JOURNALOF PATHOLOGY, VOL.

160: 3 13-320 ( 1990)

CLINICAL SIGNIFICANCE OF NUCLEAR DNA CONTENT IN PANCREATIC CARCINOMA KALLE A. ALANEN, HEIKKI JOENSUU, PEKKA J. KLEMl AND TIM0 J. NEVALAINEN

D i p i r t m m t o/'Putliologj~. Universit?. of Turku and Departments of Puthology and Radiotherapy. University Central Hospital of Turku. Turku. Finland Received 24 October 1989 Ac.c.iJpted15 December 1989

SUMMARY The nuclear DNA content of 62 pancreatic adenocarcinomas was analysed by flow cytometry from paraffinembedded material. Radical surgery could be performed in 12 of the 24cases with diploid carcinoma, but only in 3 of the 38 cases with a non-diploid tumour (P=0.0002);the radically resected carcinomas also had a lower fraction of cells in the S-phase ( P = 0 . 0 0 9 ) . Non-diploid nuclear DNA content (38 cases, 61 per cent) was associated with advanced stage ( P = 0 . 0 0 2 ) . poor histological differentiation (grade I 1 or 111. P=0.004), and primary tumour site in the body or the tail as compared with the head (P=O.OI).The median survival time of the patients with diploid carcinoma was I3 3 (SE) months, and that of the patients with non-diploid carcinoma 3 f 1 months (P=O.OOOI). The D N A index with the cutoff value 1.4 was a slightly more powerful prognostic factor than DNA ploidy, and it was the most important independent prognostic factor in Cox's multivariate analysis (P 1.3, the SPF was calculated for the nondiploid stemline only. The SPF could not be calculated in 20 non-diploid cases owing to overlapping Table I-Clinical data of the patients with pancreatic DNA stemlines. The mean coefficient of variation (CV) of the diploid peaks was 6.1 per cent (SD 1.6 carcinoma (n =62) per cent, range from 2-4 to 9-6per cent). The mean CV value of normal pancreatic tissue was 5-6 per Mean, 66 years; SD, 10 years Age cent (SD 1.4 per cent). 3 1 male, 3 1 female Sex The ploidy of the histograms was assessed Site 3 1 head, 24 body, 4 tail, 3 multiple independently by three investigators. Histogram 14 st. I, 17 st. II,4 st. III,27 st. IV Stage classification was done without any knowledge of 14Gl,36G2,12G3 Grade the clinico-pathologic or survival data. 15 radical, 47 non-radical Surgery 7 given, 55 not given Radiotherapy Statistical analysis Chemotherapy 5 given, 57 not given Frequency tables were analysed by the chisquared test or Fisher’s exact test. Comparison of age and SPF distributions in different groups was done with Kruskall-Wallis’s analysis of variance Flow cytometry and Mann-Whitney’s U-test. The survival rate was Flow cytometry was done with a FACStar flow analysed by a BMDP computer program. The cytometer (Becton-Dickinson Immunocytometry cumulative survival rate was estimated by the prodSystems, Mountain View, CA). A 488nm argon uct-limit method, and a comparison of the survival laser line run at 600 mW was used for fluorescence rate between groups was made using the generalized excitation. Fifty pm sections were cut from the Wilcoxon test. Survival rate corrected for interparaffin blocks and deparaffinized as described current deaths was used in the calculations. Patients earlier.’ DNA was stained with propidium iodide who died from other causes than pancreatic carciimmediately prior to flow cytometry. l o Before noma were withdrawn from the analysis at the date analysis, ail samples were filtered through a silk of death. The relative importance of prognostic facgauze. Prior to analysis, the cytometer was focused tors was analysed using Cox’s proportional hazard

ampullary carcinomas, 6 adenomas or cystadenomas, 8 neuroendocrine tumours (confirmed by synaptophysin immunohistochemical staining),’ 1 acinar cell tumour (confirmed by phospholipase A, immunohistochemical staining), 3 cases without clinical information available, and 14 cases with an uninterpretable DNA histogram were excluded, leaving 62 pancreatic carcinomas in the series. The epithelial origin of the poorly differentiated carcinomas was confirmed by cytokeratin immunostaining. The 62 cases consisted of 56 adenocarcinomas, 3 mucinous carcinomas, and 3 adenosquamous carcinomas. In addition, histologically normal pancreas from 22 patients was analysed for nuclear DNA content, and eight of these patients had carcinoma in another part of the gland. Histological grading was done according to the conventional criteria by one of the authors (K.A.). The hospital records were reviewed. The clinical characteristics of the patients are given in Table I. Staging was done according to the American Joint Committee.’ Sixty-one patients were followed up until death, and one patient was alive 22 months after surgery at the time of the study. Three patients died from an intercurrent disease, two of them within 1 month of surgery.

315

DNA CONTENT IN PANCREATIC CARCINOMA

2400

1 lEfB

0

Q

Fig. I-Examples of DNA histograms produced from pancreatic adenocarcinomas. The number of particlesanalysed is given on the vertical axis and the DNA content on the horizontal axis. ( A ) A diploid histogram produced from a well-differentiated carcinoma. The SPF ( S ) is 3.8 per cent and CV 4 per cent. The patient survived for 16 months after surgery. (B)An aneuploid histogram with DNA index 1.57 produced from a moderately differentiated carcinoma. The SPFof theaneuploid peak is 12.5per cent. The patient survived for 7 months. d = Diploid peak: a = aneuploid peak

model (BMDP 2L). Before entering a factor in the Cox’s model, it was studied as a single factor in a

univariate analysis, so that it could be entered in as effective a form as possible. All P values are twotailed. RESULTS

Diploid

z 10

D N A ploinv

Twenty-four carcinomas were diploid and 38 (61 per cent) non-diploid. Six of the non-diploid

carcinomas were classified as tetraploid and four as multiploid. The distribution of DNA indices is shown in Fig. 2. The mean SPF was 10.5 per cent (SD 8 . 7 per cent, range from 1.6 to 35. I per cent). In diploid cases it was 5.6 per cent (SD 3.5 per cent, I I = 24). and in non-diploid cases 17.0 per cent (SD 9.4percent.n= 15. P23

DNA-index

Fig 2-Distribution adenocarcinoma

of DNA indices in 62 cases of pancreatic

that of patients with non-diploid carcinoma (n = 38) only 3 f 1 months ( P = O . O O O l ) . Survival by DNA ploidy is shown in Fig. 3. After a series of calculations the DI value 1.4 was found to be the most effective cut-off value regarding corrected survival (i.e., it produced the smallest P value in the generalized Wilcoxon test). This cut-off value divided the series into two groups of patients with clearly different prognoses ( P < O . O O O l , Fig. 4). None of the patients with a carcinoma with DI > 1.4 (n= 34) was alive 9 months after the diagnosis as compared with 14 of the 28

316

K. A. ALANEN ET AL. 100

100 n

p = o.oO02 80

80

-

p

z

ae

3> 2

20

o !

1

0

t

I

10

h4cxmis

tI

20

Fig. 3-Survival curves corrected for intercurrent deaths of 62 patients with pancreatic carcinoma grouped by DNA ploidy. Twenty-four carcinomas were diploid, 6 tetraploid, 28 aneuploid. and 4 multiploid

m p < 0.0001

Dlc 1.4

DI> 1.4

I2L-l 0

0

10

M3NMs

20

Fig. 4-Survival curves corrected for intercurrent deaths of 62 patients with pancreatic carcinoma grouped by DNA index with the cut-off value 1.4. Twenty-eight carcinomashad DI > 1.4 and 34 DI < 1.4

patients with DI < 1.4. The most effective cut-off value regarding survival was 6 per cent for the SPF, and it was found in a similar manner as that for the DI. Carcinomas with a low SPF had a less serious prognosis than those with SPF > 6 per cent (P=0.006, Fig. 5). In addition to ploidy, DI, and SPF, several other prognostic factors were found in univariate analyses (Table 11). Survival curves by radicality of surgery and histological grade are shown in Figs 6 and 7. The patients with radical surgery survived significantly longer than those with non-radical surgery (P= 0.0003).

0.006

60-

a v)

-

40

-

20

-

04 0

I

I

10

20

Fig. 5-Survival curves corrected for intercurrent deaths of 42 patients with pancreatic carcinoma grouped by S-phase fraction. Nineteen patients had SPF < 6 per cent and 23 SPF > 6 per cent

Association of D N A ploidy with other prognostic factors The association of DNA ploidy and D I with seven clinico-pathologic prognostic variables is shown in Table 111. DNA aneuploidy was associated with non-radical surgery (P= 0.0002), advanced stage (P=0.002), poor histological differentiation (G2 or G3, P = 0-004), primary tumour site in the body or the tail as compared with the head (P=O.OI), and patient age at diagnosis greater than the median (66 years, P=0.02), but not with gender. The DNA index was associated with the same factors, and it also had a significant (P=O.Ol) association with the primary tumour size. Twelve of the 24 diploid carcinomas could be removed radically as compared with only 3 of the 38 non-diploid carcinomas (P= 0-0002). The mean SPF of the carcinomas that were excised radically was 6.3 per cent (SD 5-9per cent, n = 14) and that of the carcinomas with non-radicaI surgery was 12.6 per cent (SD 9.2 per cent, n = 28, P=0.009). Seventeen (55 per cent) of the 31 carcinomas situated in the head were diploid as compared with only seven (23 per cent) of the carcinomas found in the other sites (P=O*Ol). Carcinomas of the head were also more often well-differentiated (Gl) than carcinomas in the other parts of the organ (P= 0.03), and they tended to have an SPF below the median (P=0.09). The mean time of symptoms preceding the diagnosis was similar if the carcinoma was found in the head or if it was found elsewhere (2 & 1 months). The mean age of the patients with diploid carcinoma was 62.5 years (SD 9.9), whereas that of the

317

DNA CONTENT IN PANCREATIC CARCINOMA

Table II-Significance of prognostic factors in a univariate analysis Survival

Factor

N

Sex

31 31

Age

30 32

Stage Grade

Site

Grouping

6 months (%)

I year (YO)

P

Male vs. female

49

26 16

NS

< 66 years* vs.

52 32

33

> 66 years

14 48

I YS. II-IV

83 31

66 9

0.0005

14 36 12

GI vs. G2 vs. G3

77 40

0.0003

8

62 9 8

31 30

Head vs.

52 33

30 13

0.04

other

3s

0.0s

10

28 30

< 9 cmt vs.

67 17

32 3

0.000 I

>9cm

Ploidy

24 38

Diploid vs. non-diploid

73 24

53 3

0.000 I

DI

28

DI < 1.4t VS. DI > 1.4

73 18

49 0

< 0.000 1

34 19 23

SPF S6Yot VS. SPF >6Yo

78 46

60 9

0.006

15

Radical vs. non-radical

84 30

62 9

0.0003

Size

SPF Surgery

47 *The mean age.

t A cut-off value that gave the smallest P value was chosen.

patients with non-diploid carcinoma was 67.8 years (SD 10.2, P= 0.02). The difference in mean age at the time of diagnosis was even greater when the patients were grouped by the DI of 1.4 as the cut-off value. For carcinomas with DI ,< 1.4 the mean age at diagnosis was 61.8 years (SD 10.3). and for carcinomas with DI > 1.4. it was 69.0 years (SD 10.3, P = 0.004). Multivariate analj*ses( Table I V )

In order to determine the relative importance and independence of ploidy and DI as prognostic factors, we compared age at diagnosis, stage, grade.

site, and primary tumour size with DNA ploidy and the DI in Cox’s stepwise proportional hazard model. The internal grouping of the factors entered is given in Table 11, with the exception that age was entered as a continuous variable. The most important independent prognostic factor was the DI (with a cut-off value of 1.4, P 66 years

16 8

14 24

0.02

19

II 23

0.005

9

II

3 16

0.002

0.003

8

19

12 7 9

3

5

14 18

Grade (n=62) GI G2+G3

10 14

4 34

0.004

11 17

3 31

0.004

Site ( n = 6 1) Head Other

17 7

14 23

0.0 1

18 10

13 20

0.05

Size ( n = 58) G6cmf >6cm

II II

10 26

0.09 (NS)

14 12

7 25

0.0 1

Surgery ( n = 62) Radical Non-radical

12 12

3 35

0.0002

13

2

15

32

Stage ( n= 62) I I1 lllt IV

+

~

~

~

P

~

~~~~~

*The mean age tStages I1 and 111 werecombined because there were only four stage 111 carcinomas :A cut-offvalue that gave the smallest P value waschosen

Table IV-Results

of Cox's multivariate analysis

Factor DI ( ,< 1.4 VS.> I .4) Histological grade Age at diagnosis Tumour size Tumour site Stage DNA ploidy

Improvement chi-square

P

Coeff./SE*

18.3 4.9

< 0.00 1 0.03

* 3.7 -

3.5

Comment Independently associated with survival Not associated with survival

*Estimated regression coefficient of the hazard function divided by standard error.

P

0.0002

320

K. A. ALANEN ET AL.

control sample was aneuploid with a DI of 1.30 in repeated analyses. Barlogie et a1.22found DNA aneuploidy in seven patients with a previously established diagnosis of carcinoma among a group of 209 patients with normal histology or reactive changes, and Reid et a l . 2 3 reported aneuploidy in one patient with Barrett’s oesophagus in a sample containing specialized metaplastic epithelium. In some cases, DNA aneuploidy may be present even if no abnormality is seen by light microscopy. Another explanation for an aneuploid DNA pattern is increased binding of propidium iodide to DNA as a consequence of tissue a u t ~ l y s i sAuto.~~ lysis typically produces false aneuploid peaks with a DI < 1.4, which may partially explain why such peaks were of little prognostic significance in this study, too. We conclude that the more favourable prognosis of carcinomas located in the head of the pancreas and that of resectable carcinomas may at least partially be explained by their less aggressive biological behaviour. Flow cytometric analysis of nuclear DNA content gives important prognostic information in pancreatic carcinoma, and the DI appears to be an important independent prognostic factor. ACKNOWLEDGEMENT

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