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Ann Thorac Surg 2000;70:1629-1633
© 2000 The Society of Thoracic Surgeons

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Original articles: general thoracic

Prognostic significance of DNA ploidy in squamous cell lung carcinoma: is it really worth it?

^a Department of Thoracic Surgery, Karol Marcinkowski University of Medical Sciences, Pozna, Poland

Address reprint requests to Dr Dyszkiewicz, Department of Thoracic Surgery, Karol Marcinkowski University of Medical Sciences, 62 Szamarzewski St, 60-569 Pozna, Poland
e-mail: thorax{at}eucalyptus.usoms.poznan.pl

Presented at the Poster Session of the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Many previous studies attempted to associate DNA quantification with prognosis for lung cancer, but there is a divergence of opinion about its value. The purpose of the study was to evaluate the frequency of abnormal DNA content in squamous cell cancer (SCC) and analyze its correlation with late survival rates after surgical treatment.

Methods. A group of 110 patients surgically treated because of SCC was analyzed. Paraffin-embedded pathologic material underwent cytometric analysis. Postoperative follow-up was performed with standard follow-up visits. The statistical analysis was carried out using Mann-Whitney’s U and ² tests to compare various variables in both groups. The survival curves were drawn using the Kaplan-Meier method. Clinical staging, regional metastasis, and ploidy, were analyzed with multivariate analysis for having a great impact on survival rates.

Results. Fifty (45%) tumors were DNA aneuploid. The survival rate in patients with aneuploid cancers was worse than in those with diploid tumors and the most frequent cause of death was local recurrence (p < 0.05).

Conclusions. DNA content abnormalities were found to be an important prognostic factor in patients with SCC. The DNA quantification can select a group of high risk of recurrence even after a radical procedure and set new guidelines for adjuvant therapy.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
Lung cancer is currently one of the most frequently occurring malignant neoplasms and is the most frequent cause of death among men from malignancy. During the last 60 years, the incidence rate of this disease increased 30 times in the majority of countries all over the world. Despite intense efforts of pulmonologists, oncologists, and thoracic surgeons to improve therapeutic procedures, lung cancer is still characterized by a poor prognosis. One of the causes of treatment failure of nonsmall cell lung cancer (NSCLC) is the absence of clear prognostic criteria that could detect patients with a high risk of recurrence.

From the numerous cytogenetic studies, data reveal that inappropriate content of DNA in cell population remains in close relationship with neoplasia. In addition, the data follow that one of the important factors determining long-term prognosis could be the cytometric evaluation of the DNA content in cancer cells. However, there are only a few clinical reports and they give variable results referring to the prognostic value of DNA aneuploid patterns, but the majority of authors consider them to be an important prognostic factor in patients with squamous cell carcinoma (SCC).

The purpose of this study was to evaluate the frequency of abnormal DNA content in squamous cancer cells and analyze its correlation with late survival rates (3 years) after surgical treatment.

	Material and methods

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Between 1995 and 1996 at the Department of Thoracic Surgery at Karol Marcinkowski University of Medical Sciences in Pozna, Poland, 245 patients were operated on for lung cancer. Out of this group, 110 patients were surgically treated because of SCC. The latter group was selected and analyzed for the study. The SCC was confirmed in this group of patients by two pathologic examinations of the tumor tissue. The patient’s age at surgery, sex, type of operative procedure, clinical staging, grading, and the date of death were recorded. Postoperative follow-up and cause of death was established using the following methods: patient questionnaires, case histories, routine follow-up visit, or information from family physicians. A standard follow-up visit consisted of a review of the patient’s history, a physical examination, and chest radiographs at 3, 6, 12, 24, and 36 months after surgery. The survival time was defined as the time that elapsed from the surgical procedure to the death of the patient. Patients who died from causes other than lung cancer, or whose condition was unknown, were included in the survival analysis.

Evaluation of cancer advance was based on recently refined TNM and American Joint Committee on Cancer classifications. Tumors were also divided into one of three pathologic grades: I (well differentiated), II (moderately differentiated), and III (poorly differentiated).

Additional data such as the size of the tumor, endobronchial infiltration, presence or absence of local invasion, and status of regional lymph nodes were recorded.

Formalin fixed, paraffin-embedded pathologic material from 110 resected tumors with a diagnosis of SCC made from examination of operative specimens were included in the study. Two 50 µm sections, representative of the paraffin blocks of tumors, were examined by a pathologist to ensure that each contained tumor cells and to confirm squamous cell carcinoma. Then the samples were prepared according to the modified Hedley method. The sections were dewaxed and treated with progressively decreasing concentrations of alcohol and then digested in solutions of pepsin and ribonuclease. The DNA content was analyzed with the Cytotron Absolute (Ortho Diagnostics, Neckargemund, Germany) flow cytometer. This system is based on an argon laser with 488 nm wavelength. The results were analyzed using ModFit software. The DNA index was established in each specimen. A sample was considered DNA euploid if, on the histogram, there was a single peak in the G₀/G₁ phase and DNA aneuploid if there was at least one distant G₀/G₁ population. In those cases where there were more than 15% of cells in the G₂/M phase, the histograms were defined as tetraploid. In this study tetraploid histograms were classified into the aneuploid group.

The statistical analysis was carried out using Statistica software (Statsoft). The p value was considered statistically significant at the level of 0.05. Mann-Whitney U and ² tests were used to compare patients with diploid tumors and patients with aneuploid tumors. The following criteria were used: clinical staging, grading, tumor size, metastasis to regional lymph nodes, tumor location, type of surgical procedure, occurrence of distant metastases, local recurrence of the tumor, and survival time. Each factor that could influence in the survival in these patients was analyzed and the survival curves of the patients were drawn using the Kaplan-Meier method [1]. On the basis of this analysis three of the following variables were selected as having a considerable impact on the survival rates: clinical staging, metastasis to regional lymph nodes, and ploidy. They were also analyzed by multivariate analysis. Survival differences in these three variables were drawn using the Kaplan-Meier method and analyzed using nonparametric tests (Mann-Whitney and Kruskal-Wallis).

	Results

Top Abstract Introduction Material and methods Results Comment References

 
The study group consisted of 95 men (86.4%) and 15 women (13.6%). The age range was from 34 to 77 years (average 59.8). Of the 110 tumor samples from the patients with SCC, 49 (44,5%) were DNA aneuploid and 61 (55.5%) DNA diploid. In 11 patients (22.2%) with aneuploid tumors the regional lymph nodes were positive for cancer metastasis, compared with 14 patients (22.7%) with diploid tumors. The tumor size (diameter in cm) ranged from 3 to 15 cm (average 5.8). In aneuploid tumors the average tumor size was 5.9 cm and in diploid tumors, 5.7 cm. The AJCC clinical stage in both tumor types was similar, and there was no significant correlation between the ploidy and the clinical staging of the cancer. The tumors were located centrally (hilar) in 58 cases (52.5%) and peripherally in 42 patients (47.5%). In both types of DNA ploidy the location was similar. Tumors classified as G1 (well differentiated) were found in 10 cases (9%), G2 (medium differentiated) in 52 cases (48%), and G3 (poorly differentiated) in 48 cases (43%). In this group of patients the following surgical procedures were performed: pneumonectomy in 47 patients (42.7%), lobectomy in 52 (47.3%), bilobectomy in 7 (6.4%), and wedge resection in 4 high-risk patients (3.6%). The most prevalent surgical procedure performed in patients with aneuploid tumors was pneumonectomy (49% of all procedures in this group). In patients with diploid cancers the most common procedure was lobectomy (50.8% of all procedures).

There was no significant difference in the proportion of the group of patients with diploid and aneuploid tumors regarding factors such as age, sex, size, and localization of the tumor, grading, and the type of operative procedure. The lymph node status was considered separately (Table 1).

Three factors having a statistically significant impact (p < 0.05) on survival time were selected: clinical staging, regional lymph node metastasis, and ploidy. Multivariate analysis of these three factors revealed that after a 36-month follow-up period the survival rate observed in patients with aneuploid tumors was significantly shorter than that in patients with diploid tumors (Fig 1). This rate was independent of other variables having an impact on survival. More patients with aneuploid cancers died from cancer relapse, than did patients with diploid tumors (², p < 0.05). Distant metastasis as a cause of death was more frequent in patients with aneuploid tumors, whereas local cancer recurrence was more frequent in patients with diploid tumors (², p < 0.05; Table 2).

View larger version (35K): [in this window] [in a new window]   Fig 1. Ploidy and survival time. Kaplan-Meier estimate of survivor function.

	Comment

Top Abstract Introduction Material and methods Results Comment References

One of the important factors in determining the long-term prognosis is the cytometric evaluation of the DNA content in cancer cells. DNA content analysis of lung tumors has been studied by flow cytometric study for last 15 years. The majority of studies have demonstrated that the DNA ploidy in lung cancer cells is an important prognostic factor of survival [2, 3]. The first publications about DNA content analysis of lung tumors were those of Barlogie and Nervi [4, 5]. Volm and coworkers [6] described the clinical use of DNA content in lung cancer. They reported lower 5-year survival rates in patients with aneuploid tumors. Zimmerman and associates [7] retrospectively analyzed patients with NSLC and reported a 45% occurrence of aneuploidy and worse 2-year and 5-year survival rates in patients whose tumors had an aneuploid DNA content. The ploidy in this investigation was found to be an important prognostic factor independent of sex, age, type of surgical procedure, pathologic type, or TNM staging.

In 1989 Salvati and coworkers reported a survival advantage for patients with diploid tumors. In a later study [8] they carried out a multivariate analysis of 184 patients that showed no statistically significant correlation between ploidy and staging, grading, or type of procedure. Similar results were obtained by Dazzi and associates [9]. They stated that ploidy had a significant prognostic value of survival, independent of age, sex, pathology, size, local metastasis, or grading. Tirindelli-Danesi and colleagues [10] found that another important prognostic factor is the DNA index (DNAI). They reported that with a DNA index higher than 2 or lower than 1, both the tumor-doubling time and survival time are significantly shorter.

On the other hand many authors fail to show DNA ploidy as a prognostic factor [11, 12]. Velde and associates [13] did not find any association between the DNA content and the pathologic type, clinical stage, and type of procedure. Also the ploidy status did not have any significant impact on the survival rate. Similarly, Van Bodegom and associates [14] did not observe any worse 6-year survival rates in patients with aneuploid tumors. However, they did suggest that the greater the number of aneuploid cells, the worse the prognosis. Also Carp and coworkers [15] did not find any correlation between ploidy pattern and survival in patients with NSCLC.

It is obvious from the above data that there is a considerable divergence of opinion concerning the prognostic value of DNA ploidy. The majority of authors tend to associate ploidy with prognosis only in patients with SCC.

In this present study the percentage of aneuploid tumors was 45%, which is within the range of 31% to 84% observed in previous reports concerning paraffin-embedded specimens [16]. The group of patients surgically treated with only SCC was selected to avoid the impact of histologic type of the tumor on the survival rate. It is of considerable interest that in this study no statistically significant differences were found between ploidy status and clinical staging, positive lymph nodes, cancer infiltration of the stump, tumor size, location, grading, and the type of surgical procedure. Similar conclusions are drawn by the majority of previous authors.

In this study the prognostic value of ploidy in patients after lung parenchyma resection, namely its impact on late (3-year) survival rates, was investigated. Using multivariate analysis, a positive correlation between ploidy and survival rate was determined independently of other factors contributing to the rate. In the follow-up period significantly better survival rates were observed in patients with diploid tumors. Therefore, in this study, ploidy as a prognostic factor was found to be statistically significant.

The most common cause of death in both groups was distant metastasis. In patients with aneuploid tumors these occurred more often, whereas local recurrence was more common in patients with diploid tumors.

The variable results that were reported in the NSCLC literature may be from various factors. The analyzed groups differed one from another in pathologic type, clinical staging, and number of patients. Some analyses were made without pathologic typing, in others either SCC or adenocarcinoma predominated, and in others mixed-type cancers (both large and small cell) were included. It is difficult therefore to draw conclusions from such diverse groups.

Another issue is the type of specimen examined. Some authors examined fresh tissue specimens and others paraffin-embedded. Liewald and coworkers [2], Kallioniemi [17], and Jacobsen and associates [18] found that in both types of tissue the results were identical in 94% of cases. Both types of specimens are suitable for DNA quantification. Muguerza suggests that in those cases where there is no aneuploidy in the fresh tissue specimen it should be reexamined after paraffin-embedding. The coefficient of variation (CV) of the G₀/G₁ peaks is important for a proper evaluation of DNA histograms, and in this study it was estimated in each examination. In paraffin-embedded tumors, it is crucial to fix the tissue in 10% buffered formalin solution. At times the ploidy was not evaluated with unified criteria and the interpretation of some histograms differed depending on the examiner. Any distant G₀/G₁ peak in addition to a diploid steam-line is considered aneuploid. Also, single but displaced or distant G₀/G₁ peaks, high CV values, or the percentage of cells in phase S or G₂/M are indicative of aneuploidy.

Neoplastic heterogeneity (diploid and aneuploid elements within the same tumor tissue) also has an influence on the results. Carey and colleagues [19] state that, in lung cancer, more than 90% of tumors are heterogenic and therefore more than one specimen should be sampled. They declare that this is the main reason for the widely different aneuploid DNA content reported in various studies (31% to 95%). Accordingly, Liewald and associates [2] took three samples from each tumors (from peripheral, intermediate, and central sites). In this study samples were taken from two different parts of each tumor.

In our opinion the DNA content, as well as other factors such as clinical staging, is an important predictor in surgically treated patients with SCC. We believe that the finding of aneuploidy in neoplastic cells of patients with resected SCC is an important prognostic factor and the 3-year survival rate was significantly worse in patients with aneuploid tumors because of the frequency of distant metastases or local recurrence, independently of other factors.

DNA quantification can indicate a high risk of recurrence, even after a procedure that is oncologically radical. In the future aneuploidy could therefore be considered as an indication for adjuvant therapy.

	References

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