Incidence of intratubular germ cell

neoplasia in androgen insensitivity syndrome

Alessandra Cassio, Emanuele Cacciari, Antonia D'Errico, Antonio Balsamo, Franco W. Maria G. Pascucci, Francesco Bacci, Moreno Tacconi and Antonio M. Mancini

Departments of Pediatrics1

and Pathology2,

Abstract. Gonadal

histology was investigated by means microscopy in 6 patients with complete androgen insensitivity syndrome, in 11 with incomplete androgen insensitivity syndrome, and in 3 with 5\g=a\-reductase syndrome. Twelve subjects were prepubertal and 8 pubertal. In all patients gonadal tissue was removed as a prophylactic measure and no patients gave rise to any clinical suspicion of a tumour. Eight patients with incomplete androgen insensitivity syndrome, 5 of whom (62.5%) were prepubertal, showed intratubular germ cell neoplasia and in 6 of them it was bilateral. Histochemical and immunohistochemical analysis showed considerable agreement between atypical morphological aspects and positive response to Schiff's periodic acid and to staining with the anti-placenta alkaline phosphatase antibody. Our patients were characterized by one of the highest reported incidences of intratubular germ cell neoplasia, particularly at prepubertal age. These findings would seem to indicate that a rethink is needed concerning the general opinion that patients with androgen intensivity syndrome have practically no risk of developing malignancy, and that orchidectomy is not advisable before puberty is completed. of conventional

It has long been recognized that patients with an¬ drogen insensitivity syndrome have a higher inci¬

dence of germ cell tumours (1,2) in adulthood. Recent studies (3-5) have pointed out that some subjects with this syndrome already have intratu¬ bular germ cell neoplasia patterns in childhood, and similar cellular changes in the gonads of infer¬ tile and cryptorchid men have been shown to be a true precursor of invasive tumours (6-11). In order to investigate the incidence of this con-

Grigioni,

University ofBologna, Bologna, Italy

prepubertal patients and to evaluate its diagnostic implications, we examined the gonadal histology of 20 children and adolescents with male dition in

pseudohermaphroditism. Patients and Methods We studied 20 subjects with male pseudohermaphrodit¬ ism, 12 prepubertal and 8 pubertal (mean chronological age 10.3±4.5 years; range 1.6-18.6). Six patients had complete androgen insensitivity syn¬ drome, 11 had an incomplete variant of the syndrome, and 3 had 5a-reductase deficiency (12). All subjects had a 46,XY karyotype. None of them showed any sign of adrenal insufficiency and none were receiving hormonal therapy at the time of investigation. In all patients gonadal tissue was removed as a pro¬ phylactic measure and no cases gave rise to any clinical suspicion of a tumour. Informed consent was obtained from parents in all cases. Table 1 shows the clinical features and age at gonadectomy of the subjects examined. Table 2 shows preoperative serum levels of testosterone (T) (Diagnostic Products Corporation, Los Angeles, CA), dihydrotestosterone (DHT) (13) (both basal and after stimulation with hCG (6000 U im)), A4-androstenedione (14), LH, and FSH (Biodata, Rome, Italy). Clinical and hormonal features led to the diagnosis of androgen insensitivity syn¬ drome in all patients. In particular, the diagnosis of in¬

complete androgen insensitivity syndrome was supported by the finding of serum T levels within the male range and by a significant increase in the serum values of T and DHT after stimulation with hCG. In some of the patients (Table 2, No. 12, 14-17), part of the hormonal data which

confirming

the

lems.

diagnosis

was

lost due

to

storage prob¬

During surgery, Mullerian structures were found in subject (Table 1, No. 3) (15,16) and Wolffian struc¬ tures were identified in the 3 subjects with 5a-reductase deficiency (Table 1, No. 18-20) (12,15). Both gonads were available from 19 of the patients, but only one in the remaining patient (Table 1, No. 3). After surgery, the gonads were divided along their lon¬ gitudinal axis. One to four samples were taken from each, immediately fixed in buffered formalin and subsequently included in paraffin. Consecutive, 4 pm thick sections one

obtained from each block and stained with hematoxillin-eosin for histological diagnosis. They were also stained with Schiffs periodic acid (PAS) in order to reveal any glycogen granules. Furthermore, the sections were studied by Stemberger's immunohistochemical method using the polyclonal antibody directed against placenta alkaline phosphatase (Dakopatts cod. A 268, diluted

The panel, complete with sections, was double-blind tested by two separate groups of pathologists who as¬ sessed the maturative tendency of the total number of seminiferous tubules and the presence of atypical ele¬ ments both on the basis of strictly morphological criteria

(nuclear dimensions, prominent nucleoli, pale cytoplasm, etc.), and of the histochemical and immunohistochemical

(positive responses to PAS and to placenta alkaline phosphatase). results

Results

were

1:100).

Pre-operative hormonal levels in the various pa¬ tients are reported in Table 2. Basal levels of T were consistently within the normal male range for the subjects' age, and increased significantly after stimulation with hCG. In the subjects with 5ct-reductase deficiency, DHT levels were lower than

Table 1.

Clinical features. Patient No. Patients with 1 2 3 4 5 6

Patients with

Age at gonadectomy years

External

genitalie

(Right

Position of gonads

Pubertal

Left)

stage

complete androgen insensitivity syndrome 1.6 2.4

11.1

12.1 12.1 13.7

Normal Normal Normal Normal Normal Normal

female female female female female female

Labium Labium Absent Abdomen Abdomen Abdomen

Prepubertal Prepubertal Prepubertal Prepubertal

Labium Labium

Prepubertal Prepubertal

Inguinal canal Inguinal canal Clitoromegaly Labium Labium Clitoromegaly-hypospadia Labium Clitoromegaly Inguinal canal Labium Labium Clitoromegaly Labium Labium Clitoromegaly

Prepubertal Prepubertal Prepubertal Prepubertal Prepubertal

incomplete androgen insensitivity syndrome 1.6 Clitoromegaly 5.8 Clitoromegaly-hypospadia

Labium Labium Labium Abdomen Abdomen Abdomen Labium Labium

Pubertal Pubertal

Partial fusion of the labia 9 10 11 12 13

9.3 9.8 10.5 10.6 11.2

14 15 16

13 13.2 14.1 18.6

17

Patients with 5a-reductase deficiency 18 8.3 19 11.1 20

16

Partial fusion of the labia Mild clitoromegaly

Inguinal canal Inguinal canal

Clitoromegaly clitoromegaly Clitoromegaly

Labium Labium

clitoromegaly Clitoromegaly

Labium Labium

Mild

Mild

Partial fusion of the labia Perineal hypospadia Partial fusion of the labia

Inguinal canal

Labium Labium Inguinal canal Labium Labium

Inguinal canal Inguinal canal

Pubertal Pubertal Pubertal Pubertal

Prepubertal Pubertal

Pubertal

Table 2. Hormonal features.

A -androDHT basai nmol/1

T basai nmol/1

Patients with

complete androgen insensitivity syndrome 3.8 3.1 1.1 0.5 0.3 1.6

Patients with

9 10 11 12 13 14 15 16 17

T/DHT basai

T after DHT after hCG nmol/1 hCG nmol/1

Patient No.

17.6 7.3 0.2 0.1 0.4

4.4 4.3 4.5 5.5

3.5 2.2 3.9

0.6 0.5 0.6 1 1.3 0.3 0.3

0.1 0.3 0.3 0.6 0.5

4.3 1.5 1.8 1.7 2.4

0.2

2.2

9 4.5 4.5 7.5 3.8 4.5 10.2

17.3

2.2

7.8

41.6

1.2 1.9 1.9 1.5 1.4

7.2 2.3 2.3 4.8 2.7

1.3

7.5

IU/1

FSH IU/1

0.9 1.3 0.9 2.1 2 2.9

1.1 0.3

3.3 0.1 1 1.5 1.3 0.4 4.8

2.1 1.4 7.5 7.3

2.8 1.6 10.5

5 4.5 0.5

0.7

0.3 0.4 11.4

8.7 8 5.6 2

6.5 46.1 124.2

18.4 30.8

1.1 1.2 4.8

stene-

dione nmol/1

1.2 1.6 2.1 0.4 0.6 1.1 0.6 0.9 1.4

2.7 3.6

deficiency 0.1 0.2 0.1

7.3

was a high T/DHT ratio after stimulation with hCG. LH and FSH levels were normal or slightly raised. The results of the histological findings are sum¬ marized in Table 3. The seminiferous tubules were immature in all prepubertal and pubertal patients with complete androgen insensitivity syndrome and with 5ct-reductase deficiency, whereas patients with incomplete androgen insensitivity syndrome,

particularly two pubertal subjects (Table 3,

No. 14

15) also showed tubules with maturative

ten¬

dency in the form of spermatocytic elements. Histological analysis revealed atypical germ cells only in the tubules of patients with incomplete an¬ drogen insensitivity syndrome. These cells (Fig. 1) were characterized by a broad pale cytoplasm with a round nucleus, prominent nucleoli and irregular clumping of chromatin. Mitotic figures were rarely from

3.2 3.1 4.1

51.4

25.2

normal, and there

seen.

1.3 1.4 1.3

LH

incomplete androgen insensitivity syndrome

Patients with 5a-reductase 18 0.9 7.3 19 20 16.6

and

T/DHT after hCG

The number and location of the cells differed one tubule to another and from patient to

0.2 0.3 0.2

46.1 65.1 140.2

1.1 4.3

patient. A diagnosis of intratubular germ cell neo¬ plasia was made on the recognition of numerous atypical cells arranged in contiguous or linear groups located along the tubular membrane, which in turn appeared thickened. Eight patients, 5 of whom (62.5%) were prepubertal, showed intratu¬ bular germ cell neoplasia, and in 6 of them it was

bilateral (Table 3, No. 10, 12-14, 16, 17). Between 5 and 30% of the seminiferous tubules revealed the no patients same morphological atypia, and showed any invasion of the stroma. As regards the relationship between the inci¬ dence of intratubular germ cell neoplasia and the position of the testes, 10/24 (41.5%) of the labial testes were affected, as were 4/9 (44%) of those located in the inguinal canal. Intratubular germ cell neoplasia was not observed in the abdominally lo¬ cated testes. However, concerning the relationship between the incidence of lesions and the maturative aspects

Table 3.

Histological findings Patient No.

Patients with 1

2 3

Intratubular germ cell

Placenta alkaline Germ cells

neoplasia

phosphatase positivity

complete androgen insensitivity syndrome Few spermatogonia Spermatogonia Few spermatogonia -

Few

spermatogonia

Few

spermatogonia

Few

spermatogonia

-

Patients with

incomplete androgen insensitivity syndrome Few spermatogonia Spermatogonia

9 10 11 12 13 14 15 16 17

Few spermatocytes Few spermatocytes Few spermatocytes

+* +

Spermatogonia

+*

++ ++ +

+*

Few spermatocytes

+++

+*

Spermatocytes Spermatocytes

++

+*

Few spermatocytes Few spermatocytes

+++

+*

Patients with 5a-reductase 18 19 20

Leydig

Immature Immature

Absent Absent Absent

cells

Mainly immature Also hamartomatous nodules Also hamartomatous nodules Also hamartomatous nodules

-

-

4

Sertoli cells

++

Hyperplastic Rare

Hyperplastic

Immature Immature

Absent

Normal Normal Normal Immature Normal Normal Normal Normal Normal

Absent

Rare Rare Rare

Absent Absent Rare Rare

Normal

Hyperplastic

deficiency

-

-

Few Few Few

spermatogonia spermatogonia spermatogonia

Normal

Absent Normal

Immature Normal

Hyperplastic

-

Bilateral lesion

of the seminiferous tubules, intratubular germ cell neoplasia was found in 2/12 (16.6%) of patients with immature tubules, and in 6/8 (75%) of patients with partially matured tubules. Histochemical and immunohistochemical analy¬ sis showed considerable agreement between atypi¬ cal morphological aspects and positive response to PAS and to staining with the anti-placenta alkaline

phosphatase antibody (Figs. 2, 3). The Sertoli cells showed a degree of maturation that was correlated to the development of the se¬ miniferous tubules: in particular, in patients with

complete androgen insensitivity syndrome they were mainly immature, at times arranged in ha¬ martomatous nodules, whereas in patients with in¬ complete androgen insensitivity syndrome they were more

often

mature

(Table 3).

Leydig cells were seen in a greater number in patients over 10 years old; Leydig cell hyperplasia was

sometimes also found

(Table 3).

Discussion Our results, from examination of a large number of patients appear to confirm recent observations regarding intratubular germ cell neoplasia in sub¬ jects with androgen insensitivity syndrome (3-5,

17).

A variety of terms have been employed in the literature to define the atypical aspects of germ cells (7,8,17,18). We agree with Gondos & Migliozzi (10) that the term "intratubular germ cell neopla¬ sia" more appropriately describes the evolutionary variability of these atypical cells. In fact, immuno-

Fig.

Fig.

1. Intratubular germ cell show positivity for PAS.

neoplasia. Dysplastic

elements

(PAS stain 25x).

histochemical (19,20) and ultrastructural (20,21) observations have indicated that the atypical cells have the totipotential characteristics of germ cells at early stages of differentiation. Furthermore, recent studies in subjects with invasive tumours (22) have confirmed Waxman's findings (7) that the latter are a common intratubular precursor of various histo¬ logie types of germ cell neoplasia. Our patients were characterized by one of the highest incidences of intratubular germ cell neo¬ plasia in the literature (10,15); particularly note¬ worthy was the prevalence of neoplasia at a prepu¬ bertal age. Previous studies (4,5,17) have described similar histological findings in prepubertal subjects with incomplete androgen insensitivity syndrome, one of them being only two months old (4). Scully observed an invasive germ cell tumour in a patient with incomplete androgen insensitivity syndrome aged 14 years (23). Atypical germ cells described by Müller et al. (9) in a cryptorchid prepubertal testi¬ cle evolved into an invasive tumour after 11 years,

2.

same patient as in Fig. 1. Dysplastic elements show a ring positive stain with antibody anti-placenta alkaline phosphatase. (PAP immunohistochemical technique 25x).

The

the precancerous potential of these cells. All these findings would seem to indicate that a rethink is needed concerning the general opinion that subjects with androgen insensitivity syndrome practically have no risk of developing malignancy, and therefore that orchidectomy is not advisable until after puberty (23). Like Skakkebaek and Müller (3,4), we believe that the high incidence of intratubular germ cell neoplasiajustifies orchidectomy and that in general this should be carried out upon diagnosis of incom¬

thereby confirming

plete androgen insensitivity syndrome, particularly in patients with a female phenotype. In our expe¬ rience, delaying gonadectomy until after puberty is of little use, and, indeed, may well create psycho¬ logical problems regarding correct sexual identifi¬

cation. As regards the histological aspects of the gonads we studied, these confirm previous findings in both adolescents (1) and adults (22). It is only in patients

neoplasia does not differ substantially between located in the inguinal canal and those lo¬ cated in the labia majora, the latter location being more "physiological" than the former. Accentuated malpositioning seems at times to be almost "pro¬ tective", favouring a greater loss of germ cells and cell

testes

hence of the substratum for cancerous transforma¬ tion. In accordance with Müller (5), we did not ob¬ serve intratubular germ cell neoplasia in gonads located in the abdomen. Overall, these findings lead us to consider gonad dysgenesia as the pri¬ mary etiological factor and abnormal positioning as a secondary factor or an accompanying cause.

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1973;265:321-33.

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Fig.

with incomplete androgen insensitivity syndrome, and not in those with complete androgen insensi¬ tivity syndrome, even though pubertal, that the se¬ miniferous tubules tend to mature. This appears to express a certain degree of sensitivity to androgens. Therefore, at least in our patients, the ability to mature seems to be accompanied by the tendency to develop intratubular germ cell neoplasia. The observation of similar aspects in cryptorchidism, infertility and incomplete androgen insensitivity

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a

common

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etiopathogenetic

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Received March 5th, 1990. Accepted May 23rd, 1990. Dr Emanuele Cacciari, Department of Pediatrics,

University of Bologna, Via Massarenti 11, 1-40138 Bologna,

Italy.