Immunohistochemical Study of Adrenocortical Carcinoma
Predictive Value of the D11 Monoclonal Antibody
Eric Tartour, M.D.,*+ Bernard Caillou, M.D.,+ Florence Tenenbaum, M.D.,* Sören Schröder, M.D.,§ Sylvia Luciani, Ph.D., || Monique Talbot, M.Sc.,} and Martin Schlumberger, M.D .*
Background. The diagnosis of adrenocortical carci- noma (ACC) may be difficult with conventional light mi- croscopy, especially when the tumor is nonfunctioning. Until now, no specific adrenocortical tumor marker was available. The current study was undertaken to investi- gate the interest of the D11 MoAb for the diagnosis and prognosis of ACC.
Methods. Eighteen adrenocortical carcinomas, 10 primary adrenomedullary tumors, 20 primary hepatocel- lular carcinomas, 50 primary renal cell carcinomas, 5 primary lung carcinomas, and 18 intraadrenal metas- tases were analyzed immunohistochemically with the D11 monoclonal antibody. ACC were also evaluated for the expression of other tumor markers, including neu- ron-specific enolase , chromogranin A, S-100, Leu-7, vi- mentin, KL1, AE1AE3, and epithelial membrane antigen. Relationships between clinical features and results of immunohistochemistry were also sought.
Results. Nuclear D11 staining appears to be highly specific for normal adrenocortical cells and related tu- mors. Nuclear D11 positivity was demonstrated in 44% of ACC and was restricted to well-differentiated tumors. No cytoplasmic or nuclear D11 staining was observed in adrenomedullary tumors. D11 reactivity confined to the cytoplasm was found in 5 of 18 adrenal metastases, in all 20 hepatocellular carcinomas tested, in 3 of 5 lung carci- nomas, and in 1 of 50 primary renal cell carcinomas. Pa- tients with nuclear D11 immunostaining were initially
seen with metastases less often and survived longer than those with no nuclear D11 immunostaining (P < 0.05).
Conclusions. Nuclear D11 immunoreactivity may help to differentiate ACC from intraadrenal metastases and adrenomedullary tumors. This also selects a group of ACC patients with a more favorable outcome. Cancer 1993; 72:3296-303.
Key words: D11 monoclonal antibody, adrenocortical car- cinoma, adrenal tumors, immunohistochemistry.
The incidence of adrenocortical carcinoma (ACC) is 0.5-2 cases per million people per year, accounting for 0.2% of cancer deaths in the United States.1 Its progno- sis remains poor, with a 5-year survival rate ranging between 20% and 30%.2-4 At initial examination, 40- 60% of patients have clinical endocrine manifestations, and up to 80% of ACC appear to be functioning tumors when complete hormonal evaluation is performed.5 A high proportion of patients (50-80%) have metastatic disease at initial examination.6,7 However, adrenal me- tastases occur in 8-10% of all neoplasms8 and are partic- ularly common in the course of melanoma and lung or breast cancers. Histologically, adrenocortical carci- nomas, especially when poorly differentiated, may be confused with renal cell carcinoma, small cell lung carci- noma, melanoma, or hepatocellular carcinoma. There- fore, in the absence of hormonal overproduction, con- fusion may arise concerning patients with involvement of the adrenal gland and other organs at initial examina- tion. Consequently, many authors have investigated the immunohistochemical profile of adrenocortical car- cinomas to differentiate them from other tumors, but no specific pattern has been observed.9,10 Recently, a new monoclonal antibody (MoAb), D11, was described that recognizes an antigen present in normal adrenocor- tical cells and related tumors with a highly specific nu-
From the Departments of *Nuclear Medicine and Pathology, Institut Gustave Roussy, Villejuif, France; the §Institut für Patholo- gie, Universität Hamburg, Hamburg, Germany; and the | Department of Biostatistics, Institut Curie, Paris, France.
t Current address: Laboratoire d’Immunologie Clinique, Institut Curie, Paris, France.
The authors thank V. Mosseri for assisting with statistical analy- sis and critically reading the manuscript.
Address for reprints: Martin Schlumberger, M.D., Department of Nuclear Medicine, Institut Gustave Roussy, Rue Camille Desmou- lins, 94805 Villejuif Cedex, France.
Accepted for publication July 16, 1993.
| Patient no. | Age (yr) | Sex | Hormonal production | Tumor size (cm)* | Tumor differentiation | Surgery for primary tumor | Metastases site | Time of discovery of distant metastasest | Outcome (mo after diagnosis) | Nuclear D11 immunoreactivity |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 12 | M | A | 20 | PD | Partial | Liver/lung | (0) | Dead (11) | |
| 2 | 19 | F | GC | 20 | D | ND | Liver/lung | (0) | Dead (16) | - |
| 3 | 39 | M | A | 8 | D | Complete | Liver/lung | 47 | Alive (81) | + |
| 4 | 39 | M | GC + A | 10 | D | Complete | Liver/lung | (0) | Died (14) | |
| 5 | 54 | F | GC | 3 | D | Complete | Absent | Alive (24) | + | |
| 6 | 15 | F | GC | 15 | D | Complete | Liver/lung | 21 | Dead (41) | + |
| 7 | 31 | F | GC | 10 | D | Partial | Liver | 6 | Dead (14) | |
| 8 | 6 | M | A | NR | PD | Complete | Liver/lung | (0) | Dead (16) | |
| 9 | 35 | M | E | 22 | D | Partial | Liver | 8 | Alive (22) | |
| 10 | 17 | M | GC | 13 | PD | ND | Liver/lung | (0) | Dead (7) | |
| 11 | 60 | F | GC | 7 | D | Partial | Liver/bone, brain | 5 | Dead (20) | + |
| 12 | 67 | M | 0 | 6 | D | Complete | Absent | Alive (38) | + | |
| 13 | 38 | F | 0 | 13 | PD | Complete | Absent | Alive (72) | ||
| 14 | 66 | M | 0 | 8 | D | Partial | Absent | Alive (64) | + | |
| 15 | 62 | M | 0 | 4 | D | Complete | Absent | Alive (14) | + | |
| 16 | 41 | M | 0 | 8 | PD | Complete | Absent | - | Alive (10) | |
| 17 | 31 | F | 0 | 20 | D | Partial | Lung | 40 | Alive (96) | + |
| 18 | 42 | F | 0 | NR | D | Complete | Lung/bone | 18 | Alive (27) |
A: androgens; GC: glucocorticoids; E: estrogens; 0: no clinical or biochemical features of hormonal overproduction; ND: not done; NR: not recorded; PD: poorly differentiated; D: differentiated; (0): metastases at diagnosis.
* Greatest dimension.
t Time in months from diagnosis.
clear positivity pattern.11,12 In the current study, the im- munohistochemical features of D11 MoAb in adreno- cortical carcinoma were defined and compared with those of primary adrenal medullary tumors, intra- adrenal metastases, and primary extraadrenal neo- plasms. The prognostic value of the D11 MoAb in ACC was also investigated.
Material and Methods
Patients
Tumor tissues from 18 patients with adrenocortical car- cinoma were available for retrospective immunohisto- chemical study. All patients were treated at the Institut Gustave Roussy, Villejuif, France, from 1984 to 1992. They consisted of 10 males and 8 females, age 6-67 years at initial examination (see Table 1).
Data were collected from medical records regarding symptoms, hormonal evaluation, response to treat- ment, and mean survival. Functioning tumors were those associated with hormone overproduction. Treat- ment included partial or radical surgery followed by treatment with mitotane. Chemotherapy was given in patients with disease progression.13 Survival was con- sidered from the time of diagnosis to death or last fol- low-up.
Histology
Primary ACC were considered to be malignant on the basis of metastases, direct invasion of contiguous struc- tures, or tumor size, (> 5 cm) and histologic findings, including mitotic rate, atypical mitoses, nuclear grade, necrosis, and capsular or vascular invasion.14
Mitotic rate was evaluated in 17 ACC by counting 50 random high-power fields in the area of the greatest numbers of mitotic figures on the five slides with the greatest numbers of mitoses. The counts were per- formed on an American Optical (Leica, Germany) mi- croscope using a 45 times objective with a 0.47-mm diameter field.
ACC with less than or equal to five mitoses per 50 high-power fields are designated as low mitotic rate, and those with more than five are designated as high mitotic rate.14
A tumor was considered to be poorly differentiated when the normal adrenal architecture was lost and when 50% or more of the cells were pleomorphic, not resembling normal adrenal cortical cells.15 The adreno- cortical origin of poorly differentiated tumors was es- tablished in the presence of hormonal overproduction (Patients 1, 8, and 10) or of a single adrenal tumor with- out any other evidence of disease after a follow-up pe- riod of 10-72 months (Patients 13 and 16).
| Antibody (clone) | Type | Dilution | Recognized antigen | Source |
|---|---|---|---|---|
| D11 (14D11) | MC | 1/5 | ? | Dr. Schröder11 |
| NSE (H14) | MC | 1/75 | 146 kDa | Dako (Santa Barbara, CA) |
| Chromogranin A (LK 2 H10) | MC | 1/100 | A 60 kDa | Biogenex (Les Ullis, France) |
| KL 1 | MC | 1/500 | 55-57 kDa | Immunotech (Marseille, France) |
| Vimentin (V9) | MC | 1/10 | Vimentin | Dako |
| EMA (E29) | MC | 1/10 | EMA | Dako |
| AE1AE3 | MC | 1/50 | 30, 56.5, 58, 65-67 kDa | Boehringer Mannhein (Indianapolis, IN) |
| Leu-7 (HNK1) | MC | 1/50 | 180 kDa (CD 56) | Becton-Dickinson (Mountain View, CA) |
| S-100 | PC | 1/100 | Cow S-100 A-B | Dako |
NSE: neuron-specific enolase; EMA: epithelial membrane antigen; MC: monoclonal; PC: polyclonal.
Three normal adrenal glands obtained at nephrec- tomy for renal carcinoma were included as controls.
Tissue specimens from 10 primary adrenomedul- lary tumors were also studied: 8 pheochromocytomas with catecholamine overproduction, 1 neuroepithe- lioma, and 1 ganglioneuroma.
Seventy-five primary cancers, including 20 hepato- cellular carcinomas, 50 renal cell carcinomas, and 5 lung tumors (2 squamous cell lung carcinomas, 1 lung adenocarcinoma, 1 large cell poorly differentiated lung carcinoma, and 1 atypical carcinoid) as well as 18 in- traadrenal metastases were studied: 3 from renal cell carcinomas, 2 from melanomas, 3 from small cell lung carcinomas, 3 from large cell poorly differentiated lung carcinomas, 1 from squamous cell lung carcinoma, 1 from lung adenocarcinoma, 1 from cervix adenocarci- noma, and 4 from adenocarcinomas with typical glan- dular differentiation from an unknown primary site.
Immunohistochemistry
Tissue specimens were fixed previously in either forma- lin, Bouin solution, or formacetic alcohol and were par- affin embedded. After deparaffinization and rehydra- tion, the sections were progressively incubated at room temperature for 15 minutes, with methanol containing 3% hydrogen peroxide to inhibit endogenous peroxi- dase. The staining was then performed using the univer- sal rabbit/mouse-labeled streptavidin-biotin method (Dako LSAB Kit, Santa Barbara, CA). The source and working dilution of the primary antibodies are listed in Table 2. D11 MoAb was used as described elsewhere.11 Between each step, sections were washed with phos- phate buffered saline solution. Finally, slides were counterstained with Harris hematoxylin, dehydrated, given a coverslip, and examined by conventional light microscopy.
For each section, a negative control was performed by replacing the primary antibody with phosphate buff-
ered saline solution. Positive controls were sections of tissues and neoplasms known to contain the antigens studied.
Statistical Methods
The chi-square test, using the Yates correction or the Fisher exact test when necessary, was used to compare patient characteristics in the nuclear D11-positive and D11-negative groups. A value of P < 0.05 was consid- ered to be significant.
Survival rates were calculated according to the Kaplan-Meier method. Survival curves were compared using the log-rank test.
Results
D11 MoAb Immunostaining in Normal Adrenal Tissue and Effects of the Fixation Procedure
D11 immunostaining was analyzed in normal adreno- cortical tissue previously fixed in either formalin, for- macetic alcohol, or Bouin solution and paraffin embed- ded. A strong nuclear positivity was demonstrated in the three adrenal cortical zones, regardless of the fixing solution. This positivity was seen in all normal adreno- cortical cells. A gradient in cytoplasmic immunostain- ing was observed from the zona glomerulosa to the zona reticularis, ranging from slight granular cytoplas- mic positivity in the zona glomerulosa to diffuse cyto- plasmic staining in the zona reticularis. Conversely, no positivity with the D11 MoAb was found in the normal adrenal medulla (Fig. 1).
D11 MoAb Immunostaining and Pathologic Features of ACC
A nuclear D11 positivity was demonstrated in 8 of 18 ACC (44%) (Fig. 2 and Table 3). This positivity was focal in two cases, concerning less than 20% of cells, and diffuse in the other six cases. This nuclear positivity
was associated with varying degrees of cytoplasmic staining. A cytoplasmic immunostaining without nu- clear staining was observed in four other cases of ACC (22%) (Table 4).
D11 nuclear immunostaining was more frequently observed in well-differentiated ACC (8 of 13) than in poorly differentiated ACC (0 of 5) (P < 0.05). D11 nu- clear staining was more frequently observed in ACC with low mitotic rate, but this association was not sta- tistically significant (Table 3). As it might be expected, a low-mitotic-rate ACC was more frequently observed in well-differentiated ACC (8 of 12) than in poorly differ- entiated ACC (2 of 5).
Nuclear immunostaining was seen in both func- tioning (4 of 11) and nonfunctioning ACC (4/7). The 2
small tumors (< 6 cm) and only 6 of the 14 large tumors (> 6 cm) exhibited nuclear D11 positivity.
Nuclear D11 immunostaining in ACC did not seem to depend on fixation procedures, because it was found in four of the nine formacetic alcohol-fixed ACC, in three of seven Bouin solution fixed tissues, and in one of two formalin-fixed specimens.
Predictive Value of D11 MoAb Staining
When compared with patients with ACC without nu- clear immunostaining, those with nuclear D11 reactiv- ity had a significantly longer survival time and a lower incidence of distant metastases both at initial examina- tion and during follow-up (P < 0.05) (Table 3). Factors that may affect survival, such as sex, age, hormonal production, and complete resectability of the adrenal tumor3,16-19 did not differ significantly between the nu- clear D11-positive and D11-negative groups. Con- versely, differentiation and metastases at presentation or during follow-up are features related to nuclear D11 staining (Table 3).
Prolonged survival time was also observed in pa- tients with nonfunctioning tumors and in those without metastases at initial examination (Table 3). Survival time of patients with well-differentiated and low mi- totic rate ACC was longer than that of patients with poorly differentiated and high mitotic rate ACC, but this difference was not statistically significant. A multi- variate analysis was not feasible because of the small number of cases.
D11 MoAb Specificity Study
No nuclear or cytoplasmic D11 immunostaining was observed in eight functioning pheochromocytomas and
| Nuclear D11 positivity | P | % of patients with metastases 2 yr after diagnosis | P | Survival rate 2 years after diagnosis (%) | P | ||
|---|---|---|---|---|---|---|---|
| 1 | % | ||||||
| Nuclear D11 staining in ACC | |||||||
| Yes | 8/18 | 44 | 16.7 | 85.7 | |||
| No | 10/18 | 56 | -- | 85 | < 0.03 | 33.7 | < 0.05 |
| Sex | |||||||
| Male | 4/10 | 40 | 50 | 56.2 | |||
| Female | 4/8 | 50 | NS | 47 | NS | 62.5 | NS |
| Age at diagnosis (yr) | |||||||
| > 40 | 5/7 | 71 | 25 | 80 | |||
| < 40 | 3/11 | 27 | NS | 72.7 | < 0.03 | 45.4 | NS |
| Differentiation | |||||||
| Well | 8/13 | 61 | 52.4 | 67.7 | |||
| Poor | 0/5 | 0 | < 0.04 | 60 | NS | 26.7 | 0.08 |
| Mitotic rate (per 50 hpf) | |||||||
| 0-5 | 6/10 | 60 | 40 | 72 | |||
| > 5 | 2/7 | 28 | NS | 71 | NS | 57 | NS |
| Hormonal production | |||||||
| Yes | 4/11 | 36 | 80 | 36.4 | |||
| No | 4/7 | 57 | NS | 20 | < 0.01 | 100 | < 0.01 |
| Metastases at presentation | |||||||
| Yes | 0/5 | 0 | < 0.04 | 100 | < 0.01 | 0 | < 0.01 |
| No | 8/13 | 61 | 38 | 82.5 | |||
| Resection of primary tumor | |||||||
| Partial | 3/6 | 50 | 60 | 50 | |||
| Complete | 5/9 | 55 | NS | 40.7 | NS | 87.5 | NS |
| NS: not significant; hpf: high-power fields. | |||||||
in two other primary neural adrenal tumors (one neu- roepithelioma and one ganglioneuroma). In these cases, a positive internal control was represented by immuno- staining in normal adrenal cortical cells outside the tu- mor limits.
No nuclear D11 immunostaining was observed in the 18 adrenal metastases. However a significant focal cytoplasmic immunostaining was observed in five of these cases: three from lung carcinoma (one small cell
lung carcinoma, one lung adenocarcinoma, one large cell poorly differentiated lung carcinoma) and two from adenocarcinoma of an unknown primary site.
We conducted a general survey of D11 staining on primary neoplasms: All 20 hepatocellular carcinomas exhibited a cytoplasmic positivity without nuclear staining; only 1 of the 50 renal cell carcinomas showed a cytoplasmic D11 staining; and 3 of the 5 lung carci- nomas had a significant focal cytoplasmic immunostain-
| ACC features | Isolated cytoplasmic D11 positivity | NSE | Vimentin | KL1 | AE1AE3 | EMA | Chromogranin A | Leu-7 | S-100 |
|---|---|---|---|---|---|---|---|---|---|
| Well differentiated | 2/13 | 5/13 | 3/10 | 2/11 | 3/8 | 2/8 | 0/12 | 0/6 | 0/6 |
| Poorly differentiated | 2/5 | 0/4 | 0/3 | 3/4 | 1/3 | 0/4 | 0/4 | 0/2 | 0/2 |
| Functioning | 3/11 | 4/10 | 3/7 | 4/9 | 3/7 | 2/8 | 0/10 | 0/6 | 0/6 |
| Nonfunctioning | 1/7 | 1/7 | 0/6 | 1/6 | 1/4 | 0/4 | 0/6 | 0/2 | 0/2 |
| Metastasizing | 3/12 | 5/11 | 3/7 | 4/9 | 3/7 | 2/8 | 0/10 | 0/5 | 0/5 |
| Nonmetastasizing | 1/6 | 0/6 | 0/6 | 1/6 | 1/4 | 0/4 | 0/6 | 0/3 | 0/3 |
| Nuclear D11 negativity | 4/10 | 1/9 | 2/7 | 4/8 | 2/7 | 1/8 | 0/9 | 0/5 | 0/4 |
| Nuclear D11 postivity | 0/8 | 4/8 | 1/6 | 1/7 | 2/4 | 1/4 | 0/7 | 0/3 | 0/4 |
ACC: adrenocortical carcinoma; NSE: neuron-specific enolase; EMA: epithelial membrane antigen.
ing without any nuclear immunostaining (2 of 2 small cell lung carcinomas and 1 of 1 atypical carcinoid). One large cell poorly differentiated lung carcinoma and one lung adenocarcinoma did not show any D11 immuno- staining. Therefore, a D11 nuclear immunostaining was never observed either in intraadrenal metastases or in the primary neoplasms studied.
Immunohistochemical Profile of ACC
Immunohistochemistry revealed positive staining for neuron-specific enolase (NSE) in 29% of ACC, more often in metastasizing and well-differentiated adrenal carcinomas. Nuclear D11 immunostaining was found in 50% of ACC with NSE staining (Table 4).
Vimentin was detected in 23% of functioning ACC and metastatic tumors. Coexpression of vimentin and nuclear D11 antigen was present in one case. Cytokera- tins KL1 and AE1AE3 were expressed in 33% and 36% of ACC, respectively. Focal positivity with epithelial membrane antigen antibody was observed in 2 of 12 ACC that were well-differentiated and functioning tu- mors. Chromogranin A, Leu-7, and S-100 were nega- tive in all ACC studied.
Discussion
The data from the current study confirm and extend results from previous studies regarding the interest of D11 MoAb in adrenocortical carcinoma.11,12
Nuclear D11 staining is specifically related to adre- nal cortical tissue. It was not observed in normal adre- nal medulla, pheochromocytoma, intraadrenal metas- tases, and primary neoplasms of the liver, kidney, and lung. In a previous report, no nuclear staining was ob- served in 60 thyroid carcinomas.11 This nuclear staining was seen in all specimens of normal adrenal cortex, with an intensity gradient of cytoplasmic staining from the zona glomerulosa to the zona reticularis, as already described.11
In the current study, nuclear staining was seen in 44% of ACC and was restricted to well-differentiated adrenocortical carcinoma. In a previous study, nuclear positivity was demonstrated in all adrenocortical carci- nomas.11 This discrepancy may be related to the method of fixation or to the selection of patients. In fact, formalin-fixed paraffin-embedded tissue sections were used only in the former report, whereas in the current study, tissues were fixed in various solutions, including formalin, formacetic alcohol and Bouin solution. Never- theless, a significant nuclear positivity was found in all samples of normal adrenal cortex, regardless of the fix- ing solution. Furthermore, in adrenocortical carcinoma, no relationship between D11 immunostaining and fixa-
tion procedures was observed. This does not preclude that a weak positivity is lost with some fixing condi- tions. ACC characteristics may also differ between the two series. Adrenocortical carcinomas exhibited a more favorable behavior in Schröder’s series12 than in several other reports;2,4,6,15-19 in the current series, nuclear D11 staining was restricted to well-differentiated ACC and was associated with a better prognosis. This may ac- count for a higher D11 sensitivity in the German series and may explain this apparent discrepancy.
D11 staining confined to the cytoplasm was ob- served in 40% of ACC without nuclear staining but was also observed in some adrenal metastases from lung carcinomas or adenocarcinomas, in all primary hepato- cellular carcinomas tested, in some lung tumors, and very rarely in primary kidney carcinomas. This isolated cytoplasmic D11 staining was also demonstrated in normal hepatocytes, in various organs, and in one papil- lary thyroid cancer.11 D11 MoAb was produced using human liver membrane proteins. It recognizes an anti- genic determinant shared by different cytoplasmic 59 kDa proteins from various organs.2º One of these pro- teins represents an apoE binding protein and another one is a protein disulfide-isomerase.11 D11 adrenal cor- tex-specific nuclear recognition is due either to a cross- reaction between a specific nuclear adrenocortical pro- tein and several widely distributed 59 kDa cytoplasmic proteins or to an identity between a 59 kDa cytoplasmic protein and the nuclear adrenocortical protein, which for unknown reasons is present in the nuclei of only adrenocortical cells.
Nuclear D11 immunostaining was found in 61% of well-differentiated ACC, confirming a phenotypic het- erogeneity of these tumors also demonstrated by other tumor markers (Table 4). It represents an important diagnostic tool in patients with nonfunctioning tumors. Presence of neuroendocrine markers, such as NSE,11,21 synaptophysin,21,22 and S-10023 in ACC has already been emphasized and may be misleading. In fact, we found NSE immunostaining in 30% of ACC. Moreover, in a recent study, clusters of dense core granules were demonstrated by electron microscopy in four ACC and were consistent with neuroendocrine granules.21 This neuroendocrine differentiation in ACC may lead to confusion with pheochromocytoma, especially in poorly differentiated and nonfunctioning tumors. Nu- clear D11 immunostaining, which was present in 50% of these NSE-positive ACC, is a strong argument for the adrenocortical origin of the tumor and allows the exclu- sion of a medullary origin. Immunostaining with chro- mogranin A and Leu-7 was negative in all ACC, and this may also help to differentiate cortical from medul- lary adrenal tumors.12,23 Vimentin and cytokeratin posi- tivity in the current study was in accordance with pre-
vious reports.10,24 Unlike nuclear D11 antigen, vimentin was not present in normal adrenocortical cells. Its posi- tivity was observed in metastatic tumors only, in accor- dance with the fact that this marker is associated in some tumors with neoplastic transformation and loss of differentiation.23 Finally, D11 MoAb did not recognize renal adenocarcinoma and may be a clue to differen- tiating renal cell carcinoma from ACC. The focal epithe- lial membrane antigen positivity seen in two cases is surprising, because most studies did not find epithelial membrane antigen in adrenal cortex and in adrenocorti- cal tumors,9,10,25 although the adrenal cortex is concep- tualized as an epithelial structure.
In the current study, the survival of patients with well-differentiated ACC and low mitotic rate ACC was longer than that of patients with poorly differentiated ACC and high mitotic rate ACC, but this difference was less marked than the one between the nuclear-positive and nuclear-negative ACC groups. Moreover, in pa- tients with nuclear D11 positive staining, metastases developed less frequently. Therefore, this antibody may help to select among patients with well-differen- tiated ACC those with a more favorable prognosis. In some series, patients with differentiated ACC had a higher survival rate than those with poorly differen- tiated ACC.15,26 One can therefore argue that D11 nu- clear immunostaining reflects only a high degree of dif- ferentiation. Indeed, nuclear staining was observed in all samples of normal adrenal cortex and in all adreno- cortical adenomas of the German series11,12 and was lost in poorly differentiated ACC. Only a multivariate analy- sis including microscopic findings and D11 staining will assess relationships between morphologic parameters and presence of the nuclear structure recognized with this antibody. However, even if a low mitotic rate, a well-differentiated histologic type, and nuclear D11 staining are often associated, there is no complete over- lap between these three favorable prognostic factors. Moreover, in the current series, D11 nuclear staining provided the strongest statistical association with pa- tient outcome. Of course, further studies are needed to confirm this preliminary result.
Other factors such as sex,16 age older than 40 years,2 presence of metastases,2,3 tumor weight27 or size,17 functioning features,18 mitotic rate,7,14,28 DNA content of the neoplastic cells,29,30 and complete resect- ability of the primary tumor19 were also reported to be related to the outcome. In the current study, only non- functioning tumors and tumors without metastases at presentation were associated with a better outcome. In fact, nonfunctioning tumors, especially when poorly differentiated, may be confused with intraadrenal me- tastases, and this may explain controversies arising
concerning the prognostic value of hormonal produc- tion.2,6,18
Conclusion
A nuclear pattern staining of the D11 MoAb seems to be highly specific for adrenocortical cortex and related tu- mors. This D11 pattern represents the first ACC-spe- cific marker. Among ACC, its positivity was restricted to a well-differentiated group and was associated with a better outcome. This differentiation marker may be an important tool for ACC, because, for example, response to mitotane or chemotherapy may be dependent on tu- mor differentiation grade.26
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