Adrenocortical Tumors With Myxoid Features: A Distinct Morphologic and Phenotypical Variant Exhibiting Malignant Behavior
Mauro Papotti, MD,* Marco Volante, MD,* Eleonora Duregon, MS,* Luisa Delsedime, MD,t Massimo Terzolo, MD,* Alfredo Berruti, MD,* and Juan Rosai, MD#§
Abstract: Myxoid changes have been rarely reported both in ad- renocortical adenomas and carcinomas. The recent observation by our group of an adrenal myxoid tumor with morphologically borderline features, but aggressive clinical behavior prompted us to review a series of 196 adrenocortical lesions, comprising 122 carcinomas and 74 adenomas, to define the morphologic, pheno- typical and clinical characteristics of adrenocortical tumors with myxoid features. Fourteen cases, including 12 carcinomas and 2 borderline tumors, formed the basis of this report, and were characterized by a variably abundant myxoid component (from 5% to 90% of tumor) and 2 distinct cellular growth patterns: the first (10 cases), mostly associated with a predominant myxoid stromal component, was made of small cells with mild atypia arranged in cords and microcysts; the second (4 cases) was characterized by focal myxoid changes in tumors otherwise similar to conventional adrenocortical carcinoma, with large atypical cells having an eosinophilic cytoplasm and a diffuse or nodular architecture. The above mentioned patterns were absent in all adenomas reviewed. A peculiar reactivity to neurofilaments was seen, mostly associated to the presence of predominant rather that focal myxoid stromal changes, and in 40% of con- ventional adrenocortical carcinomas, thus representing an un- described potential pitfall in the differential diagnosis of adrenal lesions. Myxoid adrenocortical tumors probably represent a rare but histologically and phenotipically distinct entity and, although rare cases of benign lesions are on record, they seem to be generally associated to morphologic and clinical features of malignancy.
Key Words: adrenal cortex, carcinoma, myxoid, neurofilaments, chemotherapy, mitotane
(Am J Surg Pathol 2010;34:973-983)
From the *Department of Clinical & Biological Sciences, University of Turin at St Luigi Hospital, Orbassano; ¡ Division of Pathology, St Giovanni Hospital, Torino; ¿ Genzyme Genetics, New York; and §Centro Diagnostico Italiano, Milan, Italy.
Grants: Work partially supported by grants from the Ministry of University, Rome (ex-60% and PRIN to MV, MT and AB) and from the Regione Piemonte (Progetto Ricerca Sanitaria Finalizzata, D.G.R. n. 35-4231, 06.11.2006 to MV).
Correspondence: Mauro Papotti, MD, Department of Clinical & Biological Sciences, University of Turin at St Luigi Hospital, Regione Gonzole 10, I-10043 Orbassano, Torino, Italy (e-mail: mauro.papotti@unito.it).
Copyright @ 2010 by Lippincott Williams & Wilkins
V arying degrees of myxoid change have been reported in adrenocortical tumors (ACTs), either as focal cell aggregates in an alcianophilic material of an otherwise conventional adrenocortical adenoma (ACA) or carci- noma (ACC), or-much more rarely-as pure myxoid tumors made of small-sized cell cords or pseudoglands embedded in an abundant extracellular myxoid matrix. After the original report by Tang et al,26 32 functioning or nonfunctioning myxoid ACTs have been described, including 18 adenomas, 13 carcinomas, and 1 borderline case.3,4,6,7,9,10,13-17,19,23,25,30 Myxoid changes may also be present in pediatric ACT, judging from the images (Fig. 1D) shown in a recent review article.5
Although the overall immunoprofile of these tumors does not differ from that of conventional ACT (melan A, a-inhibin, synaptophysin, and vimentin being generally expressed), the morphology of myxoid ACT is challeng- ing, as several other tumor entities may enter in the differ- ential diagnosis, and the application of the so-called Weiss criteria28,29 may not be easy to apply to pure myxoid tumors because of the limited degree of atypia, lack of diffuse growth, and hard-to-assess sinusoidal invasion. To date, the treatment of these neoplasms is not different from that of conventional ACA and ACC, but the real nature of these myxoid variants is far from being under- stood. Indeed, it is not well established whether the myxoid changes represent a variant of either ACA or ACC, or if the myxoid changes by themselves point to a specific tumor type with a distinct biologic behavior within the group of primary ACT.
A few years ago, some of us (MP, MT) reported a myxoid ACT lacking morphologic signs of malignancy (Weiss score 1), which was interpreted as a myxoid ACA.3 Four years later, the patient developed a diffuse abdomi- nal neoplastic spread, which could not be resected. A fine needle aspiration biopsy of the mass showed a diffuse growth of small-medium size cells with hyperchromatic nuclei and no myxoid background. Originally, this tumor was tentatively interpreted as a primary abdominal small round cell tumor,11 based on the focal expression of CD99 and neurofilaments. However, the finding of adrenocor- tical marker expression (including melan A and a-inhibin) led to a revised diagnosis of metastatic ACT.
This prompted a review of all ACT filed at the San Luigi Hospital and University of Turin Medical School,
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which serves as a national referral center for ACC, with the aims of: (a) identifying other pure or combined myxoid ACT; (b) evaluating the immunoprofile of these neoplasms, with special reference to the expression of neural markers, in comparison with the expression pro- files of normal adult and fetal adrenal glands; (c) analyzing the clinical and pathologic features of myxoid ACT, as compared with conventional ACA and ACC.
MATERIALS AND METHODS
Sample Case (Case No. 1)
In 2002, a 58-year-old male patient underwent re- section of a well circumscribed right adrenal mass (55 g; 5 cm). Microscopically, a myxoid tumor with trabecular, microcystic, and pseudoglandular growth patterns was found having a low Ki-67 proliferation index and an immune profile consistent with an adrenocortical origin. On the basis of the tumor size and the final Weiss score28,29 of 1 (clear cell cytoplasm in less than 25% of tumor), a diagnosis of ACT of uncertain malignant potential with myxoid changes was made, and no additional treatment was administered. Two years later, this case was reported as an example of myxoid ACA.3 The patient was temporarily lost to follow-up, but on December 2007 he reappeared complaining of abdominal pain. Imaging procedures revealed a large abdominal tumor partially engulfing the small intestine, with diffuse peritoneal carcinomatosis. A fine needle aspiration biopsy revealed a small round cell tumor devoid of myxoid material. The limited immunocytochemical panel carried out at the time on the cell block was as: pancytokeratin negative, CD99, and neurofilaments positive (initially thought to support a diagnosis of intraabdominal small round cell tumor), melan A, and a-inhibin positive. The clinical and patho- logic findings were thought to be consistent with abdomi- nal spread of the myxoid ACT and the patient was treated with mitotane, but eventually died of his tumor in 2008, 68 months after the original diagnosis.
Myxoid ACT Series (Case Nos. 2 to 14)
From 1990 to 2009, 122 consecutive ACC were found in the pathology files of the University of Turin at San Luigi hospital, for which either glass slides or paraffin blocks were available for review. This series included 35 ACC resected at our hospital, 63 cases received in consul- tation from different Italian hospitals, and 24 ACC originally collected by 1 of us (MP) at the St Giovanni Hospital of Turin in years 1993 to 2003. The majority of these cases were treated at our Institution, which serves as a referral center for adrenocortical carcinoma in Italy. The group of ACA included 74 consecutive tumors diag- nosed at our Institution during the same time period. Twenty of them were characterized by the presence of either large size ( > 5cm) or a Weiss score of 1 or 2 (ie, still in the benign range). The histopathologic features of 92 ACC and of 47 ACA belonging to this data set have recently been reported.27
Case Review
All cases of ACC and ACA were reviewed by 2 of us (MP and MV) using a mean of 5.2 hematoxylin- eosin-stained slides per case (range 1 to 30) for the carcinomas and a mean of 3.2 slides per case for the adenomas (range 1 to 8). Before review, all tissue blocks were deidentified and coded by a pathology staff member not involved in the study, so that all cases were made anonymous to the investigators. In the group of ACA, no myxoid changes were identified, except for 1 case (case no. 2) represented by a 4-cm nonfunctioning tumor with a Weiss score of 1, which was diagnosed as a borderline ACT. In the group of ACCs, myxoid changes, either focal or extensive, were found in 12 cases (10%), which- together with the above described case no.1 and the borderline case (case no. 2) form the basis for this study. From all cases, clinico-pathologic data (including infor- mation on treatment and outcome) were obtained and analyzed. The study was approved by the local Review Board of our Institution.
Normal Adrenal Glands
In addition to the adrenal gland tissue present in the peritumoral peripheral areas of most cases, 2 apparently normal adrenal glands from adult patients operated for renal cell carcinoma, 1 ectopic adrenal tissue incidentally found in the spermatic cord of a patient operated for testicular seminoma, and 8 fetal adrenal glands (10 to 26 wk of gestation from cases of spontaneous or thera- peutic abortions) were also investigated, using the same markers applied to the tumors, as listed below. All tissue blocks were coded by a pathology staff member not involved in the study, so that all normal adrenal gland cases were made anonymous to the investigators.
Immunohistochemistry
Sections serial to those used for conventional hematoxylin-eosin and histochemical stains (Alcian blue and reticulin stains) were obtained from 1 or 2 represen- tative tissue blocks of most cases (only the immunoprofile of cases no. 6 and 8 could not be completely investigated). These antibodies were used: pan-cytokeratin (DakoCyto- mation, Glostrup, Denmark, clone AE1/AE3, diluted 1/100), vimentin (Dako, clone V9, diluted 1/350), melan A (Dako, clone A103, diluted 1/350), a-inhibin (Diame- dix, Miami, USA, clone R1, diluted 1/75), synaptophysin (Dako, clone SY38, diluted 1/100), chromogranin A (Abcam, Cambridge, USA, clone LK2H10, diluted 1/500), Ki67 (Dako, clone MIB-1, diluted 1/150), neuron specific enolase (NSE; Neomarkers, Fremont, USA, clone E27, diluted 1/600), CD56 (Novocastra-Leica, Milan, Italy, clone 1B6, diluted 1/150), CD57 (Zymed, San Francisco, USA, clone NK-1, diluted 1/50), CD99 (Dako, clone MIC-2, diluted 1/100), synapsin (Novocastra, clone A10C, diluted 1/150), hASH1 (BD Pharmingen, San Diego, clone 24BD72D11.1, diluted 1/150). Finally, total neurofilament proteins were tested either as a pool, inclu- ding 68 kDa, 160 kDa, 200 kDa subunits, or as each single neurofilament subunit (subunit 68 kDa, Neomarkers,
| No. | Sex/ Age | Hormonal Status | Site | Stage | Surgery | Weight (g) | Size (cm) | Diagnosis | Myxoid Area (%) | Growth Pattern (Predominant) | Weiss Score | Ki- 67 (%) | NF IHC | Mitotane/ Other | Rec/Mets | Status (mo) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Group 1-Myxoid adrenocortical tumors | ||||||||||||||||
| 1 | M/58 | Not funct | R | 1 | Radical | 55 | 5 | T-UMP | 90 | Trabecular, microacinar | 1 | 4 | + foc | mit* | Local & peritoneal (60 mo) | DOD 68 |
| 2 | F/52 | Not funct | L | 1 | Radical | 20 | 4 | T-UMP | 90 | Trabecular, microacinar | 1 | 3 | + | - | - | NED 9 |
| 3 | F/72 | Cort/andr | L | 4 | Nonrad | 200 | 9 | ACC | 70 | Trabecular | 5 | 70 | - | mit | Lung (at dx) | DOD 18 |
| 4 | M/43 | Cortisol | L | 1 | Radical | 3200 | 30 | ACC | 90 | Trabecular, solid | 7 | 15 | + | mit | - | NED 9 |
| 5 | M/47 | Not funct | L | 2 | Radical | 1000 | 25 | ACC | 85 | Trabecular, solid | 7 | na | + | EDP+mit | Local, liver, lung (6 mo) | DOD 33 |
| 6 | F/70 | Cort/andr | R | 4 | No | (Biopsy) | na | ACC | 75 | Trabecular | na | na | na | mit | Liver, lung, bone (at dx) | DOD 5 |
| 7 | M/66 | Cortisol | L | 4 | Nonrad | 250 | 9 | ACC | 15 | Conv & focal trabecular | 8 | 35 | - | mit | Lung (at dx) | DOD 8 |
| 8 | F/60 | Cortisol | L | 4 | Radical | 60 | 5 | ACC | 20 | Conv & focal trabecular | 4 | 20 | - | EDP+mit | Local & lung (14 mo) | DOD 29 |
| 9 | F/43 | Cortisol | L | 4 | Nonrad | 700 | 10 | ACC | 5 | Conv & focal trabecular | 8 | 50 | - | EDP+ mit | Lung (at dx) | AWD 8 |
| 10 | F/53 | Not funct | L | 4 | Radical+ | 3000 | 28 | ACC | 20 | Conv & focal trabecular | 9 | 30 | + foc | EDP+mit | Lung (at dx) | NED 9 |
| Group 2-Conventional ACC with focal myxoid degenerative changes | ||||||||||||||||
| 11 | M/43 | Cortisol | R | 1 | Radical | 60 | 5 | ACC | 20 | Solid, atypias | 4 | 5 | + foc | - | - | NED 9 |
| 12 | F/52 | Cortisol | L | 2 | Radicalţ | 300 | 10 | ACC | 20 | Solid, atypias | 5 | 40 | - | mit | Local (192 mo) | NED 204 |
| 13 | F/53 | Cortisol | R | 2 | Radical | 250 | 10 | ACC | 10§ | Solid, atypias | 6 | 35 | + foc | EDP+mit | Local & liver | NED 36 |
| (3 mo) | ||||||||||||||||
| 14 | F/68 | Androgens | L | 3 | Radical | 260 | 10 | ACC | 5 | Solid, atypias | 9 | 25 | - | EDP+mit | Local (6mo), liver, lung (7 mo) | DOD 15 |
*Administrated at the time of tumor recurrence and progression.
Surgery carried out after chemotherapy.
¿After local recurrence, the patient underwent a second radical surgery and adjuvant mitotane.
§Both primary and local recurrence.
ACC indicates adrenocortical carcinoma; andr, androgens; at dx, at diagnosis; AWD, alive with disease; conv, conventional ACC growth patterns; cort, cortisol; DOD, died of disease; EDP, etoposide, doxorubicin, cisplatin; F, female; foc, focal; IHC, immunohistochemistry; L, left; M, male; mit, mitotane; mo, months; na, not available; NED, no evidence of disease; NF, neurofilaments; Not funct, non functioning; nonrad, nonradical surgery; R, right; T-UMP, tumor of uncertain malignant potential.
clone NR4, diluted 1/500; subunit 160 kDa, Progen, Ancona, Italy, clone NN18, diluted 1/100; subunit 200 kDa, Progen, clone NE14, diluted 1/500). Antigen retrieval procedure using microwave heating (three 5-min passages at 750 W) in citrate buffer solution (pH 6.0) was carried out for all antibodies. A biotin-free, dextran chain detection system (EnVysion, Dako) was used according to a stan- dard procedure, and diaminobenzidine was used as a chromogen. In case no. 2 and in a fetal adrenal sample, a double immunohistochemical stain was carried out to reveal melan A and neurofilaments in the same section, using sequential immunoperoxidase and immunoalkaline phosphatase detection systems, and 3-amino, 9-ethylcar- bazole, and nitroblue tetrazolium salts, as chromogens, respectively.
The immunophenotype (namely neurofilaments) of myxoid tumors was compared with that of conventional ACC in a control group of 47 randomly selected ACC cases, using a tissue microarray (TMA).
RESULTS
Clinical Data (Table 1)
The 14 tumors affected 5 males and 9 females, with a median age of 53 years (range 43 to 72). The tumors were located in the right adrenal gland in 5 cases and in the left gland in 9. The hormonal status was known for all patients, and a functioning tumor was found in 10 cases (71%). Cortisol production was the most common event (9 patients), whereas androgen production was observed in 3 patients (2 of them had concomitant hypercortiso- lism). The tumors were generally large, ranging from 4 to 30 cm in largest diameter (mean 12.5cm). The mean weight of the 12 tumors in which this information was available was 848 g, ranging from 20 to 3200 g.
The clinical history of case no. 1 case was reported above. The other borderline case (case no. 2) was a 52- year-old female patient who was recently found to be affected by a 4-cm nonfunctioning tumor of her left adrenal gland. The patient had no additional therapies after surgery and is under close follow-up, being currently free of disease 9 months after operation.
At the time of diagnosis, 5 patients had an early stage tumor, whereas 7 had locally advanced or meta- static disease. All but 2 patients had surgery as primary treatment, attaining a radical resection in 9 cases, which became disease-free. Surgery was also carried out after diagnosis in 4 patients with metastatic disease: 1 of these had a radical resection of either primary or metastatic disease, whereas in the remaining patients surgery was carried out for tumor debulking purposes, to control the endocrine syndrome and/or favor the subsequent systemic therapy.
Except for the borderline tumors, a systemic antineoplastic treatment was administered to 11 of 12 patients (excluding case no.11). Single-agent mitotane was administered orally to 5 patients (3 of them had metastatic disease), tailoring the dose in each patient to attain and maintain circulating drug levels within the
therapeutic range (14 to 20 ng/ml). Patients with meta- static ACC had disease progression despite mitotane therapy and did not receive concomitant chemotherapy because of their poor clinical conditions. Six other patients with metastatic disease received mitotane in combination with an etoposide, adriamycin, and cisplatin (EDP-M) chemotherapy regimen.1 After 6 chemotherapy cycles, 3 patients showed a partial response (ie, tumor shrinkage greater than 30%), 1 obtained a short lived (2mo) disease stabilization and 1 progressed. The remaining patient interrupted the treatment after 3 cycles owing to disease progression. She was then addressed to a second line chemotherapy with gemcitabine, capecitabine, and mitotane combination regimen, showing a clinical complete response of liver metastases and local recurrence at CT scan. She is presently disease-free after 22 months from the start of the second-line treatment (case no. 13).
Among the 5 patients with early stage ACC who had radical surgery, 1 developed lung metastases (case no.5) and 1 (case no.12) had a local recurrence after 6 months and 16 years, respectively. Patient no. 12 under- went a radical removal of her recurrence and commenced adjuvant mitotane treatment. At the last follow-up examination (January 2010), 6 patients are alive, whereas 6 patients died of disease; the median overall survival was 30 months (range 5 to 204).
Pathologic Features (Table 1)
The gross features of myxoid ACT were not significantly different from those of conventional ACC, except that in the pure forms there were translucent gray or white areas visible on gross inspection (Fig. 1). Histologically, all 14 tumors exhibited a more or less abundant myxoid component, ranging from focal areas (approximately 5% of the tumor) to virtually the entire neoplasm. Within the myxoid background, 2 different cellular growth patterns were recognized (Fig. 2). The first was peculiar and was seen in 10 cases, mostly associated with prominent myxoid changes. It was characterized by a homogeneous growth of small regular cells arranged in cords and microcysts, in some areas very reminiscent of adenoid cystic carcinoma. The second pattern, present in the remaining cases (all of which had a focal myxoid background) was characterized by a cellular growth similar and indeed merging with that of the conventional ACC component in terms of cell size (large), cell appearance (atypia, eosinophilic cytoplasm), and cell arrangement (lack of trabeculae and microcysts).
In summary, trabecular structure, cell type, and tumor size (small vs. large) distinguished 2 groups, as:
Myxoid ACC-Group 1 (Cases no. 1 to no. 10)-Case no.1 and no.2 had small pseudoglandular structures or slightly larger microcysts containing an amorphous myxoid material. A trabecular architecture was focally recognized in both cases (10% to15% of the tumor). Myxoid lakes were prominent in case no. 1, only. The tumor cells were mostly uniform and homogeneous, with scant lightly eosinophilic cytoplasm and lack of nuclear atypia, more reminiscent of zona glomerulosa
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cells than the large foamy cells of the other fasciculate layer. The mitotic index was very low. No necrosis was present in either case. All of the unfavorable morpho- logic parameters of the Weiss scoring system were absent, except for the fact that clear cell cytoplasm was present in less than 25% of the tumor cells. Of the ACC present in this group (cases no. 3 to no. 10), a pure or predominant (> 70%) myxoid pattern was seen in 4 cases (cases no. 3 to no. 6), whereas in the other 4 tumors (cases no. 7 to no. 10), only a limited amount (5% to 25%) of myxoid background was detected. In all cases, the myxoid background was associated to a trabecular, pseudoglandular, or cribriform growth of small lightly eosinophilic cells. The extent of the myxoid background was highly variable, with extra- cellular material being abundant in some areas, in which the cells were arranged in thin cords or isolated in the myxoid material. In some cases (such as case no. 3), a more solid growth was found, with an organoid (carcinoid-like) pattern and scant myxoid material surrounding the tumor nests. In another case (case no. 8), a peculiar arrangement of cells in festoon-like structures surrounded by myxoid material was obser- ved. Cell atypia was remarkably scant and cell size was small to medium, with the exception of 1 case (case no. 4) that, despite a similar architecture and prominent myxoid background, was huge (over 30 cm, 3200 g), aggressive (Weiss score 7), and characterized by atypi- cal and deeply eosinophilic cells arranged in cords or small clusters or even isolated in a myxoid back- ground. These features were also detected in the areas of tumor invasion of the periadrenal adipose tissue. The extent of pleomorphism raised the alternative possibility of a sarcomatous tumor, such as myxoid chondrosarcoma, but the immunophenotype proved the adrenocortical origin of the tumor.
Conventional ACC with focal myxoid degenerative changes-Group 2 (no. 11 to no. 14)-This group of tumors was characterized by a myxoid component which never exceeded 20% of the lesion. It contained much larger cells with the eosinophilic cytoplasm and the nuclear atypias that one sees in conventional ACC, with which this myxoid component was actually intermingling. All of these cases were in the malignant
category, with a Weiss score ranging from 4 to 9. As outlined in Table 2, except for the presence of focal stromal myxoid changes, tumors in Group 2 are very similar to conventional ACCs and more likely repre- sent the result of degenerative myxoid changes rather than belong to the distinctive myxoid variant of ACTs described in Group 1 tumors.
Histochemical and Immunohistochemical Features
The myxoid areas were positive with the Alcian blue staining. In all malignant cases and in the morphologi- cally borderline case (no. 1), reticulin stain confirmed the stromal framework disruption, as recently reported by our group,27 whereas case no. 2 had an intact reticulin network. All tumors in this series shared the expression of the known markers of the adrenal cortex, namely melan A, a-inhibin, vimentin, and synaptophysin. Cytokeratin was expressed in 5/14 cases, always focally, except for case no.2, in which there was a diffuse and strong immuno- reactivity. The mean Ki-67 proliferation index was 31.8% in malignant cases (nos. 3 to 14) and only 3% and 4% in the borderline cases nos. 1 and 2, respectively. A peculiar expression of neurofilaments (as diffuse cytoplasmic, or small paranuclear dots) was observed in 50% of the cases, and was associated more to the extent of myxoid changes (all but 1 positive cases with predominant myxoid featu- res) (Fig. 3), rather than to the cellular subgroups (Group 1 vs. Group 2 tumors). Such reactivity was observed using an antibody to total neurofilament proteins, and it was confirmed using antibodies selective for the different neurofilament subunits. In particular, although no signal was found for the 200-kDa neurofilament subunit and only rare tumor cells reacted for the 160 kDa subunit, all cases expressed the 68-kDa subunit protein, with a pattern similar to that above described. As revealed with the double immunostaining procedure, neurofilaments were coexpressed with the adrenocortical marker melan A in the tumor cells (Fig. 3). In the control cases, 17 of 47 conventional ACCs (40.4%) were found to express neuro- filaments, with a focal paranuclear dot-like reactivity in 12 (25.2%) tumors and a granular cytoplasmic positivity in up to 25% of tumor cells of the remaining 7 neoplasms.
| Myxoid ACC (Group 1, Case Nos. 1-10) | Pathologic Feature | Conventional ACC With Focal Myxoid Degenerative Changes (Group 2, Case Nos. 11-14) |
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| Separated from conventional areas if any, extensive in 6/10 cases | Type of myxoid changes | Merged with conventional ACC areas, and always focal (< 20% of the tumor area) |
| Usually trabecular, microacinar | Growth pattern | As conventional ACC, usually solid/diffuse |
| Small, uniform | Cell size | Large, heterogeneous |
| Mild* | Nuclear atypia | Moderate to high |
| Scant, eosinophilic | Cell cytoplasm | Abundant, granular eosinophilic |
| Present in 5/9 cases, generally diffuse | Neurofilament expression | Present in 2/4 cases, focal |
| *High in case #4. ACC indicates adrenocortical carcinoma. |
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By contrast, no neurofilament reactivity was seen in ACA. As described above, CD99 was positive in the recurrent lesion of case no.1 but not in the corresponding primary lesion nor in any other case of the series tested, including the control ACC TMA cases.
The unexpected expression of a neural and neuro- endocrine marker in a mesodermally derived tumor, paralleled by the known expression of synaptophysin by ACC, prompted the investigation of further neural or neuroendocrine markers. Of these, only CD56 was inten- sely expressed in all cases (although it was also focally or diffusely expressed in up to 90% of conventional ACC; Volante & Papotti 2009, unpublished observation), whereas no reactivity was found for synapsin (except for a single case with focal reactivity), CD57, chromogranin A, or hASH1, the latter being a transcription factor of the
Notch pathway that regulates neuroendocrine differentia- tion during development.
Comparison With the Immunophenotype of the Normal Adrenal Gland
The adult and fetal adrenal glands showed the ex- pected immunoprofile, with melan A and x-inhibin expre- ssion in the cortex and neuroendocrine marker reactivity in the medulla. No neuroendocrine marker (chromo- granin A, NSE, CD57, CD56, and hASH1) reactivity was seen in the adult or fetal adrenal cortex, with the excep- tion of synaptophysin (present in both fetal and adult cortex) and of neurofilaments. The latter were expressed only in extremely rare cells of the zona glomerulosa of a single adult adrenal gland (case no. 2), all other adult adrenal gland tissue being negative. However, strong
neurofilament reactivity was found in small cells arranged singly or in small clusters in the peripheral layers of all 8 fetal adrenal cortices investigated (Fig. 3). There was a slight decrease of the number of reactive cell with increase of the gestational age. The type of neurofilaments expres- sed in individual adrenal cortical cells was mostly the 68-kDa subunit. There were very rare cells which also stained for the 160 (but not the 200) kDa subunit, paralleling the reactivity above described in the myxoid tumors.
DISCUSSION
In this study, we describe the clinico-pathologic and immunophenotypic features of 14 ACT having varying degrees of distinctive myxoid features. Most of these cases had malignant features, with a Weiss score of 1 found in 2 cases only, 1 of the cases eventually displaying an aggres- sive clinical course with fatal outcome.
In a review of a large series of ACC at our Insti- tution, myxoid features were observed in approximately 10% of the cases and accounted for 5% to 90% of the tumor cell population in individual cases, being in general associated and often intermingled with areas of conven- tional ACC. This wide variability in the extent of the myxoid component was associated to distinctive growth patterns, which have been only partially addressed in earlier reports. In the literature, the extension of myxoid changes in single tumors has only been reported by Brown et al,4 who indicated a variable percentage of myxoid areas (from 10% to 95%) in their 14 tumors, these changes being generally higher in the benign cases. In another single case report,17 large pools of myxoid material were described which involved most of the tumor.
Morphologic Features
Although the appearance of the myxoid back- ground was not different from that of a variety of human tumors with myxoid features that is, mostly characterized by more or less extensive areas of extracellular accumula- tion of light basophilic amorphous material, the cell population present within the myxoid material had distinctive characteristics in our series. In some cases (Group 2-no. 11 to 14), the tumor cells were very similar to those of conventional ACC, that is, large, irregularly shaped and deeply eosinophilic. The nuclei were large, atypical, and with prominent nucleoli. These cells were arranged in irregular nests or cords and merged with areas of conventional ACC having a diffuse growth pattern. In our view, these cases represent classical ACC having more or less extensive (5% to 20% of the tumor area) accumulation of myxoid material, probably as a result of focal intratumoral degenerative changes. The other cases (Group 1-no. 1 to no. 10) had a different architecture and apparently form a separate group of ACT having distinctive histologic features, already recognized by some workers.4 These tumors had a relatively homogeneous growth pattern made of small- to-medium sized cells with minimal atypia, arranged in thin trabeculae, pseudoglands, or small cysts, resembling
the patterns of adenoid-cystic carcinoma in a more or less abundant myxoid stroma. With regard to the malignant cases, the well-known features of classical ACC, as summarized in the 9 Weiss score parameters, were not readily apparent, with the exception of “dark” cell cytoplasm, mitotic figures, and necrosis. The other Weiss score parameters could be identified in a variable percentage, but some of these criteria were hard to assess in the myxoid areas. Among them, small vessel invasion was less apparent owing to the myxoid background, and diffuse growth pattern was never a feature in these tumors, whereas atypia was minimal or absent. For these reasons, and as already reported for other ACC variants, such as the oncocytic ACC2 or the pediatric ACCs,5 Weiss criteria might be inadequate to reliably detect malignancy in myxoid ACC. Moreover, when these distinctive morphologic features are predominant in an adrenal tumor, the differential diagnosis should include several types of metastatic tumors, including breast carcinoma, salivary, or skin adnexal carcinomas of the adenoid cystic type, and neuroendocrine tumors. All of these tumors are potentially capable of giving metastatic spread to the adrenal gland, and an immunohistochemical marker profile is, therefore mandatory.
Immunophenotype
The immunohistochemical profile of both the conventional ACC with a myxoid component (Group 2) and the pure myxoid ACT (Group 1) is similar to that of classical ACC. Thus, melan A and x-inhibin are regularly present in myxoid tumors, as is synaptophysin.12,18 A correct definition of the adrenocortical origin can there- fore, be obtained in all cases, irrespective of their myxoid material content. Focal or diffuse cytokeratin expression was observed in approximately 60% of cases, whereas in all (except case no. 2) tumors, the reticulin stain shows architectural disruption, which was recently described as a major marker of ACC.27 The Ki-67 proliferation index in myxoid ACC was not different from that of conven- tional ACC, being on average 31.8% in malignant cases as opposed to 3% and 4% in the borderline cases, respectively.
A distinctive feature was the expression of neuro- filament proteins in the small cell, pseudoglandular, or trabecular areas of cases having a predominant (ie, > 70% of tumor) myxoid component (no.1 to no.6), in a fraction of tumor cells generally not exceeding 15% to 25% of the tumor population (with the exception of case no.2). Neurofilaments were generally absent in cases with only focal myxoid stromal component, with the exception of case nos. 10, 11, and 13, which showed focal neuro- filaments reactivity in single cells. Neurofilament expres- sion in ACC has been reported earlier in the literature only once and in a limited series of cases, apparently without any association with specific morphologic featu- res.2º As a matter of fact, neurofilament expression is instead considered a marker of adrenal medullary cells and their tumors. To test whether neurofilament expres- sion was restricted to the myxoid and small cell ACC here
described, a series of conventional ACC was tested and neurofilaments were found to be expressed in 19/47 cases (40.4%). The extent of the reactivity was mostly focal (12 cases) and restricted to a small paranuclear dot, but 7 cases had a more diffuse, finely granular cytoplasmic positivity in up to 25% of the tumor cells. These findings indicate that neurofilament expression is characteristic of predominantly myxoid ACCs, but that it also occurs in a fraction of conventional ACC, thus expanding the list of neural and neuroendocrine markers detected in adreno- cortical cells and tumors, such as synaptophysin.12,18 Whether ACC in general and its myxoid variants in particular are tumors having a divergent differentiation toward endocrine (steroid-producing) and neuroendo- crine lineages is not known. Along these lines, other neuroendocrine markers were tested in the current cases (and in the fetal normal glands), including chromogranin A, synapsin, CD57, and CD56. Only the latter was found positive in all tested cases, but it is known that CD56 is highly expressed in normal adrenal cortex and derived tumors.22,24 It is worth noting, however, that in focally myxoid tumors the expression of neurofilaments and CD56 was mostly restricted to the myxoid areas. To further explore the neural/neuroendocrine phenotype of these rare myxoid tumors, the expression of transcription factors driving neuronal and endocrine differentiation during the embryonal life and in some neuroendocrine tumors was also assessed, with special reference to hASH1 (human achete scute homologue 1), but no immunoreactivity was found for this marker in any tumor.
Interestingly, although the peritumoral adrenal cortex of the current cases was totally unreactive to neurofilaments (with the exception on scattered cells in case no.2), a consistent immunoreactivity was observed in subcapsular cortical isolated cells or clusters of the fetal adrenal gland when antibodies specific to the 68 and 160-kDa neurofilament subunits were used. A similar observation had already been mentioned in an old study on fetal and adult human adrenal medullary cells, in which the researchers quoted as a “peculiar phenomenon” the expression of medium molecular weight neurofilament subunits through all adrenocortical developmental stages and also in the adult adrenal cortex.21
Clinical Features
Most myxoid ACT (71%) in our series, were functioning malignant neoplasms, with greater frequency of Cushing-related symptoms. The rate of functioning tumors of the present series is in line with the literature data on the 13 currently reported malignant ACC with myxoid features3-5,12,18,28 and is higher than that gen- erally recorded in conventional ACC, ranging from less than 50% in our experience27 to 60% in the literature.8 Unfortunately, from the literature review, it seems that some heterogeneity exists in the reported cases, in that some of these cases probably represent examples of conventional ACC having focal myxoid changes and containing large eosinophilic atypical cells. The true
clinical behavior of pure myxoid ACA and ACCs is difficult to assess. Brown et al4 reported 14 cases, comprising 6 benign and 8 malignant tumors. The latter (which had myxoid areas in 10% to 60% of the tumor mass) followed an aggressive course in 50% of cases. However, the benign tumors had frequently a large size (> 5cm in 4 cases), questionable capsular invasion (in 4 cases), and a mitotic activity potentially exceeding the cutoff of 5/50 HPF (4 cases had 1 or more mitoses in 10 HPF). In this series, a case originally considered of borderline malignancy (Weiss score 1) developed perito- neal spread and eventually killed the patient 5 years after diagnosis. The 12 ACC cases had a Weiss score of 3 to 9 and metastatic disease at diagnosis in 5 instances. With regard to the outcome data, in the absence of a comparable control group, it is difficult to state whether patients with ACT with myxoid features have a survival different from unselected ACC patients, and the respon- siveness of myxoid ACT to systemic chemotherapy8 warrants furthermore testing.
In conclusion, myxoid changes rarely occur in ACT. They are identified most frequently in malignant or borderline tumors, and are associated to 2 distinctive cellular growth patterns, one similar to conventional ACC, and the other representing a morphologically distinct entity made up of small-to-medium sized cells with minimal atypia, arranged in trabecular, pseudo- glandular, or small cystic spaces in a myxoid background which may represent up to 90% of the tumor cell population. Their immunoprofile overlaps with that of conventional ACTs. Interestingly, neurofilament expres- sion, only focally and inconsistently present in conven- tional ACC, emerged as a distinctive feature of predominantly myxoid tumors, and was paralleled by similar neurofilament reactivity in the normal fetal adrenal cortex, too.
ACKNOWLEDGMENTS
The authors thank Dr G. Fadda (Catholic University, Rome) for providing histologic material of case no. 11 and to Dr E. Bollito (San Luigi Hospital, Orbassano) for helpful discussion.
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