Respirator3' Medicine (1990) 84, 217-223

DNA analysis by flow cytometry in nonsmall cell lung cancer: relationship to epidermal growth factor receptor, histology, tumour stage and survival H. DAZZI, N. THATCHER*, P. S. HASLETONt AND R.

SWINDELL:~

CRC Department of Medical Oncology, and ~Department of Medical Statistics, Christie Hospital and Holt Radium h~stitute, Manchester M20 9BX, and t Department of Histopathology, Wythenshawe Hospital, Manchester M23 9LT, U.K.

In a retrospective study, surgical specimens of 136 patients with nonsmall cell lung cancer, were investigated for DNA content by flow cytometry. Seventy per cent of all tumours were aneuploid. Aneuploidy was a statistically significant factor for shorter survival and was independent of turnout size, histology, grade of differentiation, regional lymph node involvement, age and sex (P7"5%) was also a prognostically unfavourable factor and was observed significantly more often in tumours where the majority of cells expressed epidermal growth factor receptor.

Introduction

There are four main histological groups of lung cancer, described by the WHO 1981 classification. These are squamous, adeno, large cell anaplastic and small cell carcinomas. Lung cancer can be divided into two main groups with differing clinicopathological features, i.e. small cell (SCLC) and nonsmall cell (NSCLC) lung cancers. However, within the NSCLC group, tumour behaviour is variable and prognosis largely unrelated to the different histological subgroups. Some important clinical prognostic factors are known, e.g. performance status and stage of disease. However, different treatment results in patients with apparently comparable tumours in terms of stage, histology and clinical features do occur (1). Important aspects of the tumour's biology (e.g. cell kinetics) may now be assessed and be potentially useful in defining prognosis. Abnormality of cellular DNA content has been associated with poorer prognosis when compared with diploid tumours. The technique of flow cytometry (FCM) provides reliable and rapid detection of changes in DNA content in a large number ofcells and has been increasingly applied over the last few years. However, in NSCLC only a few previous reports are available. Furthermore, growth factors and their relationship to human tumour behaviour are only just Received12June 1989and accepted4 January 1990. *Correspondenceto: Dr N. Thatcher, CRC Departmentof Medical Oncology,ChristieHospitaland Holt Radium Institute,Manchester M20 9BX,U.K. 0954-6111/90/030217+ 07 $03.00/0

being investigated. Epidermal growth factor receptor (EGF-R) expression is seen more frequently in well differentiated tumours but this result has failed to reach statistical significance (2). The aim o f the study was to determine the relationship o f DNA analysis by FCM (using paraffin embedded specimens) to histology, tumour stage, EGF-R status and prognostic significance.

Materials and Methods

Paraffin embedded specimens of 136 patients with NSCLC who had undergone thoracotomy were investigated, the minimum follow-up time being 3 years. None of the patients in this study were known to have metastases at the time of operation. The survival time was taken from the date of operation. Where available, three samples from different parts of the tumour were studied. A 5/zm section was taken from each tumour block followed by conventional Haematoxylin and Eosin staining and when necessary other techniques. The sections were then reviewed to confirm the histological diagnosis. The tumours were classified according to W H O (1981). The method of Headley et al. (1983) was followed for preparation of a single cell suspension from the paraffin embedded specimen for analysis of cellular D N A content (3). Briefly, two 30/zm sections were cut and dewaxed in xylene for 10 rain at room temperature using two solvent changes, followed by rehydration using the sequence 100, 95, 70 and 50% ethanol for 9 1990Bailli6reTindall

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H . D a z z i e t al.

10 rain in each. The sections were then washed twice in distilled water, resuspended in 1 ml of 0.5% pepsin (Sigma) in 0.9% NaCI, adjusted to pH 1.5 with 2 N HCI and placed in a waterbath at 37 ~ for 30 min with intermittent vortex mixing. Samples now in single cell suspension were centrifuged and the pellet resuspended in staining solution, containing 1 llgml -~ if 4'6'-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) in RPM1 1640 tissue culture medium and RNAse (Sigma) at a concentration of 1 mg ml -~ was added. The suspension was filtered through a 35/zm nylon mesh. After a minimum of 30 min for incubation, the cellular DNA content was measured using an EPICS V flow cytometer. The EPICS V flow cytometer was calibrated with human lymphocytes fixed in 95% ethanol and used to align the system each time. For each histogram a total of 30 000 nuclear particles was counted. The coefficient of variation (CV) of the DNA histograms was calculated as the full width of the peak at half its maximum height divided by the channel number x2.35 using the software programme of the EPICS V system. Histograms with a CV of > 8% or where no analysis was possible due to an insufficient number of nuclei or debris were repeated and if again found to be of low quality excluded from the study. Ten per cent of all samples had to be excluded from further analysis due to the above reasons. Normal host cells in the tumour sample were used as an internal control. DNA indices were calculated as the ratios of the aneuploid peak channel to the first peak channel considered to be diploid or near diploid in each histogram. The results were expressed in four groups, i.e. diploid, near diploid, single aneuploid and multiple aneuploid. For tumours where more than one sample was analysed and different results obtained, the sample with the greatest abnormality of DNA content (i.e. multiple aueuploid > single aneuploid>near diploid>diploid) was taken for analysis. A total of 291 blocks were analysed and in these (59.8 %) the proliferation index (PI) could be calculated. None of the samples excluded from the PI analysis was diploid. Although normal diploid cells cannot be distinguished from diploid turnout cells by FCM, the samples were checked histologically before FCM analysis and contained only a small proportion of normal tissue component. As this normal lung tissue has little proliferative activity it was unlikely to substantially effect the S-phase component of the cell cycle. Estimation of the PI was calculated from the S-phase% + G 2 / M phase% of the cell cycle. The identification of EGF-R using the F 4 monoclonal antibody in paraffin embedded material has been reported previously and therefore is only briefly described here (2). Expression of E G F - R was detected

immunohistochemically using the monoclonal antibody F4, (MoAb F4), which reacts with the cytoplasmic domain of EGF-R and therefore can be used in paraffin sections. Sections (5/~m) were cut and dewaxed in xylene for 10 rain followed by rehydration in decreasing alcohol concentrations. The sections were next preincubated with normal rabbit immunoglobulin serum (Dakopatts, Denmark) diluated in TBS 1:5. After washing-off the excess, the sections were covered with the M o A b F 4 at a concentration of 1:50 and incubated overnight. Subsequent layers consisted of a rabbit antimouse immunoglobulin Z259 (Dakopatts) at a concentration of 1:25 and monoclonal mouse A P A A P D651 (Dakopatts). Finally the red enzyme reaction was developed with napthol as biphosphate and fast red in TBS 0' 1 M pH 8'2 with 1 mM Levamisole to block endogeno us alkaline phospha tase. The number of positively stained cells was estimated. Results were expressed as four groups: E G F - R -re (0-4%); E G F - R + ve (5-19%); E G F - R + + (20.49%); and E G F - R + + + v e (.~ 50%). All contingency tables were analysed using the 2': test. The P values of these analyses for the data set (expressed as percentages for ease of comparison) are given at the bottom ofeach table. Survival differences were assessed using the log rank test. Results

Of the 136 tumours, 87 were squamous cell carcinoma, 30 adenocarcinoma, six large cell carcinoma and 13 undifferentiated nonsmall cell lung cancer. Fortyone tumours were diploid (30%), 16 near diploid (12%), 59 aneuploid (43%) and 20 multi-aneuploid (15%) (Table 1). Analysis for ploidy within the four different histological groups was performed but no statistically significant differences were found within the subgroups with the exception of large cell carcinoma. Only one out of six large cell carcinomas was diploid whereas three were aneuploid and two near diploid. The grade of turnout differentiation appeared to be related to ploidy (Table 2). Well differentiated turnouts were more often diploid than poorly or undifferentiated turnouts (48% vs. 28%) but this was not significantly different (P = 0.154):'2 test). In 55 patients where measurable tumour was available from resected specimens, no statistically significant difference in ploidy status was found between the smaller TI" turnouts (less than 3 cm) and larger T2 tumours. However, the smaller tumours tended to be diploid (35% vs. 25%) (Table 3). Nor was the ploidy of the primary tmnour related to the frequency of regional lymph node involvement with tumour. In 90 primary tumours there was documented evidence of

D N A analysis in nonsmall cell lung cancer Table I

219

Histology and ploidy status Percentage of cells Diploid

Total aneuploid*

Near diploid

Aneuploid

Multi-aneuploid

Squamous (87) Adenocarcinoma (30) Large Cell (6) Undifferentiated NSCLC (13)

32 27 17 31

68 73 83 69

14 3 33 8

41 53 50 31

13 17 0 30

Total(136)

30

70

12

43

15

Histology

P = 0.834 (Z2test) *Total aneuploid = sum of near diploid, aneuploid and multi-aneuploid. Table 2 Tumour differentiation and ploidy status

Percentage ofcells Differentiation Well Moderate Poor Undifferentiated

Diploid

Total aneuploid

Near diploid

Aneuploid

Multi-aneuploid

48 22 32 22

52 78 68 78

9 14 9 11

33 56 36 39

10 8 23 28

P = 0-154 (Z2test) Table 3 Primary tumour size and ploidy status

Percentage of cells Tumour size < 3 cm .~ 3 cm

Diploid

Total aneuploid

Near diploid

Aneuploid

Multi-aneuploid

35 26

65 74

8 7

48 53

9 14

P=0'626 (2'2 test) nodal status. The primary tumour was diploid in 36% of patients who had regional lymph involvement compared with 20% of patients who were free of nodal involvement (see Table 4). Further analysis was performed to determine the E G F - R status in relation to ploidy (Table 5). There was no clear association between the percentage of tumour cells positive for E G F - R and the diploid or aneuploid status in the same specimen (Table 5). The relationship between E G F - R and ploidy was investigated separately for two larger tumour groups, i.e. squamous cell and adenocarcinoma, however, no

obvious association between the two types was found. Tumours with a low proliferation index (PI < 7'5%) were (statistically) significantly more often EGF-R positive compared with tumours with a higher PI (Table 6). Patients with diploid tumours had a statistically longer survival compared with aneuploid (Fig. 1). This relationship was independent oftumour size, histology, grade of differentiation, regional lymph node involvement, age and sex (P

DNA analysis by flow cytometry in nonsmall cell lung cancer: relationship to epidermal growth factor receptor, histology, tumour stage and survival.

In a retrospective study, surgical specimens of 136 patients with nonsmall cell lung cancer, were investigated for DNA content by flow cytometry. Seve...
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