- Email: [email protected]
Oncocytic rectal adenocarcinomas
Human Pathology (2009) 40, 478–483
www.elsevier.com/locate/humpath
Original contribution
Oncocytic rectal adenocarcinomas Runjan Chetty MB BCh, FRCPath, DPhil a,⁎, Stefano Serra MD a , Erin Kennedy MD, PhD b , Dhirendra Govender MB ChB, FCPath, FRCPath c a
Department of Pathology, University Health Network and University of Toronto, Toronto, Canada M5G 2C4 Department of Surgery, University Health Network and University of Toronto, Toronto, Canada M5G 2C4 c Division of Anatomical Pathology, University of Cape Town, Cape Town 2001, South Africa b
Received 3 July 2008; revised 2 October 2008; accepted 3 October 2008
Keywords: Rectal cancer; Oncocytes; Eosinophilic cells
Summary Oncocytic rectal carcinomas are rare and have only been documented sporadically. Oncocytes are encountered in 2 distinct settings: after preoperative chemoradiation (commoner) and without antecedent chemoradiation (uncommon). The aim of this study was to ascertain the incidence and clinicopathologic features of rectal cancers with a significant (N25%) component of oncocytes in cases not receiving chemoradiation. Of 72 cases encountered over the study period, 8 fulfilled the criteria as oncocytes. These tumors, except for the cellular component of oncocytes, were similar to conventional adenocarcinomas pathologically and immunophenotypically. Cells with eosinophilic cytoplasm are commonly seen in rectal adenocarcinomas, and they should be separated from oncocytic examples. True oncocytes may be seen in conventional adenocarcinomas as individual cells or glands, especially at the infiltrating edge of the tumor. All 8 cases appeared to have behaved aggressively with rapid local and/or distant spread over a short duration. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction Oncocytic tumors are neoplasms that are composed of large cells with abundant granular, eosinophilic cytoplasm and nuclei that are also large, vesicular, and harbor prominent acidophilic nucleoli. Cells fulfilling these criteria constitute several tumors and nontumorous conditions and are encountered in several organs. An abundance of mitochondria within the cytoplasm is the hallmark of these cells (so-called mitochondrion-rich cells) and imparts the oncocytic or deeply eosinophilic, granular appearance to the cells as observed in hematoxylin and eosin (H&E)–stained sections. Thus, the oncocytic phenotype is characterized by both cytoplasmic and nuclear features: large cells with a ⁎ Corresponding author. E-mail address: [email protected] (R. Chetty).
distinctive eosinophilic cytoplasmic, together with a vesicular nucleus and a prominent acidophilic nucleolus. However, it is must be remembered that an eosinophilic cytoplasm can also be due to other nonribosomal intracellular organelles such as neurosecretory granules, lysosomes, secretory (exocrine) granules, cytoplasmic filaments, and smooth endoplasmic reticulum [1]. Such cells, however, lack the abundant cytoplasm and nuclear features associated with oncocytes. It is generally accepted that oncocytic or oxyphilic cells are synonymous and due to intracytoplasmic accumulation of mitochondria. Tumors containing oncocytes occur more frequently at some sites, whereas they represent rare histologic variants at others. Oncocytic tumors form a distinctive subtype of tumor in the thyroid gland (Hürthle cell tumors) [2], kidney (oncocytoma) [3], salivary glands (as oncocytosis, oncocytic nodules or as part of a Warthin tumor) [4], and in endocrine
0046-8177/$ – see front matter Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.10.005
Oncocytic rectal adenocarcinomas
479
glands such as the pituitary, parathyroid, and adrenal (in adenomas and carcinomas) [4-6]. Oncocytic tumors can also occur rarely in other locations such as ovary [7,8], lung [6,9,10], breast [11], skin [12], and soft tissue [13]. Tumors with oncocytic features rarely affect the gastrointestinal tract, liver, and pancreas [14], although several tumors with prominent “pink” (eosinophilic) cytoplasm are recognizable in the digestive tract. Adenocarcinomas with oncocytic features have been described in the stomach [1517] as well as in the small intestine [6]. Tumor cells in colorectal carcinomas are known to contain eosinophilic cytoplasm and, thus, simulate oncocytes; however, these cells are not characterized by intracytoplasmic collections of mitochondria but rather other organelles that impart cytoplasmic eosinophilia. Rectal adenocarcinomas containing oncocytes have been encountered in 2 distinct settings. There have been 2 series describing oncocytic change in rectal adenocarcinomas after preoperative chemoradiation [18,19]. In addition, there have been 2 case reports of oncocytic rectal adenocarcinomas occurring without antecedent chemoradiation [20,21]. The current study was undertaken to ascertain the incidence of rectal adenocarcinomas that display a significant oncocytic phenotype without previous chemoradiation and detail their clinicopathologic characteristics.
containing 25% and more oncocytes of the total tumor cell population were labeled as “adenocarcinomas with a significant oncocytic component.” Cases containing less than 25% of the tumor population composed of oncocytes were also documented. A figure of 25% is arbitrary and was crudely determined by low-power scanning of the entire tumor. It was chosen as the threshold figure because an oncocytic tumor population of about 25% was observed readily at low magnification. None of the cases had a history (clinical or biopsy-proven of any serrated adenomas or other unusual adenoma). None of the cases were TME resections. All cases with a history of preoperative chemoradiation for any reason were excluded for the purposes of this study. In other words, none of the cases in the study group received preoperative chemoradiation for their rectal cancers or any other cancer. The following immunohistochemical panel was done on formalin-fixed tissue using the standard streptavidin-biotin complex technique: CK7, CK20, CDX-2, MSH-2 and MLH1, chromogranin, synaptophysin, MIB-1, and β-catenin (done on 7 cases). Standard transmission electron microscopy was performed on tissue from the paraffin blocks in 7 cases. Clinical follow-up was obtained on 7 of the cases. The outcome of the oncocytic cancers were compared with nononcocytic cases of the same age, grade, and stage.
2. Materials and methods 3. Results Seventy-two resection specimens for rectal adenocarcinoma received over an 8-year period at University Health Network, Toronto, Canada, were examined for this study. In addition, one case from Cape Town, South Africa, was referred with a diagnosis of oncocytic rectal adenocarcinoma for a second opinion. Relevant clinicopathologic and demographic information was collected. All slides containing primary tumor (slides examined containing primary tumor ranged from 5 to 10) and metastases were reevaluated looking specifically for the presence of oncocytes. Oncocytes were defined as large cells with voluminous eosinophilic cytoplasm and large vesicular nuclei and prominent nucleoli. In addition, the nuclei tend to be located in the center of the cell rather than basally. Cases containing cohesive aggregates Table 1
Grossly, 5 tumors appeared as ulcerating crateriform lesions and 3 were exophytic tumors with surface ulceration (see Table 1). Eight cases fulfilled the cytomorphologic and content criteria to be classified as adenocarcinomas with an oncocytic component equal to or greater than 25% of the tumor population. Four cases contained more than 80% of the tumor cells being oncocytic, whereas the remaining 4 contained 25% to 30% oncocytes. There were 4 males and 4 females ranging in age from 48 to 94 years. The tumors varied in maximal diameter from 8.5 to 2.0 cm (mean size, 4.95 cm). Three cases were T2 tumors, 4 were T3, and 1 was T4. Four cases had lymph node involvement (3 were N1 and one was N2).
Clinicopathologic and immunohistochemical results
Case
Age
Sex
Size (cm)
1 2 3 4 5 6 7 8
75 75 48 94 70 53 78 52
F F F F M M M M
2.0 × 1.0 × 5.3 × 4.2 × 2.5 × 2.5 × 3.8 × 3.0 × 6.0 × 5.5 × 8.5 × 6.0 × 5.5 × 3.3 × 6.0 × 1.0 ×
1.0 1.8 1.5 3.0 1.0 3.0 1.0 0.8
Stage
CK20
CK7
MMR proteins
Follow-up
T2 T2 T4 T3 T3 T2 T3 T3
Positive Positive Positive Positive Positive Positive Positive Positive
Positive Positive Positive Negative Negative Negative Negative Negative
Normal Normal Normal Normal Normal Normal Normal Normal
Alive with local recurrence Presumed dead Alive with local recurrence Presumed dead Died with liver metastases Alive with local recurrence Alive; no recurrence Alive with liver metastases
N0 N0 N1 N1 N1 N0 N0 N2
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Fig. 1 A and B, An example of a conventional rectal adenocarcinoma containing cells with abundant eosinophilic cytoplasm. These cells are organelle-rich and the cytoplasmic eosinophilia should not be construed as oncocytic.
Fig. 2 A and B, True oncocytic cells have both nuclear and cytoplasmic characteristic features. The cells are large, have bloated deeply eosinophilic, slightly granular cytoplasm and nuclei that have vesicular chromatin and prominent nucleoli.
The microscopic appearance in all 8 cases was similar. There was a clear distinction between conventional adenocarcinoma and the oncocytic adenocarcinoma in 6 cases, whereas in 2 cases, both components intermingled more. Conventional adenocarcinoma consisted of cells with amphophilic or weakly eosinophilic cytoplasm and hyperchromatic nuclei. Occasional cells contained more abundant, eosinophilic cytoplasm simulating an oncocytic appearance (Fig. 1A and B). On the other hand, cells that were truly oncocytic were apparent under medium power and these were large cells, with strikingly eosinophilic cytoplasm and large vesicular nuclei containing prominent acidophilic nucleoli (Fig. 2A and B). The most pronounced oncocytic change was seen at the deep infiltrating edge of the tumor either as single cells or as small glands. In 2 cases, clearly visible remnants of a preexisting tubulovillous adenoma were present, and both these adenomas contained oncocytic cells. All cases were associated with extensive intraluminal necrosis and abscess formation, and 5 cases had prominent stromal acute inflammation and suppuration. Of 8 cases, 4
Fig. 3 A consistent feature in most glands composed of oncocytic cells was intraluminal calcification. In addition, stromal calcification and intraluminal necrosis were also noted.
Oncocytic rectal adenocarcinomas also contained intraluminal calcification (Fig. 3) and a further 2 cases displayed extensive stromal calcification. In the course of examining all the cases, we noted that true oncocytes were relatively frequently encountered in many of the conventional colorectal cancers occurring either as single cells admixed with more conventional tumor cells or small glands composed of oncocytes (Fig. 4A and B). The overall impression was that oncocytic cells can be found in many cases of colorectal cancer, especially at the infiltrating edge of tumors and associated with necrosis and/or acute inflammation. Thus, in addition to organelle-rich cells that resemble oncocytes, true oncocytes can be found intermingled with conventional adenocarcinoma cells. These cells should not be mistaken for oncocytes despite the obvious eosinophilic cytoplasm. They lack the nuclear changes of oncocytes: the nuclei tend to be basally located and show coarser chromatin with inconspicuous nucleoli than oncocytes. This change was also commonly encountered in all of the conventional colorectal adenocarcinomas, and the constituent cells should
481
Fig. 5 Electron microscopic from an oncocytic rectal adenocarcinoma showing the cytoplasm of a tumor cell to contain several dilated mitochondria.
be regarded as “eosinophilic” rather than truly oncocytic. For the purposes of this article, we have referred to these cells as “eosinophilic” or organelle-rich cells.
3.1. Immunohistochemical findings All cases were CDX-2 and CK20 positive, and 3 cases displayed CK7 positivity in both conventional and oncocytic foci (2 cases) and only in the oncocytic component in 1 case (case 2). Both MSH-2 and MLH-1 proteins were retained, and all cases were chromogranin and synaptophysin negative. Interestingly, in 6 of 7 cases, the MIB-1 staining of the oncocytic component at the infiltrating edge of the tumor was less than the surrounding non-oncocytic component. Furthermore, the oncocytes cells in 5 cases displayed nuclear β-catenin immunostaining, whereas the conventional adenocarcinomatous cells retained membrane localization of this protein. In one case, the oncocytic component also displayed membrane β-catenin staining. Electron microscopy showed tumor cells with numerous cytoplasmic mitochondria in keeping with oncocytes (Fig. 5). The follow-up period ranged from 3 to 36 months. One patient is alive and well without any evidence of recurrence, 3 patients are alive and well with recurrent local disease, 1 is alive with liver metastases, and 3 patients have died.
Fig. 4 Cells fulfilling the criteria for true oncocytes are also encountered in many conventional adenocarcinomas. These cells are interspersed with non-oncocytic cells (A) or form discrete oncocytic glands (B). These cells do not form a significant component of the carcinoma and are seen only focally.
4. Discussion Tallini [4] defined oncocytes as “cells exhibiting the characteristic phenotype, which features in histology sections finely granular eosinophilic cytoplasm and ultrastructurally
482 an increase in the number of mitochondria.” He also highlighted the distinction of oncocytes from “mitochondrion-rich” cells [4]. Tremblay and Pearse [22] suggested that the distinction between these 2 similar appearing cells was based on the fact that oncocytes contain more mitochondria than “mitochondrion-rich” cells and were associated with cytoplasmic swelling. This separation is somewhat inexact and subject to sampling variation and interpretive bias. The use of antimitochondrial antibody does not really aid as the antibody is nonspecific and will stain any cells containing mitochondria. It is for this reason that we relied on the morphologic appearance of cells and applied both nuclear and cytologic criteria to cells before classifying them as truly oncocytic. It may well be that several of these eosinophilic cells may have increased mitochondria and other organelles that impart the eosinophilic tincture, rather than only large, swollen mitochondria. Hence, the term organelle-rich is perhaps preferable as it more encompassing of the variety of intracytoplasmic structures that may result in eosinophilia: neurosecretory granules, lysosomes, exocrine secretory granules, various cytoplasmic filaments, and smooth endoplasmic reticulum. Oncocytic rectal adenocarcinomas have thus far been documented in 2 clinical contexts: 2 series describing oncocytic rectal carcinomas in resection specimens with the patients having received preoperative chemoradiation [18,19] and 2 separate case reports documenting the occurrence of oncocytes in patients without preoperative chemoradiation [20,21]. In the studies where the patients received preoperative chemoradiation, the initial biopsies in several of the patients were examined for oncocytes. Ambrosini-Spaltro and colleagues [18] examined the pre-chemoradiation biopsies before definitive surgery in 27 of 28 cases in their series. On routine H&E stained sections, oncocytes were identified in 16 of the 27 cases, and these cells accounted for 3% to 20% (mean of 5.3%) of the tumor population [18]. However, when they applied the antimitochondrial antibody to the sections, as much as 100% of neoplastic cells in some biopsies displayed immunopositivity [18]. This implies that cells, which were not recognizable as typical oncocytes on the H&E stain also but containing cytoplasmic mitochondria, were also positive for anti-mitochondrial antibody. These cells would include conventional adenocarcinoma cells as well as those that were eosinophilic, mitochondrion-rich but not truly oncocytes. This highlights the non-specificity of the antibody as it stains mitochondrion-containing cells indiscriminately and thus lacks utility in detecting true oncocytes and separating them from other mitochondrion-rich cells. Rouzbahman and colleagues [19] evaluated the pretreatment diagnostic biopsies in 5 of 7 cases with post-chemoradiation oncocytic rectal adenocarcinomas. None of the 5 biopsies examined contained oncocytes; however, these were small superficial biopsies and may not have been entirely representative. Extrapolating from the series reported by AmbrosiniSpaltro et al, and from examining 72 resection specimens in
R. Chetty et al. the current study, we conclude that eosinophilic cells are not an infrequent finding in rectal adenocarcinomas. Most cases harbor single eosinophilic cells or small clusters admixed with conventional adenocarcinoma. When true oncocytes were numerous and constituted 25% of the tumor population, they were readily visible in H&E-stained sections under medium- to low-power magnification. Whether found isolated or forming a significant percentage of the tumor population, oncocytes tended to have a characteristic location at the infiltrating, deep margin of the tumor, association with intraluminal necrosis, acute inflammation and calcification, stromal acute inflammation, and calcification. The only exceptions to this were 2 cases that harbored residual tubulovillous adenomas and thus had oncocytes more superficially located as well. Eosinophilic cells, on the other hand, tended to be more randomly distributed within conventional adenocarcinomas but were also seen in glands containing pathognomonic “dirty” necrosis. From this cohort of 8 cases, rectal adenocarcinomas containing more than a 25% oncocyte population seem to have progressed aggressively with the rapid development of recurrent local disease, liver metastasis, or death compared with age- and stage-matched non-oncocytic colorectal cancers. However, this appears only to be a trend based on a very limited number of cases, and it would be very difficult to prove that the more aggressive behavior is solely due to the oncocytic phenotype. The patient reported by Piscitelli and colleagues [20] had postoperative radiotherapy was well after 22 months, whereas the patient reported by Piana et al [21] was disease-free after 3 months. Oncocytic rectal adenocarcinomas (without preoperative chemoradiation), although differing from their conventional counterparts in being composed of tinctorially distinctive cells, appear to be similar immunophenotypically with all cases being CK20 positive. The 3 cases with CK7 immunoreactivity represent anomalous CK7 immunoexpression in rectal carcinoma, a phenomenon that has been recognized previously. Interestingly, in case 2, only the oncocytic component stained with CK7, whereas the conventional adenocarcinoma component was negative. The oncocytes were always seen associated with necrosis and inflammation, suggesting that they are an adaptive response to hypoxia and/or necrosis. It is known that the mitochondria within oncocytes are abnormal, but their relationship to actual tumors is still unknown [4]. The frequent occurrence of oncocytes in post-chemoradiation rectal adenocarcinoma is noteworthy. Twenty-four of 28 cases reported by Ambrosini-Spaltro et al [18] and 5 of 7 cases described by Rouzbahman et al [19] showed rectal adenocarcinomas with oncocytes being the dominant cell type. Most pretreatment biopsies in the former study contained variable numbers of oncocytes [18], and we noted that oncocytes are present in most rectal cancers we examined [19]. It is reasonable to postulate that oncocytes are resistant to and survive chemoradiation, remaining as the dominant tumor cells in residual rectal cancers. It is also possible that conventional adenocarcinoma
Oncocytic rectal adenocarcinomas cells become oncocytic as a result of preoperative chemoradiotherapy because pretreatment biopsies have shown them to be present only focally. In 6 of the cases from the current study, the MIB-1 labeling index of the oncocytic component at the infiltrating edge of the tumor was less than the non-oncocytic component. This implies that these cells are not proliferating as much and therefore could possibly be less susceptible to treatment. Another unusual feature seen in 5 of the cases is nuclear localization of β-catenin in oncocytic tumor cells at the leading edge. In contrast, 1 oncocytic case and the conventional adenocarcinoma cells showed membrane decoration for β-catenin. It is therefore possible that the oncocytes represent a “hardier” subset of tumor cells that are resistant to chemoradiation because they proliferate less than conventional adenocarcinoma cells. This would also support the frequent observation of oncocytic cells in residual rectal cancers that were treated with preoperative chemoradiation. This needs to be explored in a larger cohort of cases, especially those cases that have received preoperative chemoradiation. Eosinophilic cells have also been described in so-called colorectal adenocarcinomas with a “dome-like phenotype” [23]. However, this rare subset of colorectal carcinomas is characterized by an absence of necrosis and association with resident lymphoid tissue [23]. We therefore wish to highlight the occurrence of oncocytic rectal adenocarcinomas without antecedent chemoradiation; these tumors appear to be no different to their conventional counterparts. Oncocytes are present in most conventional adenocarcinomas where they form an “insignificant” proportion of the tumor cell population and they should be separated from eosinophilic, mitochondrion-rich cells that are also encountered frequently. Given that oncocytes can form the dominant tumor cell population in post-chemoradiation rectal adenocarcinomas, these cells may flag those cases that will respond poorly or inadequately to preoperative treatment. In addition, all 8 cases in this series appeared to behave aggressively with death in 3 cases within 36 months, suggesting that oncocytic rectal adenocarcinoma may be more aggressive than conventional adenocarcinoma of the rectum.
References [1] Nappi O, Ferrara G, Wick MR. Neoplasms composed of eosinophilic polygonal cells: an overview with consideration of different cytomorphologic patterns. Semin Diagn Pathol 1999;16:82-90.
483 [2] Rosai J, Carcangiu ML, DeLellis RA. Tumors of the thyroid. Atlas of tumor pathology, 3rd series, fascicle 5. Washington DC: Armed Forces Institute of Pathology; 1992. p. 161-82. [3] Perez-Ordonez B, Hamed G, Campbell S, et al. Renal oncocytoma: a clinicopathologic study of 70 cases. Am J Surg Pathol 1997;21: 871-83. [4] Tallini G. Oncocytic tumours. Virchows Arch 1998;433:5-12. [5] Bisceglia M, Ludovico O, Di Mattia A, et al. Adrenocortical oncocytic tumors: report of 10 cases and review of the literature. Int J Surg Pathol 2004;12:231-43. [6] Smirnova EA, Mikhailov IG. Electron microscopic characteristics of oncocytoma of the lung, small intestine and adrenal gland. Arkh Patol 1986;48:79-81. [7] Miranda D, Leite VH, de Azevedo Jr GM. Oncocytic adenocarcinoma of the ovary. Virchows Arch 1996;429:181-3. [8] Pitman MB, Young RH, Clement PB, et al. Endometrioid carcinoma of the ovary and endometrium, oxyphilic cell type: a report of nine cases. Int J Gynecol Pathol 1994;13:290-301. [9] Fernandez MA, Nyssen J. Oncocytoma of the lung. Can J Surg 1982; 25:332-3. [10] Santos-Briz A, Terron J, Sastre R, et al. Oncocytoma of the lung. Cancer 1977;40:1330-6. [11] Damiani S, Eusebi V, Losi L, et al. Oncocytic carcinoma (malignant oncocytoma) of the breast. Am J Surg Pathol 1998;22:221-30. [12] Roth MJ, Stern JB, Hijazi Y, et al. Oncocytic nodular hidradenoma. Am J Dermatopathol 1996;18:314-6. [13] Polk P, Parker KM, Biggs PJ. Soft tissue oncocytoma. HUM PATHOL 1996;27:206-8. [14] Papotti M, Cassoni P, Taraglio S, et al. Oncocytic and oncocytoid tumors of the exocrine pancreas, liver, and gastrointestinal tract. Semin Diagn Pathol 1999;16:126-34. [15] Caruso RA, Fabiano V, Rigoli L, et al. Focal parietal cell differentiation in a well-differentiated (intestinal-type) early gastric cancer. Ultrastruct Pathol 2000;24:417-22. [16] Takubo K, Honma N, Sawabe M, et al. Oncocytic adenocarcinoma of the stomach: parietal cell carcinoma. Am J Surg Pathol 2002;26: 458-65. [17] Yang GY, Liao J, Cassai ND, et al. Parietal cell carcinoma of gastric cardia: immunophenotype and ultrastructure. Ultrastruc Pathol 2003;27:87-94. [18] Ambrosini-Spaltro A, Salvi F, Betts CM, et al. Oncocytic modifications in rectal adenocarcinomas after radio and chemotherapy. Virchows Arch 2006;448:442-8. [19] Rouzbahman M, Serra S, Chetty R. Rectal adenocarcinoma with oncocytic features: possible relationship with preoperative chemoradiotherapy. J Clin Pathol 2006;59:1039-43. [20] Piscitelli D, Ingravallo G, Resta L, et al. Oncocytic adenocarcinoma of the rectum with diffuse intra-luminal microcalcifications: the first reported case. Virchows Arch 2003;443:579-82. [21] Piana S, Asiloi S, Foroni M. Oncocytic adenocarcinoma of the rectum arising on a villous adenoma with oncocytic features. Virchows Arch 2006;448:228-31. [22] Tremblay G, Pearse AGE. Histochemistry of oxidative enzyme systems in the human thyroid with special reference to Ashkanazy cells. J Pathol Bacteriol 1960;80:353-8. [23] Asmussen L, Pachler J, Holck S. Colorectal carcinoma with dome-like phenotype: an under-recognised subset of colorectal carcinoma? J Clin Pathol 2008;61:482-6.
www.elsevier.com/locate/humpath
Original contribution
Oncocytic rectal adenocarcinomas Runjan Chetty MB BCh, FRCPath, DPhil a,⁎, Stefano Serra MD a , Erin Kennedy MD, PhD b , Dhirendra Govender MB ChB, FCPath, FRCPath c a
Department of Pathology, University Health Network and University of Toronto, Toronto, Canada M5G 2C4 Department of Surgery, University Health Network and University of Toronto, Toronto, Canada M5G 2C4 c Division of Anatomical Pathology, University of Cape Town, Cape Town 2001, South Africa b
Received 3 July 2008; revised 2 October 2008; accepted 3 October 2008
Keywords: Rectal cancer; Oncocytes; Eosinophilic cells
Summary Oncocytic rectal carcinomas are rare and have only been documented sporadically. Oncocytes are encountered in 2 distinct settings: after preoperative chemoradiation (commoner) and without antecedent chemoradiation (uncommon). The aim of this study was to ascertain the incidence and clinicopathologic features of rectal cancers with a significant (N25%) component of oncocytes in cases not receiving chemoradiation. Of 72 cases encountered over the study period, 8 fulfilled the criteria as oncocytes. These tumors, except for the cellular component of oncocytes, were similar to conventional adenocarcinomas pathologically and immunophenotypically. Cells with eosinophilic cytoplasm are commonly seen in rectal adenocarcinomas, and they should be separated from oncocytic examples. True oncocytes may be seen in conventional adenocarcinomas as individual cells or glands, especially at the infiltrating edge of the tumor. All 8 cases appeared to have behaved aggressively with rapid local and/or distant spread over a short duration. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction Oncocytic tumors are neoplasms that are composed of large cells with abundant granular, eosinophilic cytoplasm and nuclei that are also large, vesicular, and harbor prominent acidophilic nucleoli. Cells fulfilling these criteria constitute several tumors and nontumorous conditions and are encountered in several organs. An abundance of mitochondria within the cytoplasm is the hallmark of these cells (so-called mitochondrion-rich cells) and imparts the oncocytic or deeply eosinophilic, granular appearance to the cells as observed in hematoxylin and eosin (H&E)–stained sections. Thus, the oncocytic phenotype is characterized by both cytoplasmic and nuclear features: large cells with a ⁎ Corresponding author. E-mail address: [email protected] (R. Chetty).
distinctive eosinophilic cytoplasmic, together with a vesicular nucleus and a prominent acidophilic nucleolus. However, it is must be remembered that an eosinophilic cytoplasm can also be due to other nonribosomal intracellular organelles such as neurosecretory granules, lysosomes, secretory (exocrine) granules, cytoplasmic filaments, and smooth endoplasmic reticulum [1]. Such cells, however, lack the abundant cytoplasm and nuclear features associated with oncocytes. It is generally accepted that oncocytic or oxyphilic cells are synonymous and due to intracytoplasmic accumulation of mitochondria. Tumors containing oncocytes occur more frequently at some sites, whereas they represent rare histologic variants at others. Oncocytic tumors form a distinctive subtype of tumor in the thyroid gland (Hürthle cell tumors) [2], kidney (oncocytoma) [3], salivary glands (as oncocytosis, oncocytic nodules or as part of a Warthin tumor) [4], and in endocrine
0046-8177/$ – see front matter Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.10.005
Oncocytic rectal adenocarcinomas
479
glands such as the pituitary, parathyroid, and adrenal (in adenomas and carcinomas) [4-6]. Oncocytic tumors can also occur rarely in other locations such as ovary [7,8], lung [6,9,10], breast [11], skin [12], and soft tissue [13]. Tumors with oncocytic features rarely affect the gastrointestinal tract, liver, and pancreas [14], although several tumors with prominent “pink” (eosinophilic) cytoplasm are recognizable in the digestive tract. Adenocarcinomas with oncocytic features have been described in the stomach [1517] as well as in the small intestine [6]. Tumor cells in colorectal carcinomas are known to contain eosinophilic cytoplasm and, thus, simulate oncocytes; however, these cells are not characterized by intracytoplasmic collections of mitochondria but rather other organelles that impart cytoplasmic eosinophilia. Rectal adenocarcinomas containing oncocytes have been encountered in 2 distinct settings. There have been 2 series describing oncocytic change in rectal adenocarcinomas after preoperative chemoradiation [18,19]. In addition, there have been 2 case reports of oncocytic rectal adenocarcinomas occurring without antecedent chemoradiation [20,21]. The current study was undertaken to ascertain the incidence of rectal adenocarcinomas that display a significant oncocytic phenotype without previous chemoradiation and detail their clinicopathologic characteristics.
containing 25% and more oncocytes of the total tumor cell population were labeled as “adenocarcinomas with a significant oncocytic component.” Cases containing less than 25% of the tumor population composed of oncocytes were also documented. A figure of 25% is arbitrary and was crudely determined by low-power scanning of the entire tumor. It was chosen as the threshold figure because an oncocytic tumor population of about 25% was observed readily at low magnification. None of the cases had a history (clinical or biopsy-proven of any serrated adenomas or other unusual adenoma). None of the cases were TME resections. All cases with a history of preoperative chemoradiation for any reason were excluded for the purposes of this study. In other words, none of the cases in the study group received preoperative chemoradiation for their rectal cancers or any other cancer. The following immunohistochemical panel was done on formalin-fixed tissue using the standard streptavidin-biotin complex technique: CK7, CK20, CDX-2, MSH-2 and MLH1, chromogranin, synaptophysin, MIB-1, and β-catenin (done on 7 cases). Standard transmission electron microscopy was performed on tissue from the paraffin blocks in 7 cases. Clinical follow-up was obtained on 7 of the cases. The outcome of the oncocytic cancers were compared with nononcocytic cases of the same age, grade, and stage.
2. Materials and methods 3. Results Seventy-two resection specimens for rectal adenocarcinoma received over an 8-year period at University Health Network, Toronto, Canada, were examined for this study. In addition, one case from Cape Town, South Africa, was referred with a diagnosis of oncocytic rectal adenocarcinoma for a second opinion. Relevant clinicopathologic and demographic information was collected. All slides containing primary tumor (slides examined containing primary tumor ranged from 5 to 10) and metastases were reevaluated looking specifically for the presence of oncocytes. Oncocytes were defined as large cells with voluminous eosinophilic cytoplasm and large vesicular nuclei and prominent nucleoli. In addition, the nuclei tend to be located in the center of the cell rather than basally. Cases containing cohesive aggregates Table 1
Grossly, 5 tumors appeared as ulcerating crateriform lesions and 3 were exophytic tumors with surface ulceration (see Table 1). Eight cases fulfilled the cytomorphologic and content criteria to be classified as adenocarcinomas with an oncocytic component equal to or greater than 25% of the tumor population. Four cases contained more than 80% of the tumor cells being oncocytic, whereas the remaining 4 contained 25% to 30% oncocytes. There were 4 males and 4 females ranging in age from 48 to 94 years. The tumors varied in maximal diameter from 8.5 to 2.0 cm (mean size, 4.95 cm). Three cases were T2 tumors, 4 were T3, and 1 was T4. Four cases had lymph node involvement (3 were N1 and one was N2).
Clinicopathologic and immunohistochemical results
Case
Age
Sex
Size (cm)
1 2 3 4 5 6 7 8
75 75 48 94 70 53 78 52
F F F F M M M M
2.0 × 1.0 × 5.3 × 4.2 × 2.5 × 2.5 × 3.8 × 3.0 × 6.0 × 5.5 × 8.5 × 6.0 × 5.5 × 3.3 × 6.0 × 1.0 ×
1.0 1.8 1.5 3.0 1.0 3.0 1.0 0.8
Stage
CK20
CK7
MMR proteins
Follow-up
T2 T2 T4 T3 T3 T2 T3 T3
Positive Positive Positive Positive Positive Positive Positive Positive
Positive Positive Positive Negative Negative Negative Negative Negative
Normal Normal Normal Normal Normal Normal Normal Normal
Alive with local recurrence Presumed dead Alive with local recurrence Presumed dead Died with liver metastases Alive with local recurrence Alive; no recurrence Alive with liver metastases
N0 N0 N1 N1 N1 N0 N0 N2
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R. Chetty et al.
Fig. 1 A and B, An example of a conventional rectal adenocarcinoma containing cells with abundant eosinophilic cytoplasm. These cells are organelle-rich and the cytoplasmic eosinophilia should not be construed as oncocytic.
Fig. 2 A and B, True oncocytic cells have both nuclear and cytoplasmic characteristic features. The cells are large, have bloated deeply eosinophilic, slightly granular cytoplasm and nuclei that have vesicular chromatin and prominent nucleoli.
The microscopic appearance in all 8 cases was similar. There was a clear distinction between conventional adenocarcinoma and the oncocytic adenocarcinoma in 6 cases, whereas in 2 cases, both components intermingled more. Conventional adenocarcinoma consisted of cells with amphophilic or weakly eosinophilic cytoplasm and hyperchromatic nuclei. Occasional cells contained more abundant, eosinophilic cytoplasm simulating an oncocytic appearance (Fig. 1A and B). On the other hand, cells that were truly oncocytic were apparent under medium power and these were large cells, with strikingly eosinophilic cytoplasm and large vesicular nuclei containing prominent acidophilic nucleoli (Fig. 2A and B). The most pronounced oncocytic change was seen at the deep infiltrating edge of the tumor either as single cells or as small glands. In 2 cases, clearly visible remnants of a preexisting tubulovillous adenoma were present, and both these adenomas contained oncocytic cells. All cases were associated with extensive intraluminal necrosis and abscess formation, and 5 cases had prominent stromal acute inflammation and suppuration. Of 8 cases, 4
Fig. 3 A consistent feature in most glands composed of oncocytic cells was intraluminal calcification. In addition, stromal calcification and intraluminal necrosis were also noted.
Oncocytic rectal adenocarcinomas also contained intraluminal calcification (Fig. 3) and a further 2 cases displayed extensive stromal calcification. In the course of examining all the cases, we noted that true oncocytes were relatively frequently encountered in many of the conventional colorectal cancers occurring either as single cells admixed with more conventional tumor cells or small glands composed of oncocytes (Fig. 4A and B). The overall impression was that oncocytic cells can be found in many cases of colorectal cancer, especially at the infiltrating edge of tumors and associated with necrosis and/or acute inflammation. Thus, in addition to organelle-rich cells that resemble oncocytes, true oncocytes can be found intermingled with conventional adenocarcinoma cells. These cells should not be mistaken for oncocytes despite the obvious eosinophilic cytoplasm. They lack the nuclear changes of oncocytes: the nuclei tend to be basally located and show coarser chromatin with inconspicuous nucleoli than oncocytes. This change was also commonly encountered in all of the conventional colorectal adenocarcinomas, and the constituent cells should
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Fig. 5 Electron microscopic from an oncocytic rectal adenocarcinoma showing the cytoplasm of a tumor cell to contain several dilated mitochondria.
be regarded as “eosinophilic” rather than truly oncocytic. For the purposes of this article, we have referred to these cells as “eosinophilic” or organelle-rich cells.
3.1. Immunohistochemical findings All cases were CDX-2 and CK20 positive, and 3 cases displayed CK7 positivity in both conventional and oncocytic foci (2 cases) and only in the oncocytic component in 1 case (case 2). Both MSH-2 and MLH-1 proteins were retained, and all cases were chromogranin and synaptophysin negative. Interestingly, in 6 of 7 cases, the MIB-1 staining of the oncocytic component at the infiltrating edge of the tumor was less than the surrounding non-oncocytic component. Furthermore, the oncocytes cells in 5 cases displayed nuclear β-catenin immunostaining, whereas the conventional adenocarcinomatous cells retained membrane localization of this protein. In one case, the oncocytic component also displayed membrane β-catenin staining. Electron microscopy showed tumor cells with numerous cytoplasmic mitochondria in keeping with oncocytes (Fig. 5). The follow-up period ranged from 3 to 36 months. One patient is alive and well without any evidence of recurrence, 3 patients are alive and well with recurrent local disease, 1 is alive with liver metastases, and 3 patients have died.
Fig. 4 Cells fulfilling the criteria for true oncocytes are also encountered in many conventional adenocarcinomas. These cells are interspersed with non-oncocytic cells (A) or form discrete oncocytic glands (B). These cells do not form a significant component of the carcinoma and are seen only focally.
4. Discussion Tallini [4] defined oncocytes as “cells exhibiting the characteristic phenotype, which features in histology sections finely granular eosinophilic cytoplasm and ultrastructurally
482 an increase in the number of mitochondria.” He also highlighted the distinction of oncocytes from “mitochondrion-rich” cells [4]. Tremblay and Pearse [22] suggested that the distinction between these 2 similar appearing cells was based on the fact that oncocytes contain more mitochondria than “mitochondrion-rich” cells and were associated with cytoplasmic swelling. This separation is somewhat inexact and subject to sampling variation and interpretive bias. The use of antimitochondrial antibody does not really aid as the antibody is nonspecific and will stain any cells containing mitochondria. It is for this reason that we relied on the morphologic appearance of cells and applied both nuclear and cytologic criteria to cells before classifying them as truly oncocytic. It may well be that several of these eosinophilic cells may have increased mitochondria and other organelles that impart the eosinophilic tincture, rather than only large, swollen mitochondria. Hence, the term organelle-rich is perhaps preferable as it more encompassing of the variety of intracytoplasmic structures that may result in eosinophilia: neurosecretory granules, lysosomes, exocrine secretory granules, various cytoplasmic filaments, and smooth endoplasmic reticulum. Oncocytic rectal adenocarcinomas have thus far been documented in 2 clinical contexts: 2 series describing oncocytic rectal carcinomas in resection specimens with the patients having received preoperative chemoradiation [18,19] and 2 separate case reports documenting the occurrence of oncocytes in patients without preoperative chemoradiation [20,21]. In the studies where the patients received preoperative chemoradiation, the initial biopsies in several of the patients were examined for oncocytes. Ambrosini-Spaltro and colleagues [18] examined the pre-chemoradiation biopsies before definitive surgery in 27 of 28 cases in their series. On routine H&E stained sections, oncocytes were identified in 16 of the 27 cases, and these cells accounted for 3% to 20% (mean of 5.3%) of the tumor population [18]. However, when they applied the antimitochondrial antibody to the sections, as much as 100% of neoplastic cells in some biopsies displayed immunopositivity [18]. This implies that cells, which were not recognizable as typical oncocytes on the H&E stain also but containing cytoplasmic mitochondria, were also positive for anti-mitochondrial antibody. These cells would include conventional adenocarcinoma cells as well as those that were eosinophilic, mitochondrion-rich but not truly oncocytes. This highlights the non-specificity of the antibody as it stains mitochondrion-containing cells indiscriminately and thus lacks utility in detecting true oncocytes and separating them from other mitochondrion-rich cells. Rouzbahman and colleagues [19] evaluated the pretreatment diagnostic biopsies in 5 of 7 cases with post-chemoradiation oncocytic rectal adenocarcinomas. None of the 5 biopsies examined contained oncocytes; however, these were small superficial biopsies and may not have been entirely representative. Extrapolating from the series reported by AmbrosiniSpaltro et al, and from examining 72 resection specimens in
R. Chetty et al. the current study, we conclude that eosinophilic cells are not an infrequent finding in rectal adenocarcinomas. Most cases harbor single eosinophilic cells or small clusters admixed with conventional adenocarcinoma. When true oncocytes were numerous and constituted 25% of the tumor population, they were readily visible in H&E-stained sections under medium- to low-power magnification. Whether found isolated or forming a significant percentage of the tumor population, oncocytes tended to have a characteristic location at the infiltrating, deep margin of the tumor, association with intraluminal necrosis, acute inflammation and calcification, stromal acute inflammation, and calcification. The only exceptions to this were 2 cases that harbored residual tubulovillous adenomas and thus had oncocytes more superficially located as well. Eosinophilic cells, on the other hand, tended to be more randomly distributed within conventional adenocarcinomas but were also seen in glands containing pathognomonic “dirty” necrosis. From this cohort of 8 cases, rectal adenocarcinomas containing more than a 25% oncocyte population seem to have progressed aggressively with the rapid development of recurrent local disease, liver metastasis, or death compared with age- and stage-matched non-oncocytic colorectal cancers. However, this appears only to be a trend based on a very limited number of cases, and it would be very difficult to prove that the more aggressive behavior is solely due to the oncocytic phenotype. The patient reported by Piscitelli and colleagues [20] had postoperative radiotherapy was well after 22 months, whereas the patient reported by Piana et al [21] was disease-free after 3 months. Oncocytic rectal adenocarcinomas (without preoperative chemoradiation), although differing from their conventional counterparts in being composed of tinctorially distinctive cells, appear to be similar immunophenotypically with all cases being CK20 positive. The 3 cases with CK7 immunoreactivity represent anomalous CK7 immunoexpression in rectal carcinoma, a phenomenon that has been recognized previously. Interestingly, in case 2, only the oncocytic component stained with CK7, whereas the conventional adenocarcinoma component was negative. The oncocytes were always seen associated with necrosis and inflammation, suggesting that they are an adaptive response to hypoxia and/or necrosis. It is known that the mitochondria within oncocytes are abnormal, but their relationship to actual tumors is still unknown [4]. The frequent occurrence of oncocytes in post-chemoradiation rectal adenocarcinoma is noteworthy. Twenty-four of 28 cases reported by Ambrosini-Spaltro et al [18] and 5 of 7 cases described by Rouzbahman et al [19] showed rectal adenocarcinomas with oncocytes being the dominant cell type. Most pretreatment biopsies in the former study contained variable numbers of oncocytes [18], and we noted that oncocytes are present in most rectal cancers we examined [19]. It is reasonable to postulate that oncocytes are resistant to and survive chemoradiation, remaining as the dominant tumor cells in residual rectal cancers. It is also possible that conventional adenocarcinoma
Oncocytic rectal adenocarcinomas cells become oncocytic as a result of preoperative chemoradiotherapy because pretreatment biopsies have shown them to be present only focally. In 6 of the cases from the current study, the MIB-1 labeling index of the oncocytic component at the infiltrating edge of the tumor was less than the non-oncocytic component. This implies that these cells are not proliferating as much and therefore could possibly be less susceptible to treatment. Another unusual feature seen in 5 of the cases is nuclear localization of β-catenin in oncocytic tumor cells at the leading edge. In contrast, 1 oncocytic case and the conventional adenocarcinoma cells showed membrane decoration for β-catenin. It is therefore possible that the oncocytes represent a “hardier” subset of tumor cells that are resistant to chemoradiation because they proliferate less than conventional adenocarcinoma cells. This would also support the frequent observation of oncocytic cells in residual rectal cancers that were treated with preoperative chemoradiation. This needs to be explored in a larger cohort of cases, especially those cases that have received preoperative chemoradiation. Eosinophilic cells have also been described in so-called colorectal adenocarcinomas with a “dome-like phenotype” [23]. However, this rare subset of colorectal carcinomas is characterized by an absence of necrosis and association with resident lymphoid tissue [23]. We therefore wish to highlight the occurrence of oncocytic rectal adenocarcinomas without antecedent chemoradiation; these tumors appear to be no different to their conventional counterparts. Oncocytes are present in most conventional adenocarcinomas where they form an “insignificant” proportion of the tumor cell population and they should be separated from eosinophilic, mitochondrion-rich cells that are also encountered frequently. Given that oncocytes can form the dominant tumor cell population in post-chemoradiation rectal adenocarcinomas, these cells may flag those cases that will respond poorly or inadequately to preoperative treatment. In addition, all 8 cases in this series appeared to behave aggressively with death in 3 cases within 36 months, suggesting that oncocytic rectal adenocarcinoma may be more aggressive than conventional adenocarcinoma of the rectum.
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