J
ORIGINAL
* SINCE 1925*
Carcinoma of unknown primary origin with isolated adrenal metastasis: a report of two cases
Mitsuhiro Kometani1),2), Takashi Yoneda1), Yuji Maeda3), Koushiro Ohtsubo4), Yuto Yamazaki5), Hiroko Ikeda6), Shunsuke Mori1), Daisuke Aono1), Shigehiro Karashima1), Mikiya Usukura1),7), Hironobu Sasano5) and Yoshiyu Takeda1),8)
1) Department of Health Promotion and Medicine of the Future, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa 920-8641, Japan
2) Basic and Advanced Residency Training Center, Kanazawa University Hospital, Kanazawa, Ishikawa 920-8641, Japan
3) Department of Urology, Public Central Hospital of Matto Ishikawa, Hakusan-shi, Ishikawa 924-0865, Japan
4) Division of Medical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-8641, Japan
5) Department of Pathology, Tohoku University, Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan
6) Division of Diagnostic Pathology, Kanazawa University Hospital, Ishikawa 920-8641, Japan
7) Department of Internal Medicine, Houju Memorial Hospital, Nomi, Ishikawa 923-1226, Japan
8) Department of Internal Medicine, Asanogawa General Hospital, Kanazawa, Ishikawa 910-8621, Japan
Abstract. The adrenal glands are one of the most common sites of malignant tumor metastasis. However, metastatic adrenal carcinoma of unknown primary origin with localized adrenal gland involvement is an extremely rare condition. Herein, we reported two cases of carcinoma of unknown primary origin with isolated adrenal metastasis. In the first case, back pain was the trigger; while in the second case, the triggers were low fever and weight loss. Metabolic abnormalities such as hypertension and obesity were not detected in either case. Neither patient had relevant previous medical histories, including malignancy. However, both had a long-term history of smoking. Systemic imaging studies revealed only adrenal tumors and surrounding lesions. Primary adrenocortical carcinoma was initially suspected, and chemotherapy including mitotane was considered. However, due to difficulty in complete resection of the tumor, core needle tumor biopsies were performed. Histopathological examination of biopsy specimens led to the diagnosis of carcinoma of unknown primary origin with isolated adrenal metastasis. In both cases, additional laboratory testing showed high levels of serum squamous cell carcinoma- related antigen and serum cytokeratin fragment. Malignant lesions confined to the adrenal glands are rare. As in our cases, it could be occasionally difficult to differentiate non-functioning primary adrenocortical carcinoma from metastatic adrenal carcinoma of unknown primary origin localized to the adrenal gland. If the lesion is unresectable and there are elevated levels of several tumor markers with no apparent hormonal excess, core needle tumor biopsy should be considered to differentiate the primary tumor from the metastatic tumor.
Key words: Adrenal metastasis, Carcinoma of unknown primary origin, Tumor biopsy
MALIGNANT LESIONS in the adrenal glands are rare. In particular, adrenocortical carcinoma (ACC) is a very rare tumor that has an incidence of approximately 0.72 per million people [1]. Approximately 60% of ACC patients present with hormone excess, and the diagnosis is based on symptoms associated with this condition [2-8]. Complete surgical resection is the only potentially curative treatment for ACC [9, 10]. However, ACCs are localized to the adrenal glands in 30-40% of cases, and most cases are diagnosed as advanced cancer [11]. The
prognosis of advanced ACC is extremely poor, but mitotane, a dichloro-diphenyl-trichloro-ethane derivative, is used specifically for the treatment of unresectable ACC [12]. However, the adrenal glands are rich in blood flow despite their small size and are a common site for the metastasis of malignant tumors. Generally, by the time lesions suspected of being metastatic adrenal carci- noma are discovered, the primary carcinoma has already been recognized. The treatment strategy depends on the primary carcinoma, although surgical treatment may be considered if the primary tumor has regressed or is under control. In addition, the treatment options differ according to whether the adrenal carcinoma is primary or metastatic.
Here, we reported two cases of lesions initially suspected to be ACCs that were later diagnosed as
E-mail: endocrin@med.kanazawa-u.ac.jp
carcinomas of unknown primary origin with isolated adrenal metastasis via computed tomography (CT)- guided tumor biopsy. These unilateral metastatic adrenal carcinomas of unknown primary origin were localized, which is extremely rare.
Case Descriptions
Case 1
A 57-year-old man was referred to our hospital for close examination of a right adrenal tumor. He had been experiencing right back pain for approximately 3 months. He had no relevant previous medical history, including malignancy. He had smoked 50-60 cigarettes per day for 40 years. He did not have obesity (body mass index, 22.5 kg/m2) or hypertension (blood pressure, 95/69 mmHg). CT revealed a 55-mm right-sided adrenal mass, involvement of the liver and inferior vena cava, and metastasis to the lymph nodes. The adrenal mass on unenhanced images had a density of approximately 30 Hounsfield units. His laboratory data are presented in Table 1. White blood cell counts and C-reactive protein (CRP) levels were elevated, and lactate dehydrogenase, alkaline phosphatase, and y-glutamyl transpeptidase levels were slightly elevated. Endocrinological examina- tion showed only a mild increase in adrenocorticotropic hormone (ACTH) levels and renin activity. Testing with a 1-mg dexamethasone suppression test decreased the serum cortisol level to 0.7 µg/dL and the ACTH level to <0.5 pg/mL. Further, 123I-metaiodobenzylguanidine (MIBG) scintigraphy revealed no accumulations. Fluoro- deoxyglucose (FDG) positron emission tomography (PET)-CT showed remarkable FDG uptake in the right adrenal tumor (maximum standardized uptake value, 14.1) but not in any other distant organ (Fig. 1). Addi- tionally, high carcinoembryonic antigen (CEA) values were observed. Gastrointestinal endoscopy revealed a colon polyp. Initially, he was thought to have primary ACC, and surgical treatment was scheduled. However, surgical treatment was abandoned due to the fast growth rate of the tumor, extensive invasion of adjacent organs such as liver, kidney, diaphragm and inferior vena cava, and multiple lymph node metastasis. As advanced ACC was suspected, chemotherapy with mitotane was then considered. However, given the absence of endocrino- logical abnormalities and the presence of high CEA levels, the possibility of a metastatic tumor could not be excluded. A CT-guided core needle biopsy of the tumor was performed. Pathological examination revealed carci- noma with squamous differentiation. Immunohisto- chemical analysis revealed that negativity for Melan A and chromogranin and positivity for p63 and cytokeratin 5/6 (Fig. 2). In addition, immunohistochemistry showed
| Test | Case 1 | Case 2 | Reference interval |
|---|---|---|---|
| WBC (×103/µL) | 10.5 | 7.3 | 3.3-8.8 |
| RBC (×106/µL) | 4.3 | 2.7 | 4.5-5.5 |
| Hb (g/dL) | 12.8 | 7.6 | 13.5-17.0 |
| Ht (%) | 39.3 | 24.0 | 39.7-51.0 |
| Plt (×104/µL) | 263 | 294 | 130-350 |
| CRP (mg/dL) | 6.6 | 5.8 | <0.3 |
| T-Bil (mg/dL) | 0.5 | 0.4 | 0.3-1.2 |
| AST (U/L) | 14 | 14 | 13-33 |
| ALT (U/L) | 16 | 10 | 8-42 |
| LDH (U/L) | 164 | 212 | 119-229 |
| ALP (U/L) | 388 | 251 | 115-359 |
| Na (mEq/L) | 140 | 139 | 135-149 |
| K (mEq/L) | 4.3 | 4.3 | 3.5-4.9 |
| Cl (mEq/L) | 104 | 104 | 96-108 |
| Ca (mg/dL) | 9.3 | 8.2 | 8.0-10.5 |
| P (mg/dL) | 3.5 | 3.5 | 2.5-4.5 |
| BUN (mg/dL) | 14 | 5 | 8-22 |
| Cr (mg/dL) | 0.6 | 0.6 | 0.6-1.0 |
| CEA (ng/ml) | 11.5 | 4.1 | <5 |
| CA19-9 (U/mL) | 10.0 | 3.0 | <37 |
| ProGRP (pg/mL) | 29.5 | 31.7 | ≤80 |
| CYFRA (ng/ml) | 89.1 | 4.9 | <3.5 |
| SCC (ng/ml) | 7.1 | 2.8 | <1.5 |
| AFP (ng/ml) | 5 | 3 | <10 |
| ACTH (pg/mL) | 96.3 | 17.1 | ≤40 |
| Serum cortisol (µg/dL) | 7.8 | 13.1 | 6.2-19.4 |
| PAC (pg/mL) | 61 | 102 | 20-130 |
| PRA (ng/mL/h) | 3.7 | 3.3 | 0.2-2.7 |
| Plasma adrenaline (ng/ml) | <0.01 | <0.01 | <0.17 |
| Plasma noradrenaline (ng/ml) | 0.43 | 0.43 | 0.15-0.57 |
| DHEA-S (µg/dL) | 147 | — | 38-313 |
WBC, white blood cell; RBC, red blood cell; Hb, hemoglobin; Plt, platelet; CRP, C-reactive protein; T-Bil, total bilirubin; AST, aspartate transaminase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; BUN, blood urea nitrogen; Cr, creatinine; CEA, carcinoembryonic antigen; CA19-9, carbohydrate antigen 19-9; ProGRP, pro gastrin releasing peptide; CYFRA, cytokeratin 19 fragment; SCC, squamous cell carcinoma antigen; AFP, a-fetoprotein; ACTH, adrenocorticotropic hormone; PAC, plasma aldosterone concentration; PRA, plasma renin activity; DHEA-S, dehydroepiandrosterone-sulfate. Abnormal values are marked in bold.
A
B
C
(A) shows the computed tomography (CT) finding, while (B) and (C) show the PET-CT findings. No significant accumulation other than in the adrenal mass.
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B
C
D
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B
C
R
(A) shows the computed tomography (CT) finding, while (B) and (C) show the PET-CT findings. No significant accumulation other than in the adrenal mass.
negativity for steroidogenic factor (SF)-1, suggesting that the tumor was by no means of adrenal origin. Addi- tional laboratory testing showed high levels of serum squamous cell carcinoma-related antigen (SCC-Ag) and serum cytokeratin fragment (CYFRA). Based on these results, the patient was diagnosed with adrenal metastasis from squamous cell carcinoma of unknown primary origin. He refused aggressive chemotherapy and opted for palliative therapy at another hospital.
Case 2
A 64-year-old man was referred to our hospital for close examination of a 60-mm right-sided adrenal tumor. He presented with slight fever and loss of appetite and had experienced weight loss amounting to 10 kg in the past 4 months. He had no relevant previous medical his- tory, including malignancy. He had smoked 20 cigarettes a day for 40 years. He did not have obesity (body mass index, 20.2 kg/m2) or hypertension (blood pressure, 105/66 mmHg). CT showed a tumor in the right adrenal gland, as well as the formation of tumor emboli in the inferior vena cava and right pulmonary artery due to tumor invasion, extensive tumor invasion of the liver and lymph node metastasis. His laboratory data are provided in Table 1. His red blood cell count and hemoglobin level were reduced, and his CRP level was elevated.
Endocrinological examination revealed only a mild increase in renin activity. FDG-PET/CT showed remark- able FDG uptake in the right adrenal tumor (maximum standardized uptake value, 30.1) but not in any other dis- tant organ (Fig. 3). In addition, 123I-MIBG scintigraphy showed no accumulations. Gastrointestinal endoscopy revealed a colon polyp. In this case, based on the experi- ence with case 1, tumor marker levels were evaluated, and a mildly elevated level of SCC was found. A CT- guided core needle biopsy of the tumor was performed to differentiate ACC from adrenal metastasis. Poorly differ- entiated carcinoma suggestive of metastatic cancer was considered based on the examination of hematoxylin and eosin-stained tissue sections. Immunohistochemical analysis revealed that the tumor cells were positive for p63, cytokeratin 7, and cytokeratin 5/6; partially weakly positive for thyroid transcription factor-1 (TTF-1); and negative for SF-1 (Fig. 4), S100 and SOX10. These findings were consistent with squamous cell carcinoma suspected of having a focal adenocarcinoma component. Additional laboratory testing revealed high levels of CYFRA. However, bronchoscopy did not reveal any abnormal findings. Based on these results, a diagnosis of carcinoma of unknown primary tumor with isolated adrenal metastases was made; however, lung cancer was deemed the most probable. The patient subsequently
A
B
C
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started chemotherapy with carboplatin plus paclitaxel. He died approximately 2 years after the diagnosis.
Discussion
We reported two cases of carcinoma of unknown pri- mary origin with isolated adrenal metastasis diagnosed via tumor biopsy in which the lesions were initially dis- covered as localized adrenal tumors. First, one of the common features in our two cases was that the patients had a long history of smoking. However, they had no history of malignancy, and systemic imaging, including FDG-PET, revealed no findings suggestive of a primary tumor. Second, the tumors were found to be solitary uni- lateral adrenal tumors in the absence of adrenocortical hormone and catecholamine excess. Finally, the tumors were deemed difficult to resect. Tumor biopsy led to the diagnoses of carcinoma of unknown primary origin with isolated adrenal metastasis.
A carcinoma of unknown primary origin is defined as a malignancy in which the primary tumor is unknown and the lesion is found to be histologically metastatic, regardless of adequate searching. PET-CT has been reported to be useful in cases of unknown primary can- cer, particularly in cases involving solitary metastases, as in our cases [13]. However, in our cases, various imaging studies, including PET-CT, failed to identify the primary tumor. The adrenal glands are the most prone to metasta- sis from lung cancer, followed by gastric cancer [14].
Therefore, both patients underwent repeated contrast- enhanced CT and upper and lower gastrointestinal endoscopy, which did not lead to identification of the primary cancer. In the second case, additional bronchos- copy was performed, which also failed to identify the primary cancer.
Localized metastasis to the adrenal glands from carci- nomas of unknown primary origin is extremely rare. A previous study reported that 4 (0.2%) of 1,639 patients with unknown primary cancer showed involvement limited to the adrenal glands [15]. Furthermore, in only one of the four cases, the disease was confined to the unilateral adrenal gland, as in our cases; adrenal metas- tases tend to present as bilateral lesions. Another study indicated that 0 of 30 biopsies performed for incidentaloma of the adrenal gland indicated metastatic adrenal carcinoma [16]. Carcinoma of unknown primary origin with isolated adrenal metastasis is considered an exceedingly rare condition. Therefore, differentiating non-functioning ACCs from unilateral metastatic adrenal carcinomas may be extremely difficult.
Patients with functioning ACC often have high levels of adrenocortical hormones and exhibit signs of Cushing’s syndrome, such as hypertension, obesity, hyperglycemia, and moon face; these are important dis- tinctions from the manifestations of patients with adrenal metastasis. Patients should be carefully evaluated for such clinical signs. Blood and urine tests should also be performed to evaluate excess hormone levels. The ACC
working group of the European Network for the Study of Adrenal Tumours has proposed standards for diagnostic procedures in patients with suspected ACC [17]. Although the evaluation of hormonal excess is time-consuming, it should be performed along with careful assessment of the clinical stage and possibility of adrenal metastasis.
In our present cases, it was noteworthy that the serum levels of some tumor-associated biomarkers were elevated, although endocrinological tests did not show any abnormalities. The first patient exhibited a mild increase in serum CEA levels. Elevated CEA levels are mainly used as an indicator of adenocarcinomas, such as colorectal, stomach, lung, ovarian, and uterine adenocar- cinomas. However, CEA values could also increase due to smoking. In addition, both patients had high levels of SCC and CYFRA. The levels of both these markers are increased in patients with squamous cell carcinoma. Par- ticularly, CYFRA has been found to be elevated in a variety of tumors [18], but it has established its position especially in non-small cell lung cancer [19]. CYFRA evaluation may be useful in the diagnosis of non-small cell lung cancer. Non-small cell lung cancer has been reported to be closely associated with smoking. An exhaustive analysis of tumor markers does not seem to be a viable option. However, for adrenal tumors in patients without endocrine abnormalities, an evaluation of tumor markers specific to the malignancy inferred from the patient’s medical history may be useful in determining the optimal treatment plan.
Our patients were diagnosed with malignancy via tumor biopsy. However, if ACC is suspected, a tumor biopsy constitutes a risk of seeding. Therefore, the effi- cacy of tumor biopsy for the diagnosis of adrenal tumors has not been established. A previous retrospective review revealed that biopsies were not useful in patients with localized adrenal incidentalomas because of the inability to differentiate between benign and malignant primary adrenal tumors. However, the authors stated that biopsy can be used to differentiate primary adrenal tumors from metastatic tumors [16]. In particular, SF-1 has been demonstrated to be useful for distinguishing tumors of adrenocortical and non-adrenocortical origins [20]. The value of this marker has also been confirmed in a large cohort study [21]. Both our cases were considered malig- nant, given the invasion of surrounding organs. Even if it is not clear whether the tumor is primary or metastatic, adrenalectomy should be the treatment of choice if it is feasible. A previous study revealed that adrenalectomy improved the overall survival of patients with metastatic disease arising from the soft tissues, kidney, lung, and pancreas [22]. Several other studies have recommended aggressive resection, especially in cases of solitary metas- tases to the adrenal glands [23, 24]. Therefore, a biopsy
may be an option for diagnosis when the tumor cannot be resected. In such cases, a biopsy should be considered to confirm the presence of ACC before administering mitotane. It may also be useful to measure the levels of tumor markers, particularly those related to lung cancer, based on the history and frequency of ACC.
Although adrenal carcinoma is considered to have a poor prognosis, carcinoma of unknown primary origin with isolated adrenal metastasis may remain latent in some cases, particularly in those diagnosed with non- functioning ACC. The treatment strategy for adrenal tumors needs to be determined by establishing a histo- logical diagnosis. Although tumor biopsy may not be consistently effective for establishing a histological diagnosis, it should be actively considered in cases of suspected non-functioning ACC or possible carcinoma of unknown primary origin with isolated adrenal metastasis, based on the medical history and high levels of certain tumor markers, when surgical resection is difficult.
This report, particularly the first case, described a patient with squamous cell carcinoma without an estima- tion of the primary tumor. Although high CYFRA levels were found, in recent years, certain genetic tests have been used to estimate the primary site of unknown pri- mary cancers; and treatment according to the estimated primary site may now be considered. In SCC, the rela- tionship between various genetic mutations and the pri- mary tumor site has been clarified, and the development of next-generation sequencers has further accelerated this process [25].
In summary, we report two cases of carcinoma of unknown primary origin with isolated adrenal metastasis. Tumor biopsy was effective for establishing the diagno- sis, and in one case, multidisciplinary treatment was able to prolong survival, compared to that reported previ- ously. In cases of localized adrenal tumors that are unre- sectable, tumor biopsy may be actively considered to determine the optimal course of treatment. In addition, the evaluation of tumor markers, particularly those spe- cific to lung cancer, might be useful in cases involving a history of smoking.
Acknowledgements
This study was supported by a grant from the Japan Society for the Promotion of Science (Grant number: 19111212). We would like to thank Editage (www. editage.com) for English language editing.
Disclosure
None of the authors have any potential conflicts of interest associated with this research.
References
1. Bilimoria KY, Shen WT, Elaraj D, Bentrem DJ, Winchester DJ, et al. (2008) Adrenocortical carcinoma in the United States: treatment utilization and prognostic factors. Cancer 113: 3130-3136.
2. Sada A, Asaad M, Bews KA, Thompson GB, Young WF Jr, et al. (2020) Comparison between functional and non- functional adrenocortical carcinoma. Surgery 167: 216- 223.
3. Allolio B, Fassnacht M (2006) Clinical review: adreno- cortical carcinoma: clinical update. J Clin Endocrinol Metab 91: 2027-2037.
4. Luton JP, Cerdas S, Billaud L, Thomas G, Guilhaume B, et al. (1990) Clinical features of adrenocortical carcinoma, prognostic factors, and the effect of mitotane therapy. N Engl J Med 322: 1195-1201.
5. Ng L, Libertino JM (2003) Adrenocortical carcinoma: diagnosis, evaluation and treatment. J Urol 169: 5-11.
6. Crucitti F, Bellantone R, Ferrante A, Boscherini M, Crucitti P (1996) The Italian registry for adrenal cortical carcinoma: analysis of a multiinstitutional series of 129 patients. The ACC Italian Registry Study Group. Surgery 119: 161-170.
7. Icard P, Goudet P, Charpenay C, Andreassian B, Carnaille B, et al. (2001) Adrenocortical carcinomas: surgical trends and results of a 253-patient series from the French Associ- ation of Endocrine Surgeons study group. World J Surg 25: 891-897.
8. Vassilopoulou-Sellin R, Schultz PN (2001) Adrenocortical carcinoma. Clinical outcome at the end of the 20th cen- tury. Cancer 92: 1113-1121.
9. Fassnacht M, Dekkers OM, Else T, Baudin E, Berruti A, et al. (2018) European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endo- crinol 179: G1-G46.
10. Allolio B, Hahner S, Weismann D, Fassnacht M (2004) Management of adrenocortical carcinoma. Clin Endo- crinol (Oxf) 60: 273-287.
11. Ayala-Ramirez M, Jasim S, Feng L, Ejaz S, Deniz F, et al. (2013) Adrenocortical carcinoma: clinical outcomes and prognosis of 330 patients at a tertiary care center. Eur J Endocrinol 169: 891-899.
12. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, et al. (2012) Combination chemotherapy in advanced adre- nocortical carcinoma. N Engl J Med 366: 2189-2197.
13. Rades D, Kühnel G, Wildfang I, Börner AR, Schmoll HJ,
et al. (2001) Localised disease in cancer of unknown pri- mary (CUP): the value of positron emission tomography (PET) for individual therapeutic management. Ann Oncol 12: 1605-1609.
14. Lam KY, Lo CY (2002) Metastatic tumours of the adrenal glands: a 30-year experience in a teaching hospital. Clin Endocrinol (Oxf) 56: 95-101.
15. Lee JE, Evans DB, Hickey RC, Sherman SI, Gagel RF, et al. (1998) Unknown primary cancer presenting as an adre- nal mass: frequency and implications for diagnostic evalu- ation of adrenal incidentalomas. Surgery 124: 1115-1122.
16. Mazzaglia PJ, Monchik JM (2009) Limited value of adre- nal biopsy in the evaluation of adrenal neoplasm: a decade of experience. Arch Surg 144: 465-470.
17. Fassnacht M, Allolio B (2009) Clinical management of adrenocortical carcinoma. Best Pract Res Clin Endocrinol Metab 23: 273-289.
18. Rudhart SA, Gehrt F, Birk R, Schultz JD, Stankovic P, et al. (2020) Clinical relevance of CYFRA 21-1 as a tumour marker in patients with oropharyngeal squamous cell car- cinoma. Eur Arch Otorhinolaryngol 277: 2561-2571.
19. Pujol JL, Grenier J, Daurès JP, Daver A, Pujol H, et al. (1993) Serum fragment of cytokeratin subunit 19 measured by CYFRA 21-1 immunoradiometric assay as a marker of lung cancer. Cancer Res 53: 61-66.
20. Sasano H, Shizawa S, Suzuki T, Takayama K, Fukaya T, et al. (1995) Transcription factor adrenal 4 binding protein as a marker of adrenocortical malignancy. Hum Pathol 26: 1154-1156.
21. Sbiera S, Schmull S, Assie G, Voelker HU, Kraus L, et al. (2010) High diagnostic and prognostic value of steroido- genic factor-1 expression in adrenal tumors. J Clin Endo- crinol Metab 95: E161-E171.
22. Vazquez BJ, Richards ML, Lohse CM, Thompson GB, Farley DR, et al. (2012) Adrenalectomy improves out- comes of selected patients with metastatic carcinoma. World J Surg 36: 1400-1405.
23. Hirayama T, Fujita T, Koguchi D, Nishi M, Kurosaka S, et al. (2014) Laparoscopic adrenalectomy for metastatic adrenal tumor. Asian J Endosc Surg 7: 43-47.
24. Porte HL, Roumilhac D, Graziana JP, Eraldi L, Cordonier C, et al. (1998) Adrenalectomy for a solitary adrenal metastasis from lung cancer. Ann Thorac Surg 65: 331- 335.
25. Sánchez-Danés A, Blanpain C (2018) Deciphering the cells of origin of squamous cell carcinomas. Nat Rev Can- cer 18: 549-561.