNA index in the entire cohort was 1.9 (range, 1.0-2.5). Tumor stage, lymph node status, lymph node index, lymphatic invasion, and DNA index were identified as prognostic factors in the univariate analysis, but only DNA index (hazard ratio, 3.137; 95% confidence interval, 1.149-8.566; P = 0.026) and lymph node status (hazard ratio, 0.377; 95% confidence interval, 0.186-0.765; P = 0.007) were identified as independent predictive factors in the multivariate analysis. The DNA index represents an independent predictive marker in patients with pancreatic head cancer and a potential tool in designing specific treatment strategies for patients with pancreatic cancer.

Prognostic value of cell proliferation (Ki-67 antigen) and nuclear DNA content in clinically resectable, distal bile duct carcinoma.

The aim of this study was to investigate the prognostic value of cell proliferation (Ki-67 antigen) and DNA content in patients resected for distal bile duct carcinoma (DBDC). Formalin-fixed tumor specimens of 35 patients with resected DBDC and a long-term clinical follow-up were analyzed. MIB-1 antibody was used for Ki-67 antigen detection to determine the proportion of proliferating cells. DNA content was measured using flow cytometry. A significant correlation was found between a low MIB-1 index (<20%) and survival (P <.05). Of the 35 tumor specimens, 34 specimens were evaluable by flow cytometry: 22 carcinomas were diploid (65%), and 12 were aneuploid (35%). The median DNA index of aneuploid tumors was 1.36 (range, 1.09 to 1.76). No correlation of DNA-ploidy with survival time was found. In contrast to DNA-ploidy pattern, Ki-67 antigen expression showed prognostic significance in resectable DBDC. A Ki-67 positive ratio of > or =20% was associated with decreased survival time.

DNA-PLOIDY AND PROLIFERATIVE ACTIVITY IN UTERINE-TUMORS - AN IN-VIVO STUDY USING BROMODEOXYURIDINE AND FLOW-CYTOMETRY

We have studied the DNA ploidy and the proliferative activity in 102 patients with endometrial and cervical carcinoma, by flow cytometry. Samples were excised 1 hour after bromodeoxyuridine (BrdU, 250 mg/) e.v. infusion and fixed in 70% ethanol. Nuclear DNA content and BrdU incorporation, were simultaneously determined to obtain ploidy (DNA index) and proliferative activity (BrdU-labeling index, LI). No acute toxicity or side effects related to BrdU injection were recorded. The overall feasibility of the determinations was higher than 90% (93/102). Twenty-two out of 59 (37.2%) endometrial neoplasms and 23 out of 34 (67.6%) cervical neoplasms were aneuploid, with a median DNA-index of the aneuploid peak of 1.3 and 1.4, respectively. Overall median BrdU LIs were 4.8% and 7.2%. Proliferative activity was found to be higher in aneuploid tumors (p<.05). DNA ploidy and/or BrdU-LI were not significantly related either with the clinical stage or the histopathologic grading in either tumor type. The BrdU in vivo administration coupled with bivariate FCM for measurement is a simple method that can be performed in clinical settings to better evaluate the prognostic significance of proliferative parameters in gynecological tumors.

Flow cytometric analysis of DNA content in adult testicular germ cell tumors

Flow cytometric DNA analysis was carried out in 54 patients with testicular germ cell tumors (GCTs) experienced at our hospital, to evaluate the clinical relevance of DNA index (DI) and provide some insight into the pathogenesis of testicular GCTs. Histological types with their incidences were seminomas in 31 patients and nonseminomatous germ cell tumors (NSGCTs) in 23 adults. DNA ploidy and DI were analyzed by flow cytometry in 158 paraffin embedded samples; 2.9 samples per case on the average. This study revealed that 52 cases (96%) of evaluable 54 adult GCTs were DNA aneuploid, while DNA diploid tumors were observed in only each one case of NSGCT and seminoma. There was a significant difference (p less than 0.01) between the distribution of DIs in adult NSGCTs (median DI = 1.50) and that in pure seminomas (median DI = 1.85). Although we found no significant correlation between DI and clinical staging of Japanese Urological Association, on the basis of Indiana University staging system, the median DI in NSGCT patients of the advanced extent was lower than those of the other extents. DNA heterogeneity was observed only in 4 of 23 NSGCT patients (17%) and 3 of those 4 patients were assigned to advanced extent. These data suggest that the lower DI and the presence of DNA heterogeneity may have prognostic relevance for NSGCTs.

Prognostic significance of nuclear DNA content in human neuroepithelial tumors

The relationship between survival and flow cytometric DNA-ploidy and other prognostic factors such as histological subtype, anatomical tumor site, patient sex and age was investigated in 153 patients with intracranial neuroepithelial tumors who underwent surgical treatment. We found a trend toward poorer survival from anaplastic astrocytomas and glioblastomas with respect to low-grade (I and II) astrocytomas (which did not differ significantly); accordingly, patients were grouped into these 3 histologic subgroups. Thirty-seven of the 153 tumors (24.2%) were aneuploid with a median DNA-index (DI) of 1.3 (range: 1.2-2.0). DNA-ploidy correlated with histology, since anaplastic astrocytomas and glioblastomas were significantly (p = 0.041) more frequently aneuploid (around 30%) than low-grade astrocytomas (around 10%). Patients with DNA-aneuploid tumors (i.e., with DI not equal to 1.00) survived for a shorter time (31.4 weeks) than patients with DNA diploid tumors (75.1 weeks) (p less than 0.001). This difference was confirmed by Cox's multivariate analysis. Aneuploid tumors were associated with a poorer survival (p = .0002) when compared with diploid tumors, resulting in a relative risk point estimate (RR) of 2.41, 95% confidence interval (Cl) = 1.55-3.74. Histological subtype was also significantly associated with survival (p less than 0.0001), with RRs of 2.09, 95% Cl = 1.13-3.86 and 3.59, 95% Cl = 1.96-6.59 for anaplastic astrocytomas and glioblastomas, respectively, compared to low-grade astrocytomas. We therefore suggest that the flow cytometric measurement of DNA-ploidy has relevant significance in predicting survival in patients treated for intracranial neuroepithelial tumors.

DNA and RNA flow cytometric study in multiple myeloma. Clinical correlations

Flow cytometric studies of cellular DNA and RNA content using the acridine-orange technique were conducted in 81 patients with multiple myeloma (MM). All patients were treated with the M-2 protocol and clinical response was evaluated according to the criteria of the Chronic Leukemia-Myeloma Task Force. Aneuploid DNA stemlines were found in 38.2% of untreated patients with a median DNA index (DNA-I) of 1.15 in marrow aspirates and 1.22 in biopsy specimens. The median percentage of cells with abnormal DNA content was 31.5 (aspirates) and 35 (biopsy specimens) and a positive correlation with the percentage of bone marrow plasma cells was observed. Significantly higher proliferation (S-phase) was found in marrow biopsy specimens as compared with marrow aspirates. Significantly higher RNA content (RNA index [RNA-I]) was observed in aneuploid versus diploid patients in biopsy material. There was no difference in response to the Memorial Hospital M-2 protocol between diploid and aneuploid patients. In patients with DNA-I greater than 1.15 remission duration was shorter as compared with DNA-I less than or equal to 1.15. Furthermore, no difference in cellular RNA content was noted between responders and nonresponders. This study demonstrates no correlation between cellular RNA content and response, as previously described for patients treated with vincristine, Adriamycin, and dexamethasone (VAD), but DNA aneuploidy appears to be an adverse prognostic factor in MM patients treated with the M-2 protocol. It also demonstrates that prognostic models for MM are not universal but depend on the chemotherapeutic regimen used.

Flow Cytometry によるセミノーマの核DNA量解析

The DNA ploidy of testicular seminomas was studied by flow cytometry using paraffin embedded samples. The mitotic count and DNA index (DI) for 27 seminomas were analyzed in 80 samples with a mean of 3.0 samples per case. Six anaplastic seminomas which were with 3 or more mitoses per a high power field were distinguished from 21 typical seminomas. DNA ploidy pattern was aneuploid in all seminomas except one case of anaplastic seminoma, and clonal heterogeneity in DNA content was found in 3 of 20 (15%) cases of which 2 or more samples were analyzed. Although the DI had no significant difference between those two groups of seminomas classified by mitotic count, the DI in anaplastic seminomas was ranged from 1.5 to 2.0 (median DI = 1.70), otherwise the DI in typical seminomas ranged from 1.5 to 3.5 (median DI = 1.89), particularly 9 cases in 21 (43%) typical seminomas distributed in hypertetraploid region. The median DI of stage I seminomas was 1.88 and that of stages II + III seminomas was 1.75, though there was also no significant correlation between DI and clinical stages. In general, it is postulated that the higher DI is paralleled to the more malignant nature of neoplasms, nevertheless this study suggested that the higher DI in seminomas is not always related to high malignant potentiality determined by histological type and clinical stage.

Ploidy of malignant mediastinal germ-cell tumors

The ploidy of 19 primary mediastinal malignant germ-cell tumors (GCT) was measured. Six tumors were (near) diploid. The median DNA index of the 13 remaining tumors was 1.91, indicating that most aneuploid tumors were near tetraploid. Seminomas and nonseminomatous GCTs were similar with respect to proportion of diploid tumors and median DNA index of the aneuploid tumors. The ploidy of mediastinal malignant GCTs is remarkably different from that of testicular GCTs of adults, but more similar to that of testicular GCTs of infancy. The pathogenesis of malignant mediastinal GCTs is probably different from that of testicular GCTs of adults, and more similar to the pathogenesis of infantile testicular GCTs. The difference in ploidy between mediastinal malignant GCTs and GCTs of the adult testis may help to differentiate between a primary extragonadal malignant GCT and a mediastinal metastasis from a primary testicular GCT.

Diploid predominance in hereditary nonpolyposis colorectal carcinoma evaluated by flow cytometry

Fifty-nine colorectal carcinomas of patients with verified cancer family syndrome (CFS) were analyzed for DNA ploidy using flow cytometry. Sixty-eight percent of the tumors were diploid, and 32% were aneuploid. The aneuploid tumors had a median DNA index of 1.24 (range, 1.12-1.97). In 90% of all tumors the DNA index was less than 1.27. This predominance of diploid/near-diploid tumors was seen both in primary and in metachronous carcinomas. In 21 cases a cell cycle analysis was possible. Tumors with the S-phase fraction (SPF) greater than or equal to 9.8% had a worse prognosis than tumors with the SPF of less than 9.8%. These findings suggest that the predominance of diploid/near diploid DNA values is one of the characteristics of colorectal carcinomas in CFS. This might signify the existence of two or more pathogenetically different subgroups of colorectal carcinoma and explain the proposed better prognosis of colorectal carcinoma in CFS compared with other colorectal carcinomas.

Prognostic significance of DNA content in large bowel carcinoma: a retrospective flow cytometric study.

Flow cytometric (FCM) determination of DNA content was performed on surgical specimens from 44 patients with previously untreated colonic carcinoma. For each tumor, cell suspensions were prepared from 2-4 40-microns thick sections obtained from formalin-fixed and paraffin embedded tissue samples. Aneuploidy was found in 47.2% of all the tumors and the aneuploid clone had a median DNA index of 1.49 (range: 1.24-1.93). Aneuploidy was found in 26.7% of highly differentiated tumors (WHO histologic classification), in 53.8% of moderately differentiated tumors and in 100% of poorly differentiated tumors (P = 0.04). The 33.3% of stages 1 + 2 (TNM) and the 70.6% of stages 3 + 4 tumors were aneuploid (P = 0.002). Median survival from surgery was 46.4 months for all patients. It was 18.8 months for patients with aneuploid tumors and 85.7 months for those with diploid tumors (P = 0.0002). FCM determination of DNA in colon carcinomas can easily be performed on archival histological material and provides prognostic information.