Pancreatic lithiasis in the aged

GASTROENTEKOLOGY

1984:86:X31-8

Pancreatic Lithiasis in the Aged Its Clinicopathology HIDE0 Department

NAGAI of Clinical

and Pathogenesis

and KOHICHIRO Pathology,

Tokyo

OHTSUBO

Metropolitan

The incidence of pancreatic calculi in autopsy material was found to increase in proportion to age after 70 yr: 0% under 69 yr of age (0 of 134), 4.2% in the 70s (5 of 119), 7.7% in the 80s (9 of 117), and 16.7% in the 90s (8 of 48). Most of the stones were found scattered throughout the peripheral ducts. Pancreatic lithiasis in the aged was clinically characterized by lack of signs and symptoms, absence of alcoholism, and was unassociated with hypercalcemia. Extensive parenchymal atrophy and fibrosis were limited to the areas upstream from the calculi. The stones were found in the ducts just above the sites of obstruction where squamous metaplasia was invariably present. Immunohistologic study showed intense staining for lactoferrin in the protein plugs and in the cytoplasm of cuboidal or squamous cells of ducts containing the plugs. Ductal stenosis, either primarily caused or secondarily exacerbated by squamous metaplasia, and lactoferrin-positive cells appeared to play a role in the pathogenesis of pancreatic Jithiasis in the aged. It is generally believed that pancreatic lithiasis is an extremely rare disorder (l-3) and that it represents a terminal sequel of chronic pancreatitis (4-6). The present study demonstrates that it is not rare to find calcified stones in the pancreas of elderly people, and that they seem to be associated with different clinical and pathologic features from those seen in

Received April 4. 1983. Accepted September 9, 1983. Address requests for reprints to: Hideo Nagai, M.D., First Department of Surgery. University of Tokyo School of Medicine, 7-3-l Hongo, Bunkpo-ku, Tokyo 113, Japan. The authors thank Dr. H. Shimada of the Department of Pathology, Tokyo Metropolitan Geriatric Hospital, and Dr. S. Saiki of the Division of Pathology, St. Luke’s International Hospital, Tokyo, for their permission to use the materials. The authors also thank Professor Y. Morioka and Dr. A. Kuroda of the First Department of Surgery, Tokyo University Hospital, for their advice on this study, and Mrs. K. Sato. Mr. N. Aikyo, and Mr. K. Nakamura for their technical assistancr,. e 1984 by the American Gastroenterological Association 0016-5085/84/$3.00

Institute

of Gerontology,

Tokyo,

Japan

younger patients with chronic calcifying pancreatitis. This study also refers to the pathogenesis of pancreatic lithiasis found in the aged.

Materials and Methods We used pancreases extirpated from 418 patients (age, >30 yr) autopsied at the Tokyo Metropolitan Geriatric Hospital and St. Luke’s International Hospital, Tokyo, Japan. All the pancreases were subjected to plain soft x-ral roentgenography soon after their removal. The specimens were exposed for 2 min on Fine Grain Softex Films (Fuji Photo Film Co., Tokyo, Japan) at 70 cm, using 30 kVP, 10 mA irradiation. Eighty-five cases were analyzed further by postmortem pancreatic ductography with 2-3 ml of warm gelatin-mixed barium sulfate (60%) wt/vol) injected from the papilla Vateri through a vinyl canula. After fixation in 10% formalin, we looked for calcified stones by cutting the specimens into slices, with reference to their roentgenograms. Calcifications of arterial walls were excluded. Calcified stones were removed whenever possible and were submitted to infrared spectroscopic analysis for chemical composition. Clinical records of the patients with pancreatic calculi were reviewed for a history of alcoholism, signs and symptoms related to the digestive system, and laborator! findings (glucose tolerance and serum calcium in particular]. Histologic sections of the pancreas. including the areas that had contained calculi, were stained with hematoxylin and eosin, periodic acid-Schiff, and Weigert’s elastic fiber stains. The pancreases of noncalculous patients were also studied microscopically. We paid special attention to the presence of stonelike substances and metaplastic changes of ductal epithelium. Various types of stonelike substances and lining epithelial cells are shown in Figure 1. For those calculous specimens subjected to pancreatic: ductography, a 5-pm-thick serial section study was performed after decalcification to search for clues to the pathogenesis of the calculi. Immunohistochemical staining was performed for localization of lactoferrin, which is supposed to play an important role in the formation of pancreatic stones (6,7). We studied the calculous and noncalculous specimens from

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I. Various types of stonelike substances and lining epithelial cells observed in microscopic slides without decalcification. Hematoxylin and eosin. (a). Squamous metaplasia lining a dilated duct that contained a calcified stone. x170. (b). Several noncalcified stones, which we define here as discrete substances with a lamellar structure that are similar to calcified stones. The duct containing the noncalcified stones is lined mostly by squamous metaplasia. ~40.(c). Plugs in ductules lined by oneto two-layered cuboidal or squamous cells, which we provisionally call “oligolayered squamous cells.” Plugs are amorphous, irregular, and condensed intraductal materials that are usually smaller than noncalcified stones. x170.

patients who had been autopsied within 12 h postmortem. The unlabeled antibody peroxidase-antiperoxidase technique of Sternberger (8) was used after pronase treatment of deparaffinized sections (9) (Table 1). Rabbit antihuman lactoferrin, swine antirabbit IgG, and peroxidase-antiperoxidase complex (rabbit) were purchased from Dako Corporation, Santa Barbara, Calif. and Pronase P from Kaken Kagaku Co., Tokyo, Japan.

ducts were filled with stones, l-10 mm in diameter, from the papilla Vateri up to the tail. Another man, 89 yr old, had a single, yellowish stone, 5 mm in diameter, in Wirsung’s duct. The stone consisted of protein and calcium phosphate. Clinical Findings Retrospective ed that no patient

Results

study of clinical charts indicathad a history of pancreatitis,

Incidence Calcified pancreatic stones were found in 22 cases (5.3%); these subjects ranged in age from 72 to 97 yr. As shown in Figure 2, the incidence of pancreatic lithiasis increased in proportion to age after 70 yr: 0% (none of 134 cases) under 69 yr, 4.2% (5 of 119) in the 70s, 7.7% (9 of 117) in the 8Os, and 16.7% (8 of 48) in the 90s. Of the 22 patients, 14 were men and 8 were women. Characteristics

of Calculi

Twenty of the 22 patients had small calculi of 1-3 mm in diameter and 2-100 in number (Figure 3). All these small stones were located in the peripheral branches and were almost exclusively composed of calcium carbonate. In a specimen from a 79-yr-old man, the pancreatic

Table

1. Procedure Lactoferrin

of Immunohistochemical

Staining

Deparaffinization Phosphate-buffered saline (PBS) 15 min I Pronase P (O.l%, pH 7.4)37"C,30 min 1 PBS 15 min 3% H,O, 5 min 1 PBS 15 min Normal swine serum 20 min Rabbit antihuman lactoferrin (1:40) 30 min 1 PBS 15 min Swine antirabbit IgG (1:50) 20 min i PBS15 min Peroxidase-antiperoxidase (rabbit) (1:20) 20 min I PBS 15 min Diaminobenzidine 4 HCl (0.5mg/ml) + O.Ol"hH,O, 5-15 min

of

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-

1 16.7%

1.2: a #'

Figure

ma and inter- and intralobular fibrosis, were exclusively confined to the areas in the vicinity of the stones. Only 1 case had a marked atrophy and fibrosis of the whole pancreas. The ducts encasing the calculi were lined by epithelium with squamous metaplasia in more than one-half of the sites examined microscopically (Table 2). Epithelium was desquamated in one-fourth of them. All of the calculi that seemed embedded in the parenchyma proved to be intraductal by elastic fiber staining. Squamous metaplasia was observed not only in the vicinity of the calcified stones, but also at a long distance from them. Foci of squamous metaplasia were significantly more frequent in calculous specimens than in age- and sex-matched, noncalculous controls (Table 3). Mucous cell metaplasia was also found in the specimens, but it was mostly situated in sites unrelated to the calculi. The extent of mucous cell metaplasia in pancreatic lithiasis was the same as in noncalculous pancreases (Table 3).

.?. Histogram of patients with pancreatic calculi (striped bars] alid those without stones (unshaded bars). The vertical axis shows the number of patients and the horizontal axis indicates the age in years. The broken line shows the incidence of pancreatic lithiasis. The difference between the group below 69 yr and each successive age group was statistically significant by the x2 method (p < 0.05-O.OOl), as was the difference between the patients in their 70s and 99s (p < 0.05). The difference between the 70s and 80s or between the 80s and 90s was not statistically significant.

pancreatic trauma, or abdominal irradiation. A history of alcoholism was confirmed in 3 patients. None of the 22 patients had abdominal pain or steatorrhea ascribable to pancreatic abnormality. Diabetes mellitus was confirmed in 6 cases. Intractable diabetes under poor control even with insulin therapy was present in only 1 patient who had innumerable stones throughout the pancreatic ductal system. The other 5 diabetic patients were appropriately treated with diet therapy. Eight patients showed normal glucose tolerance, and the remaining 8 had no record concerning glucose tolerance. Serum calcium values were available for all the patients, and there were no cases of hypercalcemia. Pathologic Almost all moderate atrophy differed little in matched subjects histologic changes,

Findings the pancreases showed mild-toin gross appearance, but they this respect from those of agewithout calculi. In most cases, such as atrophy of the parenchy-

Figure

3. (a). Plain roentgenogram of the pancreas lvith small calcified stones. Calcifications are seen scattered throughout the pancreas. (h). Gross appearance of slices of the same pancreas. Calcified stones (orro~~~ heuds) are found in the peripheral pancreatic dmts. The pancreas shows moderate atroph!,.

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2. Lining Epithelium Calcified Stone+’

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of Ducts Containing No. of sites

Squamous metaplasia Oligolayered squamous cells Columnar epithelium

26

8

Desquamation

2 0 12

Total

48

Mucous

cells

" Forty-eight

sites from 22 cases were microscopically

examined,

Four patients had stones in the gallbladder, and 1 patient had had a cholecystectomy 6 yr before. None had stones in the common bile duct. Marked sclerosis of the papilla Vateri was found in 1 case, but the pancreatic ducts of this patient were not dilated. Another patient had a large periampullary diverticulum, although there was no evidence for stasis of pancreatic secretion. There was no case of pancreas divisum or pancreatic cancer.

Study

of Serial

Figure

Sections

Four available calculous specimens with pancreatic ductograms were used for the 5-pm-thick serial section study [Figure 4). In all 4 cases, the pancreatic stones were lodged just above the stenotic ducts, where squamous metaplasia was invariably observed (Figures 5 and 6). Plugs and noncalcified stones were also found scattered throughout the peripheral ducts. Plugs were located in the ducts lined by columnar cells or oligolayered squamous cells. Noncalcified stones were mostly found in ducts lined either by stratified squamous metaplasia or by oligolayered squamous cells. Stenosis of ducts below plugs and noncalcified stones was also present, but was not so remarkable as in the ducts below calcified calculi. Squamous metaplasia was also found in areas other than those related to the presence of the calculi. When the metaplasia was not Table

3. The Number CaJcuJous

of Foci

Group

of Squamous

Metaplasia

roentgenogram calcified stones of the same site. the area studied arrow indicates

of the (case

pancreas body with Figure 5). (b). hset: schematic drawing to by 5-wrn-thick serial secthe stone examined in this 1 of

associated with ductal stenosis, as was often seen in the larger ducts, plugs or calculi were rarely found there. Extensive parenchymal atrophy and fibrosis were limited to the area upstream from the site of marked ductal stenosis (Figure 5). At the site of obstruction, below all of the calcified stones and a few of the noncalcified stones, there was a moderate-to-marked periductal fibrosis, which was always continuous with the fibrotic lobules above.

ImmunohistochemistrJl Lactoferrin

for Localization

of

We studied 14 patients with pancreatic calculi and the same number of age- and sex-matched noncalculous controls (Table 4).

and

and in the Noncalculous

4. ((1). Plain scattered Ductogram illustrate tions. The case.

Control

Mucous

Cell Metaplasia

per

Microscopic

Slide

in the

Group’ Pair

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

0032691214 0 0 2 0

0

0

0

0

0

1

3 o

1 1

8 0

12 o

5 o

3 0

3 0

0 0

4 1

5 0

0 0

2 3

1 2 13

0 10 1 0 1 0 452010143

o

7

3

4

3

10

2 08211

2

0

6

1 Squamous metaplasia group Calculous group Noncalculous Mucous cell metaplasia group Calculous Noncalculous group

2

3

0 0

0 7

14 01-I

a We made up 22 completely age- and sex-matched pairs by arbitarily selecting 1 noncalculous patient of the same age and sex for each calculous patient. Slides of the calculous group were taken from the sites where calcified stones had existed, and those of the control group were from the corresponding areas of the pancreas. Foci of squamous metaplasia were significantly more frequent in calculous specimens (p < 0.005, Wilcoxon’s t-test). There was no significant difference in muc.ous cell metaplasia.

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L

c

-

0

squamous

1

metaplasia

LlTHIASlS

L

atrophic

1N THE

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4

and fibrotic

lobule

Figurr,

5. Schematic drawings of the sites near calcified stones in 4 patients (l-4). reconstructed by serial sections. In at1 4 cases, the pancreatic Lalculi are located just above ductal stenosis where squamous metaplasia is observed. I’arenchymal atrophy and tihrosis are dkerned in the lobules upstream from att of the calcified stones and some ot the noncalcified stones, Foci of ducts. LVhere squamous metaplasia is associated with squamotls metaplasia are found scattered throughout the peripheral riuc.tal stenosis. noncalcified stones often occur above the site of stenosis. Th? length of the sc.Jr und~?r each drawing is equivalent to 1 mm.

Figure

6. A site of ductal stenosis in case 2 of Figure 5. Apparent stricture of the duct is demonstrated just below the stone. The upper duct is connected with the lower one by an extremely narrowed duct (large arrowheads). Foci of squamous metaplasia (small medium (barium). Hematoxylin and eosin. arrowheads] are observed at the site of stenosis. *Decalcified stone. **Contrast x 111.

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4. The Number of Ducts or Ductules Lined by Lactoferrin-Positive Cells per Microscopic Slide in the Calculous Group and in the Noncalculous Control” Pair 1234

Calculous group Noncalcuous group

5

6 7 8 9 10 11 12 13 14

1 13 0 22 111 67 3 8 3 0 0 0 0 3 0000

0 0

1 0

3 5

6 2 10

a Of the 22 calculous patients, 14 with postmortem time of ~12 h were investigated for immunohistochemical evidence of lactoferrin. The pair numbers and the methods of selecting the control group and microscopic slides were the same as used in the study of squamous metaplasia and mucous cell metaplasia (see Table 3). Ducts or ductules lined by lactoferrin-positive cells were found significantly more often in calculous specimens (p < 0.005, Wilcoxon’s t-test].

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sitions similar to those found in younger patients with chronic calcifying pancreatitis (2-41, the clinical and pathologic features were quite different. Pancreatic lithiasis in the aged was rarely associated with symptoms or with a history of alcoholism. Moreover, parenchymal atrophy and fibrosis were usually limited to the areas upstream from the sites of ductal stenosis. Thus, the clinicopathology of our cases was far different from that reported for the terminal stages of chronic pancreatitis (4-6,lO). Similar cases have not been reported up to the present, except by Stobbe et al. (2). Reviewing 27,787 autopsy records, they found 43 cases with pancreatic lithiasis, 22 of which were classified into “Group 1,” that is, those with calculi in localized sectors of the pancreas. They stated that the patients in Group 1,

Intracellular lactoferrin was demonstrated in 12 calculous specimens, all of which showed intense staining in the cytoplasm of oligolayered squamous cells of dilated ducts (Figure 7). In these cases, the plugs within the lactoferrin-positive ducts were also stained for lactoferrin. Epithelium with squamous metaplasia usually showed little or no staining, but large quantities of lactoferrin were sometimes observed in the cells of the most superficial layers of squamous metaplasia. Most calcified and noncalcified stones were either unstained or were faintly stained. In addition, columnar cells of regenerative ductules were strongly stained in 2 cases and acinar cells in 3 cases. Metaplastic mucous cells were not stained in any case. In the noncalculous group, there were 3 patients who had lactoferrin-positive ductal cells. The number of ducts or ductules lined by lactoferrin-positive cells, however, was much greater in the calculous group than in the noncalculous controls (Table 4). Discussion The incidence of pancreatic lithiasis in autopsy material has been reported to be 0.034% (l), 0.15% (21, o.o~%-0.33% (3), and 0.43% (6). The present study has shown that calcified pancreatic stones were found in 22 cases (5.3%) out of 418 autopsied patients, and that the incidence of pancreatic lithiasis increased in proportion to age after 70 yr. There are probably two reasons for the higher incidence of stones in our material than in other investigations. One is that we subjected all the specimens to soft x-ray roentgenography for the detection of calcifications, followed by a meticulous search for stones. The other reason is that about twothirds of our patients were >70 yr of age. Although these calcified stones found in elderly people had gross appearances and chemical compo-

Figure

7. Immunohistologic demonstration of lactoferrin in a calculous pancreas. Peroxidase-antiperoxidase method. (n). Lower magnification (X32). Staining for lactoferrin is observed in the plugs and in the lining epithelium of dilated ducts. The pancreatic lobules, other than the areas around the dilated ducts, show almost normal appearance except for moderate atrophy, as is often seen in the pancreas of the elderly. (b). Higher magnification (X 160). Lactoferrin is localized in the cytoplasm of oligolayered squamous cells, as well as of the cells of the most superficial layers of squamous metaplasia.

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19 of whom were >i’O yr old, were symptomless and rarely had a history of alcoholism, and that pathologically most of them had narrowing or occlusion of the pancreatic ducts caused by squamous metaplasia. Stobbe’s Group 1 might correspond to our cases of pancreatic lithiasis in the aged, which were similar in clinical and pathologic features, and in age distribution. With regard to the pathogenesis of pancreatic stones in the aged, Stobbe et al. (2) mentioned ductal narrowing by squamous metaplasia as a cause of yr of age. pancreatic stones in patients of >70 Involvement of squamous metaplasia was sugggested in our study as well. First, most noncalcified stones, which are likely to be precursors of calcified stones, were located in the ducts lined by metaplastic squamous epithelium. Second, ductal stenosis below the calculi appeared to be caused by squamous metaplasia. Third, there were many foci with squamous metaplasia in areas of the pancreas unrelated to and far away from the calculi; hence, it is less likely that the metaplastic change is secondary to the presence of calculi. Nevertheless, squamous metaplasia under these circumstances could still be regarded as a secondary change in response to the presence of plugs or calculi (5,ll). For the present, it would seem more appropriate to think that the squamous metaplasia preceded the process of calcification and that the presence of stones, calcified or noncalcified, might further promote the growth of the metaplastic epithelium as a result of mechanical stimuli (5). Once metaplasia has occurred, it would be possible for a duct, especially one of small caliber, to become narrowed by the intraluminal proliferation of the metaplastic epithelium, causing stasis of pancreatic secretion (12). Stasis could then elicit the formation of calculi upstream from the stenosis by the same mechanism seen in canine experimental pancreatic lithiasis induced by ligation of the main pancreatic duct (13). Parenchymal collapse caused by stenosis of draining ducts or ductules, due either to stones or to metaplastic epithelial changes, might result in periductal fibrosis and produce deformity of the ducts, which would further aggravate the obstruction. The possibility of ductal stenosis having been primarily caused by periductal inflammation or fibrosis or both. is less likely because the strictures below noncalcified stones were mostly unassociated with inflammation or fibrosis. Obstructive lesions of pancreatic ducts are known to be observed in various pancreatic diseases, such as cystic fibrosis or pancreatic tumors. Since these disorders are usually unassociated with calcified stones, it might be suggested that some other factor(s), besides ductal obstruction, cause(s) calcified shows that, in addistones to be formed. Our study

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tion to ductal obstruction, lactoferrin might play a role in the pathogenesis of pancreatic lithiasis in the aged. According to Sarles et al. (7), increased lactoferrin concentrations are regularly found in the pancreatic juice of chronic calcifying pancreatitis, even in the early stage, whereas its concentrations in normal juice are much lower. Lactoferrin has the property of binding acidophilic proteins and would thus form protein precipitates (14). Colomb et al. (15) studied the localization of lactoferrin in pancreatic tissues using an indirect immunofluorescence method. They reported that lactoferrin was detected in the acinar lumen and in the cytoplasm of acinar cells. In our study, lactoferrin was observed in acinar cells to some extent, but it was present in much greater concentration in the protein plugs and in the cytoplasm of oligolayered squamous cells of dilated ducts encasing the plugs. Calculi. calcified or noncalcified, and lining epithelium with squamous metaplasia, however, showed little or no staining. If lactoferrin has anything to do with the formation of pancreatic stones, therefore, it seems to take part in the initial stage of the formation of protein plugs. Further study is needed to elucidate the relationship between ductal obstruction and squamous metaplasia, the process of the appearance of lactoferrin-positive cells. and the mechanism of calcification of plugs and noncalcified stones. Immunohistochemical study of lactoferrin will also be necessary for other pancreatic diseases associated with chronic ductal obstruction.

References 1. Ishii

2.

3.

4.

5. 6. 7.

a. 9.

K, Nakamura K, Takeuchi T. et al. Chronic calcifying pancreatitis and pancreatic carcinoma in Japan. Digestion 1973:9:429-37. Stobbe KC, KeMine WH, Baggenstoss AH. Pancreatic lithiasis. Surg Gynecol Obstet 197~:131:109()-9. Edmondson HA, Bullock WK. Mehl JW. Chronic pancreatitis and lithiasis. II. Pathology and pathogen&s of pancreatic lithiasis. Am J Path01 1949;26:37-49. Owens JL Jr, Howard JM. Pancreatic: calcitication: a late sequel in the natural history of chronic. alcoholism and alcoholic pancreatitis. Ann Surg 1958;147:326-38. Bank PA. Pancreatitis. New York and London: Plenum, 1979. Sarles H. Chronic calcifying pancreatitis--chronic alcoholic pancreatitis. Gastroenterology 1974:66:604-16. Sarles H, Figarella C. Tiscornia 0. et al. Chronic calcifying pancreatitis (CCP). Mechanism of formation of the lesions. New data and critical study. In: Fitzgerald PT. Morrison AB. eds. The pancreas. Baltimore. London: \L’illiams & Wilkins. 1980:4&6fi. Sternberger LA. Immunocytoc.hemistry. end ed. New York. Chichester, Brisbane, Toronto: Wiley, 1979. Huang S-N. Immunohistochemical demonstration of hepatitis B core and surface antigens in paraffin swtions. Lab Invest i975:33:8a--95.

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10. Gambill EE, Comfort MW, Baggenstoss AH. Chronic relapsing pancreatitis: an analysis of 27 cases associated with disease of the biliary tract. Gastroenterology 1948;11:1-33. 11. Becker V. Morphology of chronic pancreatitis. In: Scuro LA, Dagradi A, eds. Topics in acute and chronic pancreatitis. Berlin, Heidelberg, New York: Springer-Verlag, 1981:161-71. 12. Blumenthal HT, Probstein JG. Pancreas, a clinical-pathologic correlation. Springfield, 111.: Charles C. Thomas, 1959. 13. Konishi K, Izumi R, Kato 0, Yamaguchi A, Miyazaki 1.

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AM. Association of lactoferrin with other proteins as demonstrated by changing in electrophoretic mobility. Biochim Biophys Acta 1971;251:380-7. 15. Colomb E, Pianetta C, Estevenon JP, Guy 0, Figarella C. Sarles H. Lactoferrin in human pancreas. Immunohistological localization in normal and pathological pancreatic tissues. Digestion 1976:14:242-g.