Methods of internal standards’ preservation for genome size assessments: a comparative study

The prognostic value of tumor cell nuclear DNA content and various pre‐ and postoperative prognostic factors (pre‐ and postoperative TNM, tumor size, resection margins involved by tumor, age, and treatment) have been evaluated by means of bivariate correlations and multiple linear regression analyses in 182 patients with either papillary, follicular, or medullary thyroid carcinomas, or follicular adenomas. Fifty‐one patients died of diseases and 131 patients were alive at least 10 years after diagnosis. The strongest correlation between tumor cell nuclear DNA content and survival was obtained in the group with papillary carcinoma, followed by follicular and medullary carcinoma. Nuclear DNA content alone had a predictive power equivalent to and, in papillary carcinoma, significantly greater than, that of all other prognostic factors combined. When DNA was added to all other prognostic factors, a statistically significant increase of the prognostic value in the present 3 kinds of thyroid carcinomas, both pre‐ and postoperatively, was obtained.

C-value was applied as a new criterion to investigate the relationships within the genus Agave L. (Agavaceae). Samples representing nearly 40 taxa were investigated, augmented with data from the literature. Species of Agave have the same basic chromosome number, x = 30, but there are several polyploids. Their somatic nuclear DNA contents (2C), as measured by flow cytometry with propidium iodide falls in a narrow range around 8 pg for the diploids. A comparison is made with nuclear DNA amounts of other related genera. Hosta, with a chromosome complement identical to that of Agave, with 5 large and 25 small chromosomes in the haploid set, is at the base of the Agavaceae clade and has two to three times the amount of nuclear DNA of the other genera.

The prognostic value of nuclear DNA content in papillary thyroid carcinoma was studied retrospectively in 90 patients. Eighty survived for at least 10 years, and 10 died, of papillary thyroid carcinoma, between 6 months and 12 years after diagnosis. Clinical data as well as morphological tumor characteristics were examined. DNA measurements in morphologically identified single tumor cells were performed either on fine‐needle aspiration material or on histologic sections. The tumors of the survivors were composed of cells with a DNA content comparable to that of normal cells, whereas the tumors of the non‐survivors had significantly higher DNA values. The data suggest that DNA measurements in papillary thyroid carcinoma offer a valuable adjunct to standard clinical and microscopic analysis.

The predictive value of nuclear DNA content in breast cancer in relation to clinical and morphologic factors was studied in 227 consecutive cases of invasive breast adenocarcinomas with follow-up periods of 8 to 13 years. The results show that, with the use of Cox multivariate analysis nuclear DNA content provided significant prognostic information additional to that given by all other clinical and histomorphologic variables taken together. This fact indicates that the DNA content of breast cancer cells reflects biological properties, associated with the malignant behavior of the tumor, other than those determining the stage of the disease. Nuclear DNA content was strongly correlated to histopathologic grading of the ductal carcinomas, with poorly differentiated tumors more likely to be aneuploid. On the other hand, no clear correlation was found to exist between nuclear DNA content and axillary node status, indicating that these two factors are independent prognostic parameters. It is noteworthy that DNA content provided additional prognostic information within both the node-negative and node-positive patient groups. In summary, the results shown here indicate that nuclear DNA content, as an objective biological marker of tumor aggressiveness, can significantly improve our prognostic capabilities within the currently designated stages.

This retrospective study evaluates several prognostic factors in 63 patients with invasive ductal breast cancer. Special attention is paid to the additional prognostic value of cellular DNA content to the previously developed and evaluated quantitative features mitotic activity index (MAI) and multivariate morphometric prognostic index (MPI). Follow-up was monitored for at least 50 months (median survival, 78 months) and only patients who died of distant metastases were included. The results show that the MAI is the strongest prognostic factor of all single features (Mantel-Cox, P = 0.008). Although patients with a diploid or tetraploid tumor tended to have a better prognosis than those with an aneuploid cancer, the DNA index as a single parameter was a weak prognosticator in the univariate survival analysis (Mantel-Cox, P = 0.24). Within the diploid and tetraploid tumors the MAI could distinguish patients with a favorable and unfavorable prognosis prediction (chi-square, P = 0.01). For aneuploid tumors this was not possible. Analysis of combined features revealed that the MPI has a high prognostic value (Mantel-Cox, P = 0.0015), thus confirming other studies. A linear combination of the nuclear DNA index, MAI, nodal status, and mean nuclear area showed only a slight improvement in prognosis prediction compared with the MPI (Mantel-Cox, P = 0.0005); with this rule, the classification of the patients was more in agreement with the actual outcome in 4% of the cases. The gain was in the poor prognosis group. These results suggest that the additional prognostic value of nuclear DNA content is restricted when compared with the morphometric prognostic factors. Further studies on a larger number of patients are required to confirm these findings.

Buffel grass is a fodder grass that reproduces mainly via apomixis. Using in vitro tissue culture in apomictic species provides alternatives that can be applied in a breeding program. The aims of this work were to establish a protocol for in vitro generation of apomictic buffel grass genotypes using anthers as explants and to evaluate the genetic stability of regenerated plants via flow cytometry (FCM). Three genotypes were able to induce embryogenic calli in Murashige and Skoog (MS) medium supplemented with 6 mg/l of 2,4-dichlorophenoxyacetic acid. Seedling regeneration occurred in a MS medium supplemented with 0.5 mg/l napthaleneacetic acid + 1 mg/l 6-benzylaminopurin. Seedlings were derived from somatic embryos and the morphogenic process was induced using the somatic tissue of the stamens. Induction and regeneration efficiency depended on the genotype and was affected by date of tiller collection, different pretreatments or the interaction of these variables. FCM analyses in in vitro regenerated plants showed genetic instability in their nuclear DNA content. Plants with lower nuclear DNA content may indicate DNA aneuploids (8.6 %), whereas plants that had twice the value of nuclear DNA content (4.7 %) suggest in vitro polyploidization. This variation observed in apomictic genotypes provides an opportunity to obtain new variants, which may then be included as sources of genetic variability in buffel grass breeding programs.

The relative importance of prognostic factors in papillary and follicular thyroid cancer was studied in 113 patients using Cox's proportional hazards model. Prognostic factors studied were: histology, tumor grade, extrathyroidal growth, nodal involvement, distant métastases at diagnosis, nuclear DNA content, age at diagnosis, and sex. Nuclear DNA content was measured in primary tumors by flow cytometry. Total thyroidectomy and postoperative131I ablation was the standard treatment. The results showed that nuclear DNA content correlated significantly with histologic type and, in papillary cancer, also with tumor grade. The presence of distant metastases at diagnosis was, by far, the most important prognostic factor. In the patient group without distant metastases (n=91), multiploidy (i.e., presence of 2 or more aberrant stemlines) was the only significant prognostic factor for overall survival. With respect to diseasefree survival, multiploidy was second only to the age factor. In the patient group with distant métastases (n=22), all 6 patients with multiploid tumors died compared to 8 (50%) of 16 of those with other ploidy tumors. However, the small number in this group precluded significant results. The present study demonstrates that nuclear DNA content is a prognostic factor in those patients with papillary and follicular thyroid cancer without distant metastases at diagnosis.

It has been known for over 50 years that the amount of nuclear chromatin (DNA) in malignant neoplasms differs from that of homologous normal cells (1). More recently, it has been shown that nuclear DNA content correlates with the clinical outcome of various human neoplasms including urologic malignancies (2-10). An important problem in the care of patients with renal cell carcinoma (RCC) is the prediction of the neoplasms malignant potential, and in turn the patient's prognosis. Various parameters have been used to assess the malignant potential of renal cell carcinoma, including clinical and pathologic stage, histologic grade, tumor size, nuclear morphology, immunohistochemistry, age, elevated erythrocyte sedimentation rate, and hypercalcemia. To date, the most important predictors of prognosis in patients with RCC have been tumor pathologic stage, histologic grade and type (11,12). However, it has been shown that patients within a specified stage and grade may differ in their disease progression and survival (13,14). Furthermore, none of these variables alone or in combination has shown to provide total reliable prognostic information for the individual patient. These reasons led several groups to evaluate the prognostic value of nuclear DNA content in patients with renal cell carcinoma.

Using flow cytometry, spherulating nuclei of Physarum isolated at the beginning of spherule wall formation were found to exhibit a DNA content corresponding to the G2 phase of the cell cycle, although 8% lower. Before the first mitosis after spherule germination, a very slight incorporation of 3H thymidine into DNA was observed that was too weak to correspond to S phase, strongly suggesting that nuclei are stopped in G2 phase inside the spherules. The lower value of nuclear DNA content found using flow cytometry of germinating spherules may not be related to DNA quantity, but may be due to a difference in chromatin organization during growth or spherulation, resulting in interference with the staining.

The prognostic value of nuclear DNA content was studied retrospectively using flow cytometry in 203 cases of resected hepatocellular carcinoma. The occurrence of DNA aneuploidy, which was detected in 50% of patients, correlated significantly with tumor size and the presence of vascular invasion or intrahepatic metastasis. Overall, patients with DNA aneuploid tumors had a significantly worse prognosis than those with DNA diploid tumors (P less than 0.001) and, also in subdivided groups by tumor size (P less than 0.01). Among DNA aneuploid patients, the survival times were significantly shorter for patients with a low DNA index (less than 1.5) than for those with a high DNA index (greater than or equal to 1.5) (P less than 0.05). In a Cox multivariate analysis, nuclear DNA content provided significant prognostic value (P = 0.008), as did vascular invasion (P = 0.001) and intrahepatic metastasis (P = 0.005). These results indicated that nuclear DNA content has an important prognostic value in hepatocellular carcinoma.

When Hymenopteran Males Reinvented Diploidy

The prognostic value of nuclear DNA content in medullary thyroid carcinoma was studied in 36 patients. Eleven patients are still living 13–35 years after diagnosis. Twenty‐five patients died of medullary thyroid carcinoma between 10 months and 23 years following diagnosis. DNA measurements in morphologically identified single tumor cells were performed either on fine‐needle aspiration biopsy material or on histologic sections from the primary tumor. Metastases or local recurrences were also examined in 18 cases. In the survivors, the majority of tumors exhibited cells with DNA amounts approximating the normal diploid value, whereas the tumors of the nonsurvivors had significantly higher DNA values. The primary tumors and their metastases or local recurrences exhibited in all cases essentially similar DNA content. The data suggest that DNA measurements in medullary thyroid carcinoma contribute valuable prognostic information that adds to standard clinical and morphological parameters.

Abstract: Poa densa Troitsky is a characteristic species of the high steppe to low alpine vegetation of Turkey. It was overlooked in the Flora of Turkey, and although subsequently reported from a few stations in W part of C Anatolia, is actually relatively common and widespread across the semi-arid C and N steppes. Here we provide a key to related species, a full description, photographs, 2C value of nuclear DNA content, and a distribution map, and discuss its classification in the genus. The synonym Festuca conferta is lectotypified. DNA phylogenetic analysis and morphology data indicate P. densa is allied to species of P. subg. Poa sect. Macropoa, rather than to the P. bulbosa complex of P. subg. Ochlopoa sect. Arenariae, and support multiple origins of the basal bulb in the genus Poa. Citation: Cabi E., Soreng R. J., Gillespie L. & Amiri N. 2016: Poa densa (Poaceae), an overlooked Turkish steppe grass, and the evolution of bulbs in Poa. — Willdenowia 46: 201–211. doi: http://dx.doi.org/10.3372/wi.46.46...

The prognostic value of nuclear DNA distribution pattern in relation to tumor size, axillary lymph node status, and estrogen receptor (ER) content was studied in 464 patients with primary, operable mammary adenocarcinoma. The median follow-up time was 3 1/2 years. Slide cytophotometric DNA analysis was performed on morphologically identified Feulgen-stained tumor cells. The tumors were classified into four subgroups according to their DNA histogram type. DNA content was significantly related to tumor size and ER level but not to nodal status. When all variables were stimultaneously introduced into Cox's proportional hazards model, tumor size, nodal status, and DNA profile remained as significant predictors of recurrence. Restricting the analysis to node-negative patients, both DNA profile and tumor size showed a significant prognostic value. DNA did not contribute significant prognostic information in node-positive patients. However, the trends in recurrence-free survival were similar to those in the node-negative subgroup: patients with aneuploid tumors tended to fare worse than those with euploid carcinomas.

13565 Background: We assessed the prognostic value of nuclear DNA content measured in the primary tumors of 123 patients with stage II or stage III colorectal cancer (CRC). Methods: Isolated nuclei from paraffin sections were stained with Feulgen and DNA was measured using a computer-assisted image analysis cytometry system (Ahrens ACAS). We applied 4 different approaches in analysis of DNA histograms: ABCDE approach, histogram range, peak evaluation, and DNA cut-off values. Results: Using the histogram range, narrow range was rare (3.7%) in patients who died of disease as compared with 16.4% among those alive (p=0.017). Modal peak evaluation was a significant predictor of disease free survival (DFS) (Kaplan-Meier log-rank p=0.0235). In the range evaluation, the first set (low-start gates) was a significant predictor of DFS (log-rank p=0.0121), where disease recurrence was closely associated the widest range (1.8c->10c) gates. Recurrence-free survival was markedly better among patients with narrow gate histograms than wide range histograms than among patients with wide range histograms (p<0.03). The first set also proved to be significant predictor of disease specific survival (DSS) (log-rank p=0.0045), being markedly better (78–90.0%) among the patients with the narrow-gate histograms. Grading of the histogram range into two categories (with 6.0c as cut-off for low and wide range), was a powerful predictor of both DSS (log-rank p= 0.0092) and 5-year DFS (p=0.0106) in the whole series, and separately in Stage III (but not Stage II) disease; p=0.0131 and p=0.0201, respectively. Conclusions: The DNA image cytometry with careful analysis of the histograms may provide valuable prognostic information in CRC, with potential clinical implications in patient management, particularly in predicting the patients at high risk for recurrence who should be considered as candidates for adjuvant therapy. No significant financial relationships to disclose.