Oncocytic Adrenocortical Tumors: Diagnostic Algorithm and Mitochondrial DNA Profile in 27 Cases
Eleonora Duregon, MD,* Marco Volante, MD,* Susanna Cappia, BSc(Med),* Alessandra Cuccurullo, PhD,* Michele Bisceglia, MD,; Daniel D. Wong, BSc(Med), MBBS,¿ Dominic V. Spagnolo, MBBS, FRCPA,¿ Sylwia Szpak-Ulczok, MD,§ Enrico Bollito, MD,* Fulvia Daffara, MD, | Alfredo Berruti, MD, Massimo Terzolo, MD, | and Mauro Papotti, MD*
Abstract: The pathologic diagnosis of adrenocortical carcinoma (ACC) relies on microscopic features that are sometimes equivocal in special variants, including oncocytic adrenocortical tumors (OACTs). We report a series of 27 unpublished OACTs (15 pure and 12 mixed or focal) and assess for the first time in OACTs the diagnostic utility of an algorithm recently proposed by our group (“reticulin” algorithm) for conventional ACCs on the basis of a combination of reticulin staining and assessment of only 3 Weiss parameters. Overall, 12 cases were malignant according to the Lin-Weiss-Bisceglia (L-W-B) system for pure tumors and the original Weiss system for mixed or focal tumors; extensive or focal disruption of the reticulin network was found in 16 of 27 OACTs. This was associated with either a high mitotic index, presence of necrosis, and/or vascular invasion in 14 of these, which were thus considered malignant according to our algorithm. From a clinical standpoint, OACTs, at least in the pure form, are “low grade” lesions with a low mean Weiss score, mitotic and Ki-67 indices, and uncommon capsular or vascular invasion. They, including unequivocal morphologically malignant cases, generally pursue an indolent clinical course. In addition, the 4977 bp mitochondrial DNA “common deletion” was detected using real-time polymerase chain reaction in 54% of cases from this study and an additional validation series of 23
From the *Division of Pathology; |Division of Medical Oncology; | Division of Internal Medicine, Department of Clinical and Biological Sciences, University of Turin at San Luigi Hospital, Orbassano, Torino; * Department of Pathology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy; ¿Department of Anatomical Pathology, PathWest Laboratory Medicine WA, Nedlands, Western Australia, Australia; and §De- partment of Nuclear Medicine and Oncological Endocrinology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland.
Conflicts of Interest and Source of Funding: This article received financial support by grants from the Italian Ministry of University (PRIN, 20085P5SC49 to MT), the Italian Association for Cancer Research (AIRC, Milan, grant no. IG/10795/2010 to MP), and from the Regione Piemonte (Progetto Ricerca Sanitaria Finalizzata, D.G.R. n. 35-4231 dated 06.11.2006 to M.V.). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.
Correspondence: Marco Volante, MD, Department of Clinical & Biological Sciences, University of Turin at San Luigi Hospital, Regione Gonzole 10, 10043 Orbassano, Torino, Italy (e-mail: marco.volante@unito.it).
Copyright @ 2011 by Lippincott Williams & Wilkins
OACTs, with a heterogenous (heteroplasmic) intratissue and intracellular distribution (as detected by a modified FISH procedure) and a marked association with the presence of intact reticulin framework.
Key Words: adrenal cancer, oncocytic variant, mitochondrial DNA, reticulin framework, classification
(Am J Surg Pathol 2011;35:1882-1893)
A drenocortical carcinoma (ACC) is an extremely rare (< 2 cases per million individuals per year) and highly aggressive malignant tumor of the adrenal gland.5,18,24,60,64 A large fraction of cases present with metastatic disease at diagnosis, and the behavior is unpredictable, even in cases that have undergone radical surgery.3,24,60 Progressing or advanced cases may benefit from chemotherapy, including new protocols based on mitotane,56 or from multiagent therapy.5,26,40,54 Disease relapse is, however, a major problem in many cases, and to date only radical surgery offers some prospect for better disease control. 10,48 Molecular studies have identified several genes, either related to control of tumor cell growth (cyclin D, IGF2, ß-catenin) or to other cell functions, which are differen- tially regulated in ACC compared with adenomas [adre- nocortical adenoma (ACA)] or borderline tumors. Most of these may play a role in malignant progression, but so far none has proven to be sufficiently specific or sensitive for diagnostic purposes. 2,22,24,25,39,50,51
Further, the correct classification of borderline adrenocortical tumors incompletely expressing the classi- cal Weiss parameters is problematic, and there is the concrete risk of overestimating or underestimating the real biological potential of individual cases. In this respect, oncocytic adrenocortical tumors (OACTs) repre- sent the most challenging group, as some of the classical Weiss parameters, such as eosinophilic cyto- plasm, nuclear atypia and diffuse growth, are intrinsically present in the vast majority of these cases irrespective of their biological and clinical behavior. Therefore, the presently accepted cutoff value for malignancy, that is, Weiss score 363 and subsequent modifications,3 may not be as accurate for OACT as for conventional ACC. In fact, at least a proportion of such cases would be
overdiagnosed as carcinomas, as a Weiss score 3 is easily attained because of the innate morphology of OACT.
This issue has been extensively discussed in the literature,35 and alternative classification systems have been proposed, of which the Lin-Weiss-Bisceglia system (L-W-B) is nowadays the most widely accepted.
The L-W-B system is a modified Weiss scoring system for OACT7,8,35,65 and is based on major and minor criteria to more accurately classify these tumors as either benign, borderline malignant, or frankly malignant potential. There still exist some controversies with regard to this group of tumors. One is the existence of a borderline diagnostic category, which-according to some35-largely reflects the scarcity of clinical information relating to the biological behavior. A second is the issue of any clear cell component in such tumors, specifically its pathobiological and clinical significance.7,8 Further, OACTs are very uncommon, with an unknown incidence worldwide and with only a total of 110 cases recorded so far.65
Recently, an algorithmic approach, the “reticulin” algorithm, was proposed for the diagnosis of conven- tional ACC61 on the basis of a combination of reticulin staining and 3 malignancy-related parameters (mitotic index, necrosis, vascular invasion), all of which had already been considered in the classical Weiss system, and none related to those intrinsically present in OACT, irrespective of their malignant potential. The performance of this algorithmic approach has never been tested specifically in OACT.
Moreover, the molecular features of OACT, namely, the presence of cytogenetic or mitochondrial DNA (mtDNA) alterations reported in oncocytic tumors of other locations, 21,23,36,37,42,44,55,59 have never been investigated in OACT.
Therefore, the aims of this study were 2-fold: (i) to describe the clinicopathologic features-including reticulin staining and the potential application of the newly proposed diagnostic algorithm-in an unpublished series of 27 adrenocortical tumors having more or less extensive oncocytic features; and (ii) to determine the presence and tissue distribution of the most frequent mtDNA alteration, the 4977 bp “common deletion,” examine its relationship to pathologic and clinical parameters in individual tumors comprising these 27 cases, and compare the findings with those in an external control series of OACTs, most of which have been previously published.7,65
We show that OACTs represent a heterogenous group of tumors with morphologic features resembling those of oncocytic neoplasms of other locations, including the presence of the mtDNA 4977 bp “common deletion,” and confirm OACTs, in the pure form, as low-grade tumors with a more indolent clinical course compared with conventional ACCs.
MATERIALS AND METHODS
OACT Turin Series (Table 1)
A total of 194 consecutive adrenocortical tumors having a Weiss score ≥ 362,63 obtained between 1990 and
2010 from the pathology files of the University of Turin were reviewed independently by 3 of us (E.D., M.V., and M.P.) using a mean of 5 hematoxylin and eosin-stained slides per case (range, 1 to 22). This series included 35 cases resected at San Luigi Hospital, 24 cases originally collected by 1 of us (M.P.) at the San Giovanni Hospital of Turin from 1993 to 2003, and 135 cases received in consultation from different hospitals. The majority of these patients were treated at our institution, which serves as a referral center for ACC in Italy. The histopathologic features of 92 conventional ACCs and 14 cases of the myxoid variant belonging to this data set have already been reported.47,61 Moreover, a series of 116 ACAs, all resected at San Luigi Hospital between 1994 and 2008, with paraffin material and follow-up information avail- able was also reviewed.
From this review, 27 cases were identified as OACT, of which, according to the criteria of Bisceglia et al,8 15 were purely oncocytic (> 90% oncocytes). The remaining had a variable oncocytic component (30% to 80% oncocytes) combined with areas of conventional ACC or adenoma (10 were classified as mixed OACTs and 2 as focal OACTs; Table 2). These 27 cases formed the basis of the current study. For all cases, clinicopathologic data (including information on treatment and outcome) were obtained and analyzed. Six additional cases with onco- cytic features accounting for < 10% of the neoplasm were excluded. The study received ethical approval from the local review board of our institution.
OACT Validation Series (Table 3) and Control Cases
With the aim of verifying the prevalence of the mtDNA “common deletion” (see below), a separate vali- dation series of 23 OACTs, classified using the L-W-B scoring system7 retrieved from the files of the Casa Sollievo della Sofferenza Hospital of San Giovanni Rotondo (Italy) and PathWest Laboratory Medicine WA (Australia), was also investigated. This series included 16 pure oncocytic, 4 mixed, and 3 focal OACT cases. The clinicopathologic features of some of these tumors (#16-15 pure OACTs and 1 mixed OACT) were reported previously (cases B6 to B9 and B23,7,8 and W1 to W3, W5, and W10 to W1665) and thus were not considered further in this study.
In addition, the mtDNA status was also investi- gated in a series of control cases, including 18 normal adrenal glands obtained from nephrectomy specimens, 13 ACAs (none having oncocytic features, except for 3 showing < 10% oncocytic changes), and 50 conventional ACCs. All the pathologic material from the Turin, validation, and control series was deidentified and coded by a pathology staff member not involved in the study before any type of analysis in order to make all specimens anonymous to the investigators.
Immunohistochemistry and Histochemistry
Paraffin sections of 5 um thickness, serial to those used for conventional hematoxylin and eosin staining,
| TABLE 1. Clinicopathologic and Mitochondrial DNA Alterations in 27 OACTs | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sex/ Age (y) | Hormone Production | Location/ Weight (g)/ Size (cm) | Oncocytic Area (%) | Classification System | Reticulin Stain | Reticulin Algorithm | PCR mtDNA Deletion | FISH mtDNA Pattern | Follow-up (mo) | |
| Pure OACT | LWB | |||||||||
| classification | ||||||||||
| 1 | F/45 | Cortisol | L/9/3 | >90 | Benign | Intact | ACA | Yes | Deleted | NED 36 |
| 2 | F/47 | Not funct | R/30/3 | >90 | UMP | Intact | ACA | No | Normal | NED 72 |
| 3 | F/45 | Not funct | R/NA/5.5 | >90 | UMP | Intact | ACA | Yes | Deleted | NED 24 |
| 4 | F/45 | Not funct | R/NA/4 | > 90 | UMP | Intact | ACA | Yes | Deleted | NED 36 |
| 5 | F/61 | Androgens | L/1252/17 | > 90 | UMP | Disrupted | ACA | Yes | Deleted | Alive 60 |
| 6 | F/49 | Not funct | L/400/13 | >90 | UMP | Disrupted* | ACA | No | Normal | Alive 124 |
| 7 | F/61 | Not funct | L/400/12 | >90 | UMP | Disrupted* | ACC (necrosis) | Yes | Deleted | NED 165 |
| 8 | F/41 | Not funct | L/NA/11 | >90+ | UMP | Disrupted | ACC (necrosis) | No | Normal | AWD 72 |
| 9 | F/42 | Not funct | L/530/16 | >90+ | UMP | Disrupted | ACC (necrosis) | No | NA | DOD 15 |
| 10 | F/31 | Hypertens | L/255/9.5 | >90 | Malignant | Disrupted* | ACC (venous | No | Normal | Alive 113 |
| invasion) | ||||||||||
| 11 | M/60 | Not funct | L/8/1.6 | >90 | Malignant | Disrupted* | ACC (mitoses) | No | Normal | NED 48 |
| 12 | F/68 | Not funct | R/NA/17 | > 90+ | Malignant | Disrupted* | ACC (necrosis, | No | Normal | NED 36 |
| venous invasion) | ||||||||||
| 13 | F/66 | Not funct | NA | >90+ | Malignant | Disrupted | ACC (necrosis, venous invasion) | Yes | Deleted | NED 11 |
| 14 | M/46 | Not funct | L/950/18 | >90 | Malignant | Disrupted | ACC (necrosis, | ND | ND | NED 4 |
| venous invasion) | ||||||||||
| 15 | M/32 | Not funct | R/NA/23 | >90 | Malignant | Disrupted | ACC (necrosis, mitoses, venous invasion) | ND | ND | DOD 24 |
| Mixed/Focal | Classical WSS | |||||||||
| OACT | ||||||||||
| 16 | F/53 | Hypertens | L/135/8 | 60 | Benign | Intact | ACA | Yes | Deleted | NED 108 |
| 17 | F/66 | Aldosteron | R/52/5 | 60 | Benign | Intact | ACA | Yes | Deleted | NED 72 |
| 18 | M/50 | Cortisol | L/18/4 | 50 | Benign | Intact | ACA | Yes | Deleted | NED 92 |
| 19 | F/55 | Cortisol | R/86/11 | 50 | Benign | Intact | ACA | No | Normal | NED 55 |
| 20 | F/37 | Cortisol | L/7.3/2.8 | 50 | Benign | Intact | ACA | Yes | Deleted | NED 47 |
| 21 | F/40 | Aldosteron | R/10/4 | 30 | Benign | Intact | ACA | Yes | Deleted | NED 43 |
| 22 | F/44 | Cortisol | R/NA/8 | 60 | Malignant | Disrupted | ACC (necrosis) | No | Normal | DOD 90 |
| 23 | M/35 | Not funct | L/40/8 | 50 | Malignant | Intact | ACA | ND | ND | NED 60 |
| 24 | F/67 | Cortisol | L/1050/15 | 60 | Malignant | Disrupted | ACC (necrosis, | No | Normal | NED 156 |
| venous | ||||||||||
| invasion) | ||||||||||
| 25 | M/44 | Cortisol | R/1300/20 | 40 | Malignant | Disrupted | ACC (necrosis, | Yes | Deleted | DOD 20 |
| venous invasion) | ||||||||||
| 26 | F/46 | Not funct | L/270/9.9 | 70 | Malignant | Disrupted | ACC (necrosis, | ND | ND | NED 64 |
| mitoses, venous invasion) | ||||||||||
| 27 | M/28 | Cortisol | L/210/11 | 80 | Malignant | Disrupted | ACC (necrosis, | ND | ND | DOD 23 |
| mitoses, venous invasion) | ||||||||||
*Focal reticulin disruption.
*Small-sized oncocytes predominant.
AWD indicates alive with disease; DOD, died of diseasel; F, female; Hypertens, hypertension; L, left; LWB, classification of pure OACT according to Lin-Weiss-Bisceglia; M, male; na, not adequate; NA, not available; ND, not done; NED, no evidence of disease; Not funct, non functioning; R, right; UMP, uncertain malignant potential; WSS, Weiss scoring system.
were obtained for immunohistochemical and reticulin stains from 1 or 2 representative tissue blocks of all cases. The following antibodies were used: melan A (DakoCy- tomation, Glostrup, Denmark; clone A103, diluted 1/50), a-inhibin (Diamedix, Miami, Florida; clone R1, diluted 1/75), and Ki-67 (Dako, clone MIB-1, diluted 1/150). A biotin-free, dextran chain-based detection system (EnVy- sion, Dako) was used according to a standard protocol,
using diaminobenzidine as the chromogen. Reticulin histochemical staining was performed to define the status of the reticulin framework using a commercially available silver impregnation-based kit (Bio Optica, Milan, Italy). Disruption of the reticulin/basal membrane network was defined as the loss of continuity of the reticular fiber network evaluated at high magnification (×400) in more or less extensive tumor areas, as previously described.61
| Composition | Pure OACT (%) | Mixed OACT (%) | Focal OACT (%) | (Pure) Conventional ACT (%)* |
|---|---|---|---|---|
| Oncocytic | >90 | 50-90 | 10- < 50 | <10 |
| Clear cell | < 10 | 10- < 50 | 50-90 | >90 |
*Excluded from the study.
ACT indicates adrenocortical tumor.
Mitochondrial DNA Deletion Screening by PCR
Molecular analysis was performed in all but 5 cases lacking informative material from the residual paraffin- embedded tissue. Total DNA of OACT cases was obtained from macrodissected oncocytic areas by scraping five 10- um-thick dewaxed sections into an Eppendorf tube using a sterile scalpel blade. A DNA extraction kit (QIAamp DNA Mini kit, Qiagen srl Italy, Milan, Italy) was used to isolate the DNA according to the manufacturer’s recommenda- tions. Polymerase chain reaction (PCR) primers used to
| Classification System | Oncocytic Area (%) | Reticulin Stain | PCR mtDNA Deletion | FISH mtDNA Pattern | |
|---|---|---|---|---|---|
| Pure | LWB | ||||
| OACT | classification | ||||
| W1 | Benign | >90 | Disrupted | No | NA |
| W2 | Benign | >90 | Disrupted* | Yes | Deleted |
| W3 | UMP | >90 | Intact | Yes | NA |
| W4 | UMP | >90 | Disruptedt | No | NA |
| W5 | UMP | >90 | Disrupted* | Yes | Deleted |
| B6 | UMP | >90 | Disrupted | No | Normal |
| B7 | Malignant | >90 | Disrupted+ | Not | NA |
| B8 | Malignant | >90 | Disrupted | No | Normal |
| B9 | Malignant | >90 | Disrupted* | No | NA |
| W10 | Malignant | >90 | Disrupted+ | Not | Normal |
| W11 | Malignant | >90 | Disrupted | Yes | Deleted |
| W12 | Malignant | >90 | Disrupted | No | Normal |
| W13 | Malignant | > 90 | Disrupted | No | Normal |
| W14 | Malignant | >90 | Disrupted | No | Normal |
| W15 | Malignant | >90 | Disrupted | Yest | Deleted |
| W16 | Malignant | >90 | Disrupted ** | Not | Normal |
| Mixed/ | Classical | ||||
| Focal | WSS | ||||
| OACT | |||||
| W17 | Benign | 60 | Intact | Yes | Deleted |
| W18 | Benign | 10 | Intact | Yes | Deleted |
| W19 | Malignant | 60 | Intact | Yes | Deleted |
| W20 | Malignant | 60 | Disrupted* | No | Normal |
| W21 | Malignant | 70 | Disrupted | No | Normal |
| W22 | Malignant | 40 | Disrupted | No | NA |
| B23 | Malignant | 75 | Disrupted | No | Normal |
*Focal reticulin disruption.
+In these cases, tissue from recurrence (B7, W10, W16) and metastasis (W15) was also tested with similar results as in the primary tumor, except for mtDNA detection of case W15, which was restricted to the primary.
LWB indicates classification for pure OACT according to Lin-Weiss-Bisceglia; NA, not adequate; UMP, uncertain malignant potential; WSS, Weiss scoring system.
detect a range of mtDNA deletions in the deletion-prone region (P1 and P2) and a mtDNA-conserved sequence (P3 and P4) are listed in Table 4. PCR was performed using Maxima Hot Start Polymerase (Fermentas, Part of ThermoFisher Scientific, Waltham, MA) and EvaGreen Dye, 20X, in water (Biotium, Hayward, CA) on the Rotor- Gene Q (Qiagen) apparatus. The cycling conditions were as follows: 1 cycle at 95℃ for 5 minutes; 40 cycles at 95℃ for 60 seconds, at 56℃ for 30 seconds, and at 72℃ for 90 seconds; 1 cycle at 72℃ for 7 minutes; 5 cycles of fluorescence acquisition at 60℃; and 1 melt ramp from 65℃ to 95℃. P3 and P4 primers generated the specific PCR product in all cases, whereas samples with wild-type mtDNA were not amplifiable using P1 and P2 primers that yielded the specific 380 bp PCR product only in the presence of the mtDNA common deletion.
Mitochondrial DNA FISH Analysis
Fluorescent in situ hybridization (FISH) analysis was performed in all cases analyzed using PCR. To assess the mtDNA status, a 2-color FISH procedure was performed as follows. Probes specific for both the deletion-prone and the rarely deleted regions of mtDNA were generated by means of PCR using a Long Template PCR System (Roche, Mannheim, Germany) according to the manufacturer’s recommendations using total DNA extracted from human umbilical cord fibroblasts. Primer sequences for FISH are listed in Table 4. The first probe [common (‘COM’)] bound to mtDNA at a rarely deleted region, and the second probe [deleted (‘DEL’)] bound in the vicinity of the deletion-prone region. PCR products were purified in Quantum Prep PCR Kleen Spin Columns (Bio-Rad Lab, Hercules, CA), and 1 µg was labeled with digoxigenin (COM probe) or biotin (DEL probe) using a nick-translation labeling kit (Roche). The probes were tested before use on cultured human fibroblasts. FISH experiments were carried out on dewaxed 5-um-thick sections according to Lewis et al36 and van de Corput et al,58 with minor modifications. Slides were pretreated in a pressure cooker for 5 minutes at 125°℃, followed by a 10-second step at 90℃ using citrate buffer (pH 6). They were then digested with 0.1% (wt/vol) pepsin (Sigma, Steinheim, Germany) for 20 minutes at 37℃. RNAse A digestion [0.1 mg/mL in phosphate-buffered saline (PBS), Sigma] was carried out for 1 hour at 37℃ and subsequently blocked in PBS. Finally, the sections were fixed in 1% formaldehyde in PBS for 20 minutes at room temperature, dehydrated in 70%, 90%, and 100% ethanols, and air dried. Both the digoxigenin-labeled COM and the biotinylated DEL probes were diluted in equal amounts to a final concentration of 5 ng/uL in LSI/WCP Hybridization Buffer (Abbott Molecular, Des Plaines, IL), applied to sections, and covered with a glass coverslip sealed with rubber cement. Negative control sections included omission of the probe from the hybridization mixture. Target DNA and probe were denatured simultaneously on an 80℃ hot plate for 5 minutes and then incubated overnight at 37℃ in a
| TABLE 4. PCR Primers (mtDNA Accession Number NC-012920) | |||||
| Detection | of mtDNA common deletion by means of PCR | ||||
| P1 | Nucleotides | 8286-8305 | 5'-TCTAGAGCCCACTGTAAAG-3' | ||
| P2 | Nucleotides | 13662-13643 | 5'-GTTAGTAAGGGTGGGGAAGC-3' | ||
| P3 | Nucleotides | 4504-4526 | 5'-CCATCTTTGCAGGCACACTCATC-3' | ||
| P4 | Nucleotides | 4955-4977 | 5'-ATCCACCTCAACTGCCTGCTATG-3' | ||
| Primers for the generation of FISH probes | |||||
| COM | Probe | Forward | Nucleotides | 13972-13949 | 5'-CCTATCTAGGCCTTCTTACGAGCC-3' |
| COM | Probe | Reverse | Nucleotides | 4468-4440 | 5'-AGTACGGGAAGGGTATAACCAACATTTTC-3' |
| DEL | Probe | Forward | Nucleotides | 8848-8873 | 5'-TTATGAGCGGGCACAGTGATTATAGG-3' |
| DEL | Probe | Reverse | Nucleotides | 12864-12839 | 5'-ACGGTTGTATAGGATTGCTTGAATGG-3' |
ThermoBrite System (Abbot Molecular, Des Plaines, IL). Coverslips were carefully removed, and slides were washed 3 times for 5 minutes each in 2X saline-sodium, citrate and 3 times for 10 minutes each in Tris-HCl, NaCl, Tween [100 mM Tris ± HCl, 150 mM NaCl (pH 7.5), 0.05% Tween-20] at 45°℃. Before probe detection, slides were incubated with a blocking solution (0.5% bovine serum albumin in 4 x saline-sodium, citrate) for 45 min- utes at 37℃. The slides were then incubated simulta- neously with antidigoxigenin-fluorescein Fab fragments (Roche, Mannheim, Germany) diluted 1/250 and with Streptavidin Alexa Fluor 594 (Molecular Probes, Invitro- gen, Eugene, OR) diluted 1/200 in blocking buffer for 45 minutes at 37℃. Finally, sections were washed 3 times (10 min each) in Tris-HCl, NaCl, Tween, dehydrated in 70%, 90%, and 100% ethanols, air dried, and counter- stained with DAPI (Abbott Molecular, Des Plaines, IL). Sections were analyzed using an Olympus BX61 fluorescence microscope, and images were captured using the Cytovision 4.02 software package (Olympus Italia srl, Milan, Italy). Using this detection system, the green fluorescent probe (COM) bound all mtDNA molecules, whereas the red fluorescent probe (DEL) bound the deletion-prone region of mtDNA only in the absence of deletion. Therefore, wild-type mtDNA was defined by the presence of distinct yellow fluorescent dots or by a ratio of 1:1 green and red dots, whereas the presence of an excess (at least 2:1 ratio) of green dots indicated the occurrence of the mtDNA deletion.
Statistical Analysis
Clinical and pathologic variables were compared with mtDNA status and patient outcome using the Fisher exact or x2 and the Student t tests. A level of P < 0.05 was
considered statistically significant. All data were analyzed with STATISTICA for Windows software version 6.1 (StatSoft Italia, Vigonza, Padova, Italy).
RESULTS
Clinicopathologic Data of the OACT Turin Series
The 27 OACTs affected 7 men and 20 women, with a median age of 48 years (range, 28 to 68 y). The tumors were located in the right adrenal gland in 10 cases and in the left gland in 16, whereas in the remaining case the location was not reported. The hormonal status was known for all patients, of whom a functioning tumor was found in 13 (48%). Cortisol production was the most common event (8 patients); aldosteron production was observed in 2 cases, hypertension was present in 2 cases, and androgen secretion was found in the remaining case (Table 1). Adjuvant mitotane treatment was administered to 5 patients, who subsequently showed no disease progression, whereas first-line chemotherapy (epirubicin, docetaxel, cisplatin protocol or mitotane alone)3 was used in 4 further cases at the time of disease progression.
The tumors were generally large, with a mean diameter of 10 cm (range, 1.6 to 23 cm). The mean weight of the 20 tumors for which this information was available was 141.5 g (range, 8 to 1300 g). The neoplastic oncocytes were similar in all cases, meeting their definition as cells with a homogenously deeply eosinophilic and granular cytoplasm. They had frequently atypical or bizarre nuclei with prominent nucleoli (Fuhrman grade 3 or 4), and eosinophilic nuclear pseudoinclusions were common. As these Turin cases exhibited a more or less extensive population of oncocytes, 2 subgroups could be identified.
FIGURE 1. OACT. (A, B, case 4) Pure form with homogenous growth of large atypical cells. In a serial section (B), the reticulin framework appears to be entirely preserved. The tumor has a low proliferative index (1%, inset). This case was scored as being of UMP according to the L-W-B classification. A malignant OACT according to L-W-B is shown in C and D (case 13), in which a diffuse growth of medium/ small-sized oncocytes (C) is associated with a reticulin network disruption (D). The proliferative index was 10% (C, inset). In mixed OACTs (E, F, case 22) 2 cell populations coexist, with oncocytic cells (E, bottom left) having a very low proliferative index (E, inset bottom left) adjacent to conventional carcinoma areas (E, top right) with a proliferative index up to 40% (E inset, top right). These 2 components have a different reticulin framework pattern, appearing intact in oncocytic areas (F, bottom left) and disrupted in conventional carcinoma (F, top right). In pure OACTs, either classical, large, granular oncocytes (G case 4) or small, mildly atypical oncocytes (H, case 13) can be observed [hematoxylin and eosin, original magnification x200 in A, C, E; x400 in G, H. Insets: immunoperoxidase for Ki67; original magnification x400 in A, C; x200 in E. Histochemical reticulin stain, original magnification x200 in B, D, F].
Pure OACT (15 cases)
This main subgroup included OACTs (cases 1 to 15) in which oncocytes accounted for > 90% of the tumor area (Fig. 1). The tumor cells were arranged predominantly or entirely in a diffuse, “patternless pattern,” with trabecular,
alveolar, and microcystic areas occasionally observed as a minor component. A variable cell size was observed, with 4 of 15 cases (cases 8, 9, 12, and 13) being composed predominantly of small oncocytes. These cells had a deeply eosinophilic cytoplasm but a reduced volume and small,
A
B
C
D
E
F
G
3
G
H
centrally located, mildly atypical nuclei. Bizarre multi- nucleated giant cells, patchy mononuclear inflammatory cell infiltrates, and fibromyxoid stroma were occasionally observed. One tumor was associated with a myelolipoma- tous component. Additional pathologic parameters included necrosis in 7 patients; mitotic index was < 5/50 high-power fields in 14 cases, and only in 1 case (# 15) was there a relatively high mitotic count (15/50 HPF) with atypical mitotic figures. Invasion of vessels and/or capsules was a feature of 5 and 3 cases, respectively. As a result, on the basis of the proposed L-W-B system for OACT (which excludes the so-called “definitional criteria” of the classical Weiss parameters), a diagnosis of malignancy was con- firmed in 6 tumors only. These fulfilled ≥ 1 of the “major criteria” of the L-W-B system (mitotic rate > 5/50 HPF, atypical mitoses, and venous invasion). Of the remaining cases, 8 were classified as OACT of uncertain malignant potential (UMP), as only 1 or more “minor criteria” (necrosis, capsular invasion, sinusoidal invasion, diameter > 10 cm, and/or weight > 200 g) were identified, and 1 was classified as a benign case. Five of the UMP cases defined by L-W-B were benign on the basis of the “reticulin” algorithm (see below).
Mixed and Focal OACTs (12 cases)
This second subgroup included 12 cases (#16 to 27), in which oncocytes constituted 30% to 80% of the tumor area. The cytoarchitectural features of the oncocytic component were comparable to that of pure OACT. The oncocytic cell population was either separate from, or intermingled with, the non-oncocytic component (Fig. 1E). At variance with pure OACT cases, a prevalence of medium-sized to small-sized oncocytes was observed. The non-oncocytic cell population was composed of pleo- morphic atypical cells having a diffuse growth pattern, as seen in conventional ACC. As the L-W-B system was originally devised for pure OACT only and could not be applied to this mixed/focal oncocytic tumor subgroup, we applied the classical Weiss scoring system here, which recognized 6 benign and 6 malignant cases. Using the “reticulin” algorithm, 7 cases were scored benign and the remaining 5 were qualified as malignant (see below).
Immunohistochemical and Histochemical Features
Immunoprofile and Tumor Proliferation Fraction
All tumors in this series shared the expression of at least 1 of the more reliable markers of the adrenal cortex, namely, melan A and a-inhibin. The mean Ki-67 OACTs (range, 1% to 20%), with mean values of 1% for the benign case, 2% for borderline tumors, and 6.5% for malignant ones. Among mixed/focal OACTs it was 6.8% (range, 1% to 40%), being 1.3% for benign and 12.3% for malignant cases.
Evaluation of Reticulin Framework and Application of the “Reticulin” Diagnostic Algorithm
As recently reported by our group, a histochemical staining for reticulin was performed in each case. This demonstrated disruption of the stromal framework in 16 of 27 (11 of 15 pure and 5 of 12 mixed) OACT cases of the Turin series and in 19 of 23 (15 of 16 pure and 4 of 7 mixed and focal) cases of the validation cohort (Tables 1 and 3). The reticulin disruption was either focal (13 cases) or extensive. In mixed/focal OACTs, disruption of the reticulin network was more evident in conventional (non-oncocytic) carcinoma components, whereas an intact reticulin frame- work, if present, was mostly recognized in the oncocytic component (Fig. 1).
An intact reticulin framework in the present series was observed in 11 cases (4 pure OACTs, 7 mixed OACTs), all composed of large-sized oncocytes. Three of 4 pure OACTs with an intact reticulin framework were classified as being of UMP by L-W-B criteria but as benign according to the “reticulin” algorithm. In addition, 1 mixed OACT was classified as malignant by the classical Weiss scoring system and as benign by the “reticulin” algorithm. Thus, all cases with intact reticulin were benign by the “reticulin” algorithm, and, indeed, this finding was associated with a nonmalignant condition in our cases. However, it should be noted that in this series of OACTs, apparently at variance with conven- tional ACCs, some malignant cases had only focal reticulin disruption that might be underestimated or missed in single sections. Using the reticulin stain as an inclusion criterion of the “reticulin” algorithm in the Turin series only, 13 cases (6 pure and 7 mixed OACTs) were categorized as benign and 14 as malignant (disrupted reticulin framework together with at least 1 of the following criteria: necrosis, high mitotic rate, and vascular invasion). Two cases with reticulin disruption but lacking any of the additional criteria for malignancy were classified as benign (Table 1).
Mitochondrial DNA Alterations
Prevalence of mtDNA “Common Deletion” as Detected by PCR
The presence of the mtDNA “common deletion” was successfully analyzed using real-time PCR in OACT cases having informative tissue available. In the Turin series, it was present in 12 of 22 cases (54%), including 6 of 13 pure and 6 of 9 mixed cases (Fig. 2). A similar positive proportion was observed in the validation cohort: 8 of 23 cases (35%), including 5 of 16 pure and 3 of 7 mixed (2) or focal (1) tumors. Overall in the 2 series, according to the L-W-B classification for pure OACT or according to the Weiss system for mixed and focal OACTs, the mtDNA deletion was found in 15 benign or UMP cases and in only 5 malignant OACTs. When compared with reticulin staining, 12 of 14 cases (86%) with an intact reticulin framework had the mtDNA common deletion, whereas a lower proportion of the reticulin-disrupted cases harbored the deletion (8 of 31, 26%); the difference was statistically significant (the Fisher test; P = 0.0003). In control cases of non-oncocytic adrenal
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tissues/tumors, the mtDNA common deletion was detected in 4 of 18 normal adrenal and 5 of 13 ACAs but in none of 50 conventional ACCs.
Tissue Patterns and Distribution of mtDNA “Common Deletion” by Means of FISH
FISH analysis was performed in all PCR-tested cases, and the results were reliable in all but 7 cases (mostly because of poor tissue preservation). There was 100% concordance between an altered FISH pattern and the PCR detection of the mtDNA common deletion (Fig. 2). The tissue and cellular distribution of such mtDNA alterations was not present uniformly in all tumor cells in any individual case. Rather, the alteration was limited to only a fraction of neoplastic cells in the majority of cases (intratissue heteroplasmy); it was not present in all mitochondria of deletion-bearing oncocytes (intracellular heteroplasmy). Interestingly, the presence of intracellular heteroplasmy was more commonly asso- ciated with a diagnosis of “malignancy,” whereas all 11 cases with intracellular homoplasmy were classified as benign or being of UMP, according to the L-W-B criteria.
Correlation of OACT Classification and mtDNA Alterations With Clinical Outcome
Follow-up data were available for all 27 cases in the Turin series. Although the number of cases is too small for survival analysis, the disease status was compared using the alternate classification systems (to test their ability to identify cases following a clinically malignant behavior) and with reticulin staining alone and with the presence of the mtDNA “common deletion.” Two clinically aggressive cases were classified as being of UMP, rather than malignant, using the L-W-B proposal for OACT classification. When the reticulin stain alone or the “reticulin” algorithm was applied, specificity values of 57% and 62% were observed, respectively, but both methods were 100% sensitive in identifying cases with recurrence or disease-related deaths. A statistical correla- tion between the reticulin algorithm and the L-W-B system could not be performed because of possible selection bias and small sample size. However, the “reticulin” algorithm, which was initially proposed for conventional ACC,61 appeared to be diagnostically applicable in the oncocytic group of adrenocortical
lesions as well. By contrast, the presence of the mtDNA “common deletion” was not invariably associated with a more favorable clinical outcome, as it was also detected in a single patient who died of disease 20 months after diagnosis.
DISCUSSION
In this study, we reviewed the pathologic features of an unpublished series of 27 adrenocortical tumors, the large majority having more or less extensive oncocytic features, and confirmed the diagnostic value of reticulin staining and of a diagnostic algorithm recently proposed by our group in distinguishing clinically malignant from benign OACTs. In addition, we traced the mtDNA deletion commonly described in oncocytic tumors of other organs in a proportion of OACTs, mostly in cases with an intact reticulin framework, although rare clinically aggressive cases also bore the deletion.
Diagnostic Features of OACT
A large series of 194 ACCs and 116 ACAs was reviewed to search for oncocytic features. In all, 27 cases (approximately 10% of the whole series) met the classic morphologic criteria for the diagnosis of oncocytic tumors of other organs,4,12 and of the adrenal gland in particular.7,8,35,65
These include a tumor cell population of large cells with eosinophilic and granular cytoplasm, generally showing a diffuse growth pattern and high-grade nuclear atypia.7,8,35,65 By definition (pure), OACTs were com- posed (almost) exclusively (> 90%) of oncocytic cells; however, a minor proportion of cases (12 of 20 in the Turin series and 7 of 23 in the validation group) showed a variable component of (non-oncocytic) adrenocortical clear tumor cells (mixed OACT and focal OACT). Interestingly, a number of cases, all pure OACTs, were composed of predominantly small cells. This was an occurrence that had been already observed7,65 and that, in contrast to the findings reported in poorly differentiated oncocytic carcinomas of the thyroid gland,46 did not predict a different behavior in the OACT. Further unusual findings were: (i) the presence of stromal myxoid foci, which were seen in a single case of the Turin series, although not to the extent to allow their classification as the OACT counterpart of the myxoid variant of conven- tional ACC47; and (ii) the presence of a myelolipomatous component in a case of pure OACT. Both fibromyxoid stromal foci and a lipomatous component in pure OACT were also described in the validation series.6
The definition of malignancy in OACT is problematic if the classical Weiss criteria are applied for classification, as first suspected by Lin et al.38 In fact, 20 of 27 cases from the Turin series, having significant numbers of cells with eosinophilic cytoplasm, nuclear atypia, and diffuse growth (“definitional criteria”7,8), achieved the minimal Weiss score 3 for a diagnosis of malignancy, thus suggesting a risk of overestimating potential malignancy in the OACT tumor group if the Weiss scoring system is used as a
diagnostic tool. Therefore, following our recent proposal of a newly designed algorithm for the diagnosis of conven- tional ACC,61 we tested its diagnostic usefulness in the group of OACTs. The recognition of the presence of a disrupted reticulin network, together with the presence of any of 3 clear-cut morphologic signs of malignancy (high mitotic index, necrosis, and vascular invasion), was able to segregate clinically benign from malignant cases with optimal performance, with no cases in the “benign” group following an aggressive course included, provided that an accurate search for even focal reticulin disruption was conducted. Indeed, although in conventional adrenocortical tumors all malignant (ie, Weiss score ≥3) cases had disrupted reticulin framework, in OACTs the reticulin pattern was more heterogenous, possibly because of the association with (residual?) low-grade “oncocytoma” areas in a fraction of cases. Reticulin distribution pattern or loss in OACTs (both pure, mixed, and focal) likely deserves the same attention that pathologists devote when examining difficult or equivocal tumoral hepatocellular lesions. Thus, a careful search for even focal reticulin disruption is necessary in more than one paraffin block before reliably excluding any reticulin alteration. Notably, in mixed and focal OACTs, reticulin disruption was more evident in conven- tional ACC areas, whereas in some cases the oncocytic component showed a preserved reticulin framework. In any case, this simplified algorithmic approach has the advantage of reducing the evaluation of multiple Weiss parameters, which in OACTs are either not applicable (“definitional criteria” according to the L-W-B scheme) or may be of equivocal and/or subjective definition (such as sinusoidal and capsular invasion).
Apart from these practical diagnostic considerations, it should be stressed that pure OACTs are “low-grade” lesions, with a low mean Weiss score, frequent preservation of the reticulin network, low mitotic and Ki67 proliferation indices, and uncommon capsular or vascular invasion. Most tumors followed an indolent clinical course even when only un- equivocally morphologically malignant cases were consid- ered. Although a direct comparison with conventional ACC was not possible because of the limited number of OACT cases, a better outcome was observed, with only 3 of 14 pure OACTs in our series developing recurrence or metastases (although in 2 cases follow-up was still short, < 24 mo). These outcomes approximate those of “oncocytomas” of some other organs (kidney and salivary gland), which generally follow an indolent course and are in any case associated with a lower grade compared with their conven- tional carcinoma counterparts. These data are in accordance with the previous study by Wong et al,65 who provided the first preliminary statistical evidence of an improved prognosis of malignant OACT as compared with conventional ACC (overall median survival of 58 mo vs. 14 to 32 mo).
By contrast, whether mixed and focal OACTs share the same favorable behavior remains to be proven, although we strongly suspect that this will not be the case. In our very limited data set of 5 malignant cases with follow-up (all from Turin; range 16 to 165 mo) 3 patients died of disease within 20 to 97 months, 2 of whom died in < 24 months.
Prevalence of Mitochondrial DNA “Common Deletion” in OACT
The identification of pathologic and clinical simila- rities with oncocytic tumors of other organs led us to question whether OACTs may also display molecular abnormalities in the mtDNA. We focused on the analysis of the presence of the mtDNA 4977 bp “common deletion” using 2 alternative methods, a PCR-based technique and a FISH procedure, to establish the cell and tissue localization of the molecular changes. The “common deletion” is the most common somatic mtDNA abnormality detected in oncocytic tumors of various organs.37,44,59 It occurs between two 13-bp direct repeats (at positions 13447 to 13459 and 8470 to 8482) of the human mtDNA. This portion of mtDNA encompasses 5 transfer ribonucleic acid genes and 7 genes encoding subunits of cytochrome c oxidase, complex I, and ATPases.53 By means of PCR, the mtDNA “common deletion” was detected in approximately 50% of cases, both in the Turin cases and in the external validation cases. Interestingly, such mtDNA alteration was slightly more prevalent in pure OACTs and in cases classified as benign or of UMP, according to both the L-W-B classification and the “reticulin” algorithm. However, although these findings are of potential pathogenetic interest, to date, the analysis of the presence of the mtDNA “common deletion” has no practical diagnostic value. Further, to answer the question whether the “common deletion” of mtDNA was a feature of all cells of individual oncocytic tumors (as a result of a “clonal” event) or a heterogenous event, FISH analysis was performed with probes specific for the preserved (green) and deleted (red) regions, as previously described in the Warthin tumor model of salivary glands.36 The con- cordance between PCR and FISH methods was 100%. There was a relatively high degree of intercellular and intracellular heteroplasmy, with the deletion being detected in some, but not in other, tumor areas and heterogenously within single tumor cells in most cases. This observation probably indicates that this feature is not clonally transmitted to all neoplastic cells and probably represents an early change in the mtDNA, which is maintained in a subpopulation of oncocytes only and likely has no role in malignant progression. Rather, it could act as a tumor suppression mutation. 7 In support of this hypothesis, the same mtDNA alteration was detected at a comparable rate in control cases of normal adrenal cortex and ACA but not in conventional ACC. It should be noted that such mtDNA deletion is specific not only for oncocytic tumors but has also been reported in normal tissues and in several pathologic conditions not specifically associated with an oncocytic phenotype, including non-neoplastic9,14,15,19,27,28,30,31,41,49,66 and neo- plastic conditions. 1,13,16,20,29,32,33,43,52,57
In conclusion, (a) OACTs represent a heterogenous group of tumors with morphologic features resembling those of oncocytic neoplasms of other locations; (b) the “reticulin” diagnostic algorithm seems to recognize clinically aggressive cases well and seems to overcome
the risk of underestimating malignancy in this tumor group; (c) malignant OACTs, especially in their pure form, are low-grade tumors with a more indolent clinical course as compared with conventional ACCs; (d) the mtDNA “common deletion” described in other oncocytic human tumors is also present in a fraction of OACTs (approximately 50%); (e) such mtDNA alteration is also present in the normal adrenal cortex and in ACAs but has never been identified in conventional ACCs.
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