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Urinary gas-chromatographic steroid spectra and testicular tumours
77
CLINICA CHIMICA ACTA
URINARY
GAS-CHROMATOGRAPHIC
TESTICULAR
STEROID
SPECTRA
AND
TUMOURS
E. L. FRENSDORF
Department
of Pathology, Onze Lieve Vrouwe Gasthuis, Amsterdam
E. J. VAN
KAMPEN
AND W.
Clinical Chemical Laboratory,
( Received
October
HOEK
Diakonessenhuis,
Groningen (The
Netherlands)
15. 1969)
SUMMARY
A first attempt is made to correlate the pathological and histochemical findings with the urinary excretion of differentiated r7-keto- and ketogenic steroids (steroid spectrum) in cases of testicular tumours, to support an early diagnosis. In this light, differentiation between seminoma and embryonal tumours is, naturally, important.
A simple method of obtaining essential information about urinary steroid excretion spectra has been presented recently’. At first, it was merely attempted to obtain recognition patterns in clinically suspected endocrine disorders, however, by refining the method through strict standardization it has since also become possible to obtain quantitative information as regards, e.g., the excretion of total r7-ketosteroids, r7-ketogenic steroids, etiocholanolone and pregnanedio12. In the course of a morphologic and histochemical enzyme investigation of the histogenic factors of germinal testicular tumours *, a preliminary study of possible hormonal influences was also undertaken using the above method. In cases of testicular tumours (seminoma 49% of cases, embryonal carcinoma 257o, choriocarcinoma 3%, teratocarcinoma 17%, teratoma 5% and interstitial cell tumours I%), little is known of hormonal relations. This has also been the provisional conclusion of the English Testicular Tumour Panel, summarized by Symington and Wallace3 as follows : “Regarding the gonadotrophins, there seems to be a relation with testicular tumours; possibly also with their prognosis. For ketosteroid hormones a similar relation could not be shown.” There are, however, a number of arguments for the involvement of hormones: I. The testis is an organ which is strongly hormone-dependent, as regards development and function and besides, it has an important endocrine function in its own right; 2. There are sufficient reasons to assume a relation between testicular atrophy and the genesis of some testicular tumours; l
E. L. Frensdorf;
Thesis, in preparation. Clin. Chim. Acta, 28 (1970) 77-81
FRENSDORF
78 TABLE
I
CLINICAL
CHEMICAL -
OF THE
TUMOURS
OBSERVED
Weight
Diagnosis
A.&F
Registration NV.
UATA
[years)
.~
et d.
Rest of testis
L
Metastases
Sparse. Complete spermatogenesis Very sparse. Total atrophy Atrophy. Rest of spermatogonia and -cytes. Some atypia No rest
l;lr
Dubious prelumbar Dubious retroperit.
Very sparse. Atrophy sporadically. Sertoli cells From total atrophy to sparse, complete spermatogenesis
f
Not demonstrable
rt
Dubious lumbar 2, 3
Sparse, mainly Sertoli cells. Some spermatogonia, sporadically -cytes From extensive atrophy to complete spermatogenesis
-f-+
Cerebrum ?
+ i-
b.a. Paraaortal
++
Not demonstrable
++
Not demonstrable
(g)
Normal values Group A T 265387 EH
24
Seminoma
375
T
253300
EM
32
Seminoma
290
T
255436
EM
26
Embryonal teratoma (MTIA)
200
T
273128
EH
3’
12.5
T
263617
EH
67
Embryonal (MTIB) Seminoma
T 246829 EH
30
Seminoma
100
EH
43
Seminoma
150
T 258453 EH
33
85
T 218301 EH
4o
Embryonal teratoma + seminoma. Also “chondroma” (MTIA) Seminoma (mamma virilis)
T 257628 EH
32
Embryonal (MTIB)
50
Ca.
“5
& &
Generalized
Group B T
252005
L:
ao: po: d: m: EH
:
number of Leydig cells before operation after operation days months Enzyme-histnchemistry also
Ca. MTIA MTIB i: + -+-: 1: t:
70
Hyaline atrophy. Sporadically retained full spermatogenesis Rest of full spermatogenesis
following classification of English testicular panel Malignant Teratoma Intermediate, group A or B no distinct aberration distinct increase living trace
:
3. Elevation of gonadotrophins could well play a part in the genesis of testicular tumours; 4. An obvious increase of Leydig cells in some testicular tumours could well argue for an elevation of lutinising hormone (LH), a hormone which may originate extra-testicularly as well as be produced by the tumour itself (human chorionic gonadotrophin, HCG) ; 5. Testicular tumours may be accompanied by an elevation of ketosteroid levels, which decrease after extirpation of the tumour. The purpose of this paper is to report the preliminary results of investigations of IO cases of testicular tumours. Unfortunately, most results have been obtained from material sent to the Pathology Department for diagnosis and urinary hormonal assays could only be performed some days, or even some months, after semi-castration. In only one case have assays been obtained both before and after operation. The tumours referred to, together with available clinical chemical data, have czin. Claim.A&,
28
(1970) 77-k
URINARY
AOIPO (days/months)
STEROIDS
17-K
3 d PO 3 d PO
29
IO
17-OH
TESTICULAR
O&r.
5 h
IOk7 13 f 5 mg/*4 h Icd24 16
h
dpo
12
AP
LDH
Fractions isoenzymes
o-3 u (Ressey)
TOO-300 u (King)
44-46-7-2%
640 =9o
.+43-8-r-t
815
56-39-4-1-t
-
4.3
4.3 I2
8
ZI
I2
0.1
co.1
-
2.3
*40 I55
45-45-8-1-1
1 14 yr.
500
65-32-3-t
4 yr. moribund
m PO 16
5 ~PO 4 m PO
I5
2 Yr PO
20
28
3 d PO
23
I9
0.1
-
1.6 3.6
-
Q6-6-2-t-O
140
18
if0
1330
Course
1 I yr.
IO-20 u
29
5m
5
HCG
d po
5 d PO
79
TUMOURS
PVtp
13 i W24 20 13 I9
;
AND
-
dead 7 m 460
170
3.4
1 3 yr. l7m
been tabulated according to size in Table I. Fig. I presents the normal urinary steroid excretion of the mature male using the procedure described previously19 solidline( with the results of this investigation drawn in dotted line(. . .) The successive peaks or areas in the normal chromatogram represent the following: I. breakdown products, e.g. monohydroandrosterone, of, among others, pregnanediol and dehydroisoandrosterone; IIa. etiocholanolone; IIb. androsterone, dehydroisoandrosterone and androstenedione; III. rr-oxy-r7-ketosteroids; IV. pregnanediol, pregnanetriol, etc. ; V. steroids of the @-cortol series; VI. cholesterol. Post-operatively two main types of spectrum of ketosteroids have been found; they have been called type I and type 2. Type I is characterized by elevation of etiocholanolone (peak IIa) and subnormal androsterone excretion (peak IIb). Type z is characterized by elevation of etiocholanolone (IIa), elevation of androsterone (III?) and an increase in zones I and IV. Patients with types I and 2 urinary steroid excretion patterns were classed as group A and B respectively in Table I. The pre-operative C&z. Chim. Acta, 28 (x970) 77-81
FRENSDORF et di.
Fig. I. Steroid metabolism am1 urinary steroitl in the case of seminoma (type I) and embryonal is given by solid lines; deviations by the dashed
spectra obtained by means of gas chromatography carcinoma (type 2). The normal chromatogram lines.
For I to VI. see text.
steroid spectra given are open to argument as they are based on a single observation, Although this small series is merely sufficient to put forward some suggestions, an attempt has been made to correlate the two types of urinary steroid spectra with the clinical and morphological manifestations. In group A, which comprised mainly large tumours, four of the six were seminoma. The two embryonal tumours in this group were poorly differentiated. Except for T 273128, already showing generalized me&stases when diagnosed, the cases of group A have heen in good condition up till now. However, the cases are too few and too recent to allow any definite conclusions. In those cases where LDI-I values remain elevated, especially where the first fraction is concerned, strong reservation as to prognosis would seem justified. Ctin. Chim
A&z,
PX
(1970) 77-81
URINARY
STEROIDS AND TESTICULAR
TUMOURS
81
The tumours of group B are on the whole smaller in size, which might imply development in previously atrophic testis and/or an earlier stage of the clinical symptoms. In clear contrast to group A, the testicular parenchyma of the cases comprising group B shows an increase of Leydig cells. The persistent elevation of peak IIb in the urinary steroid spectrum after operation could therefore possibly be correlated with an elevation of LH in the patient, resulting in a sustained elevation of IT-ketosteroid production by the remaining testis and/or adrenals. The two early deaths in group B would seem to support the unfavourable prognosis of Leydig cell hyperplasia suggested in the literature4. This is quite conceivable in the case of HCGproducing chorial tumour elements; however, elevated gonadotrophin production of extra-tumoural origin also would seem to influence the prognosis. Further investigation of the material presented is in progress to trace possible distinct histological or enzyme-histochemical characteristics other than Leydig cell hyperplasia, which could determine whether a tumour belonged to group A or group B. So far, our enzyme-histochemical tindings (enzyme-histochemical determinations were performed by Dr. M. J. Hardonk of the Histochemical Division, Department of Pathology, IJniversity of Groningen) argue against the direct production of steroid hormones by tumour cells in cases of germinal testicular tumours. Up till now, 30 of these tumours have been investigated, 8 of which (indicated by EH in Table I) have been included in the material presented. Without exception, the enzymes directly involved in the production of steroid hormones (e.g., /3-hydroxysteroid dehydrogenase) were absent, or present in very low concentration only, in the tumour cells. Production of the steroid hormones involved must therefore be sought in the intact testicular interstitium, possibly the adrenals. The preliminary results are reported at an early stage, hoping to stimulate research in this field of the rather rare testicular tumours in other centers also. The techniques applied can make, in our opinion, a valuable contribution in the investigation of these tumours which, till now, have been hardly accessible due to their polymorphism and varied biological aspect. REFERENCES I z 3 4
W. HOEK AND E. J. VAN KAMPEN, C&z. Chim. Acta, rg (1968) 371. E. J. VAN KAMPEN AND W. HOEK, Clin. China. Acta, 16 (1967) 442. T. SYMIPUGTON AND N. WALLACE. Suppl. &it. J. Ural., 34 (1964) z, 103. F, J. DIXON AND R. A. MOORE, AFJP Atlas of Tumor Pathobgy (x952), CEin. Clrim.
pp.
32-59.
Acta, 28 (1970) 77-81
CLINICA CHIMICA ACTA
URINARY
GAS-CHROMATOGRAPHIC
TESTICULAR
STEROID
SPECTRA
AND
TUMOURS
E. L. FRENSDORF
Department
of Pathology, Onze Lieve Vrouwe Gasthuis, Amsterdam
E. J. VAN
KAMPEN
AND W.
Clinical Chemical Laboratory,
( Received
October
HOEK
Diakonessenhuis,
Groningen (The
Netherlands)
15. 1969)
SUMMARY
A first attempt is made to correlate the pathological and histochemical findings with the urinary excretion of differentiated r7-keto- and ketogenic steroids (steroid spectrum) in cases of testicular tumours, to support an early diagnosis. In this light, differentiation between seminoma and embryonal tumours is, naturally, important.
A simple method of obtaining essential information about urinary steroid excretion spectra has been presented recently’. At first, it was merely attempted to obtain recognition patterns in clinically suspected endocrine disorders, however, by refining the method through strict standardization it has since also become possible to obtain quantitative information as regards, e.g., the excretion of total r7-ketosteroids, r7-ketogenic steroids, etiocholanolone and pregnanedio12. In the course of a morphologic and histochemical enzyme investigation of the histogenic factors of germinal testicular tumours *, a preliminary study of possible hormonal influences was also undertaken using the above method. In cases of testicular tumours (seminoma 49% of cases, embryonal carcinoma 257o, choriocarcinoma 3%, teratocarcinoma 17%, teratoma 5% and interstitial cell tumours I%), little is known of hormonal relations. This has also been the provisional conclusion of the English Testicular Tumour Panel, summarized by Symington and Wallace3 as follows : “Regarding the gonadotrophins, there seems to be a relation with testicular tumours; possibly also with their prognosis. For ketosteroid hormones a similar relation could not be shown.” There are, however, a number of arguments for the involvement of hormones: I. The testis is an organ which is strongly hormone-dependent, as regards development and function and besides, it has an important endocrine function in its own right; 2. There are sufficient reasons to assume a relation between testicular atrophy and the genesis of some testicular tumours; l
E. L. Frensdorf;
Thesis, in preparation. Clin. Chim. Acta, 28 (1970) 77-81
FRENSDORF
78 TABLE
I
CLINICAL
CHEMICAL -
OF THE
TUMOURS
OBSERVED
Weight
Diagnosis
A.&F
Registration NV.
UATA
[years)
.~
et d.
Rest of testis
L
Metastases
Sparse. Complete spermatogenesis Very sparse. Total atrophy Atrophy. Rest of spermatogonia and -cytes. Some atypia No rest
l;lr
Dubious prelumbar Dubious retroperit.
Very sparse. Atrophy sporadically. Sertoli cells From total atrophy to sparse, complete spermatogenesis
f
Not demonstrable
rt
Dubious lumbar 2, 3
Sparse, mainly Sertoli cells. Some spermatogonia, sporadically -cytes From extensive atrophy to complete spermatogenesis
-f-+
Cerebrum ?
+ i-
b.a. Paraaortal
++
Not demonstrable
++
Not demonstrable
(g)
Normal values Group A T 265387 EH
24
Seminoma
375
T
253300
EM
32
Seminoma
290
T
255436
EM
26
Embryonal teratoma (MTIA)
200
T
273128
EH
3’
12.5
T
263617
EH
67
Embryonal (MTIB) Seminoma
T 246829 EH
30
Seminoma
100
EH
43
Seminoma
150
T 258453 EH
33
85
T 218301 EH
4o
Embryonal teratoma + seminoma. Also “chondroma” (MTIA) Seminoma (mamma virilis)
T 257628 EH
32
Embryonal (MTIB)
50
Ca.
“5
& &
Generalized
Group B T
252005
L:
ao: po: d: m: EH
:
number of Leydig cells before operation after operation days months Enzyme-histnchemistry also
Ca. MTIA MTIB i: + -+-: 1: t:
70
Hyaline atrophy. Sporadically retained full spermatogenesis Rest of full spermatogenesis
following classification of English testicular panel Malignant Teratoma Intermediate, group A or B no distinct aberration distinct increase living trace
:
3. Elevation of gonadotrophins could well play a part in the genesis of testicular tumours; 4. An obvious increase of Leydig cells in some testicular tumours could well argue for an elevation of lutinising hormone (LH), a hormone which may originate extra-testicularly as well as be produced by the tumour itself (human chorionic gonadotrophin, HCG) ; 5. Testicular tumours may be accompanied by an elevation of ketosteroid levels, which decrease after extirpation of the tumour. The purpose of this paper is to report the preliminary results of investigations of IO cases of testicular tumours. Unfortunately, most results have been obtained from material sent to the Pathology Department for diagnosis and urinary hormonal assays could only be performed some days, or even some months, after semi-castration. In only one case have assays been obtained both before and after operation. The tumours referred to, together with available clinical chemical data, have czin. Claim.A&,
28
(1970) 77-k
URINARY
AOIPO (days/months)
STEROIDS
17-K
3 d PO 3 d PO
29
IO
17-OH
TESTICULAR
O&r.
5 h
IOk7 13 f 5 mg/*4 h Icd24 16
h
dpo
12
AP
LDH
Fractions isoenzymes
o-3 u (Ressey)
TOO-300 u (King)
44-46-7-2%
640 =9o
.+43-8-r-t
815
56-39-4-1-t
-
4.3
4.3 I2
8
ZI
I2
0.1
co.1
-
2.3
*40 I55
45-45-8-1-1
1 14 yr.
500
65-32-3-t
4 yr. moribund
m PO 16
5 ~PO 4 m PO
I5
2 Yr PO
20
28
3 d PO
23
I9
0.1
-
1.6 3.6
-
Q6-6-2-t-O
140
18
if0
1330
Course
1 I yr.
IO-20 u
29
5m
5
HCG
d po
5 d PO
79
TUMOURS
PVtp
13 i W24 20 13 I9
;
AND
-
dead 7 m 460
170
3.4
1 3 yr. l7m
been tabulated according to size in Table I. Fig. I presents the normal urinary steroid excretion of the mature male using the procedure described previously19 solidline( with the results of this investigation drawn in dotted line(. . .) The successive peaks or areas in the normal chromatogram represent the following: I. breakdown products, e.g. monohydroandrosterone, of, among others, pregnanediol and dehydroisoandrosterone; IIa. etiocholanolone; IIb. androsterone, dehydroisoandrosterone and androstenedione; III. rr-oxy-r7-ketosteroids; IV. pregnanediol, pregnanetriol, etc. ; V. steroids of the @-cortol series; VI. cholesterol. Post-operatively two main types of spectrum of ketosteroids have been found; they have been called type I and type 2. Type I is characterized by elevation of etiocholanolone (peak IIa) and subnormal androsterone excretion (peak IIb). Type z is characterized by elevation of etiocholanolone (IIa), elevation of androsterone (III?) and an increase in zones I and IV. Patients with types I and 2 urinary steroid excretion patterns were classed as group A and B respectively in Table I. The pre-operative C&z. Chim. Acta, 28 (x970) 77-81
FRENSDORF et di.
Fig. I. Steroid metabolism am1 urinary steroitl in the case of seminoma (type I) and embryonal is given by solid lines; deviations by the dashed
spectra obtained by means of gas chromatography carcinoma (type 2). The normal chromatogram lines.
For I to VI. see text.
steroid spectra given are open to argument as they are based on a single observation, Although this small series is merely sufficient to put forward some suggestions, an attempt has been made to correlate the two types of urinary steroid spectra with the clinical and morphological manifestations. In group A, which comprised mainly large tumours, four of the six were seminoma. The two embryonal tumours in this group were poorly differentiated. Except for T 273128, already showing generalized me&stases when diagnosed, the cases of group A have heen in good condition up till now. However, the cases are too few and too recent to allow any definite conclusions. In those cases where LDI-I values remain elevated, especially where the first fraction is concerned, strong reservation as to prognosis would seem justified. Ctin. Chim
A&z,
PX
(1970) 77-81
URINARY
STEROIDS AND TESTICULAR
TUMOURS
81
The tumours of group B are on the whole smaller in size, which might imply development in previously atrophic testis and/or an earlier stage of the clinical symptoms. In clear contrast to group A, the testicular parenchyma of the cases comprising group B shows an increase of Leydig cells. The persistent elevation of peak IIb in the urinary steroid spectrum after operation could therefore possibly be correlated with an elevation of LH in the patient, resulting in a sustained elevation of IT-ketosteroid production by the remaining testis and/or adrenals. The two early deaths in group B would seem to support the unfavourable prognosis of Leydig cell hyperplasia suggested in the literature4. This is quite conceivable in the case of HCGproducing chorial tumour elements; however, elevated gonadotrophin production of extra-tumoural origin also would seem to influence the prognosis. Further investigation of the material presented is in progress to trace possible distinct histological or enzyme-histochemical characteristics other than Leydig cell hyperplasia, which could determine whether a tumour belonged to group A or group B. So far, our enzyme-histochemical tindings (enzyme-histochemical determinations were performed by Dr. M. J. Hardonk of the Histochemical Division, Department of Pathology, IJniversity of Groningen) argue against the direct production of steroid hormones by tumour cells in cases of germinal testicular tumours. Up till now, 30 of these tumours have been investigated, 8 of which (indicated by EH in Table I) have been included in the material presented. Without exception, the enzymes directly involved in the production of steroid hormones (e.g., /3-hydroxysteroid dehydrogenase) were absent, or present in very low concentration only, in the tumour cells. Production of the steroid hormones involved must therefore be sought in the intact testicular interstitium, possibly the adrenals. The preliminary results are reported at an early stage, hoping to stimulate research in this field of the rather rare testicular tumours in other centers also. The techniques applied can make, in our opinion, a valuable contribution in the investigation of these tumours which, till now, have been hardly accessible due to their polymorphism and varied biological aspect. REFERENCES I z 3 4
W. HOEK AND E. J. VAN KAMPEN, C&z. Chim. Acta, rg (1968) 371. E. J. VAN KAMPEN AND W. HOEK, Clin. China. Acta, 16 (1967) 442. T. SYMIPUGTON AND N. WALLACE. Suppl. &it. J. Ural., 34 (1964) z, 103. F, J. DIXON AND R. A. MOORE, AFJP Atlas of Tumor Pathobgy (x952), CEin. Clrim.
pp.
32-59.
Acta, 28 (1970) 77-81