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ALK-rearranged adenocarcinoma with extensive mucin production can mimic mucinous adenocarcinoma: clinicopathological analysis and comprehensive histological comparison with KRAS-mutated mucinous adenocarcinoma
Pathology (June 2016) 48(4), pp. 325–329
A N ATO M I C A L PAT H O L O G Y
ALK-rearranged adenocarcinoma with extensive mucin production can mimic mucinous adenocarcinoma: clinicopathological analysis and comprehensive histological comparison with KRAS-mutated mucinous adenocarcinoma YOON JIN CHA1,2, JOUNGHO HAN1, SOO HYUN HWANG1, TAE BUM LEE1, HOJOONG KIM3 AND JEA ILL ZO4 1
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 2Department of Pathology, Yonsei University College of Medicine, 3Division of Pulmonary and Critical Care Medicine, and 4Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
Summary We aimed to investigate clinicopathological features and histology of ALK-rearranged adenocarcinomas with extensive mucin production (AEM) that mimic mucinous adenocarcinoma (MA). Retrospectively, 12 cases of AEM and 25 cases of MA harbouring KRAS mutation were retrieved. The clinicopathological profile and detailed histological features were analysed and compared based on the ALK and KRAS status. AEMs occurred in younger patients (p = 0.044) and were characterised by floating tubulopapillary pattern (p < 0.001), prominent nucleolus (p < 0.001), and apical cytoplasmic snouts (p < 0.001). In contrast, KRAS-mutated MAs lacked ALK-specific histological patterns (p < 0.05). Instead, tumour-infiltrating leukocytes (p = 0.018) and smooth cytoplasmic borders (p < 0.001) with vesicular nuclei (p = 0.004) were prominent in KRAS-mutated MAs. AEMs demonstrated characteristic tubulopapillary pattern and apical snouts, which were distinguishing features from MAs with KRAS mutation. Apical snouts can be a useful histological surrogate for ALK rearrangement in the pulmonary adenocarcinomas showing extensive mucin that mimic MA. Key words: Adenocarcinoma of lung; anaplastic lymphoma kinase; adenocarcinoma; mucinous; histology. Received 21 December 2015, revised 29 January, accepted 8 February 2016 Available online 22 April 2016
INTRODUCTION Anaplastic lymphoma kinase (ALK) gene rearrangement in pulmonary adenocarcinomas was first reported by Soda et al. in 2007.1 ALK rearrangement accounts for about 5–6% of pulmonary adenocarcinomas and correlates with non- or light smoking and young age.2,3 Histologically, extra/intracytoplasmic mucin, cribriform, tubulopapillary, and solid
signet-ring cell patterns are the most well-known indicators of ALK rearrangement.2–4 ALK-rearranged adenocarcinomas can rarely present with extensive mucin that mimic mucinous adenocarcinomas (MAs). Shim et al. reported unexpected ALK rearrangement in approximately 3% of MAs, which would have been ALK-rearranged adenocarcinomas with extensive mucin production (AEM).5 Histologically, pulmonary MA displays columnar cells with basally located nuclei and intracytoplasmic mucin and mucin pool. KRAS mutations are the most frequent driver mutation in pulmonary MA, followed by NRG1 rearrangement.5,6 KRAS mutation in lung cancer has been considered undruggable, in spite of many efforts to improve progression-free survival. Thus, it is important to suspect the possibility of AEM in tumour with extensive mucin production, because patients with ALKrearranged adenocarcinoma respond to treatment with the ALK inhibitor crizotinib.7 In this study, we compared the clinicopathological features and detailed histological features of AEMs and KRASmutated MAs, and aimed to describe the distinguishing features of AEMs.
MATERIALS AND METHODS This retrospective study was approved by the Institutional Review Board of the Samsung Medical Center. Case selection Among 1777 cases of surgical resection of primary lung adenocarcinomas at Samsung Medical Center from 2009 to 2014, ALK-rearranged adenocarcinomas accounted for 76 cases (4.3%), and MAs accounted for 109 cases (6.1%). KRAS mutation was present in 41 of the 109 MAs (37.6%). Among the 76 ALK-rearranged adenocarcinomas, 12 cases were classified as AEM, based on the mucinous histological features (goblet or columnar tumour cells) and extensive mucin production, and were selected for analysis. For comparison, 25 available cases of KRAS-mutated MA were selected. Clinicopathological analysis The following clinicopathological parameters were recorded: age, gender, smoking status (never, ex-, and current smoker), pack-year smoking history (defined as packs of cigarettes per day multiplied by years of smoking), tumour size, lymphovascular invasion, lymph node metastasis, pathological
Print ISSN 0031-3025/Online ISSN 1465-3931 © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved. DOI: http://dx.doi.org/10.1016/j.pathol.2016.02.016
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stage (American Joint Committee on Cancer seventh edition), death, and recurrence. Histological analysis For each case, whole tumour sections were re-reviewed by two pathologists (Han and Cha). Using a modification of the histological scoring system proposed by IASLC/ATS/ERS8 and described in previous studies,2,9 predominant histological pattern and histological features of ALK rearrangement (cribriform, solid signet-ring cell, tubulopapillary patterns) previously described,2,4 and presence of psammomatous calcification were recorded. Tubulopapillary pattern was defined as ring-shaped, floating micropapillary structures having dilated central luminal space without central fibrovascular cores, that sometimes coalesced to each other4 (Fig. 1A). Cytological features including nuclear membrane irregularity and nucleolar prominancy were checked and categorised into marked, moderate, or absent/ minimal, according to the previously described criteria.9 Tumour-infiltrating leukocytes (TILs) and peritumoural muciphages were evaluated according to the criteria used in the previous study:9 predominant = prominent at low power (×4 objective) or easily noticeable at low power; not predominant = none or inconspicuous at low power. In addition, the presence of apical snouts, which were defined as accumulation of secreted granules in the apical portion of mucin-containing tumour cells, was evaluated (Fig. 1B, arrowheads). ALK immunohistochemistry and fluorescent in situ hybridisation Immunohistochemical staining for ALK [NCL-ALK (clone 5A4), 1:40; Novocastra, UK] was performed using a biotin-avidin peroxidase complex on a BOND-MAX autostainer (Leica, Germany) after retrieval with ER2 solution. As described in previous studies, diffuse strong ALK-positivity (3+) can expect ALK rearrangement.10 Faint cytoplasmic (1+) staining was considered
Pathology (2016), 48(4), June
negative for ALK rearrangement. Cases with moderate, smooth cytoplasmic staining (2+) were further analysed by fluorescent in situ hybridisation using an ALK break-apart probe (Vysis LSI ALK Dual Color, break-apart rearrangement probe; Abbott Molecular, USA) to confirm the ALK rearrangement. Only diffuse, strong ALK-expressing (3+) cases were selected (Fig. 1C,D). Detection of EGFR and KRAS mutations The mutational analyses of EGFR (exons 18–21) and KRAS (exons 2, 3) were performed using directional sequencing of polymerase chain reaction (PCR) fragments amplified with genomic DNA from paraffin embedded tissue. PCR was performed in a 20 mL volume containing 100 ng of template DNA, 10x PCR buffer; 0.25 mMdNTPs, 10 pmol primers, and 1.25 U Taq DNA polymerase (iNtRON, Korea). PCR products were electrophoresed on 2% agarose gels and purified with a QIAquick PCR purification kit (Qiagen, Germany). Bidirectional sequencing was performed using the BigDye Terminator v 1.1 kit (Applied Biosystems, USA) on an ABI 3130xl genetic analyser (Applied Biosystems). Statistical analysis The comparison of clinicopathological and histological parameters based on ALK and KRAS status were assessed using the Chi-square (for categorical parameters) and Mann-Whitney U (for continuous parameters) tests. Statistical analyses were performed using SPSS 19.0 (IBM, USA), and p value less than 0.05 was considered statistically significant.
RESULTS Clinically, the mean age of patients with AEM was 56.8 years, which was approximately 10 years younger than
Representative pictures of floating tubulopapillary pattern (A) and apical snouts (B, arrowheads). Note diffuse strong cytoplasmic ALK expression in the attenuated cytoplasm due to the prominent intracytoplasmic mucin (C). Apical snouts are also positive for ALK immunohistochemical staining (D).
Fig. 1
ALK-REARRANGED ADENOCARCINOMA WITH EXTENSIVE MUCIN PRODUCTION
patients with KRAS-mutated MA. Tumour size, smoking history, and pathological stage showed no significant difference between AEMs and KRAS-mutated MAs. There was no gender predilection in both groups. One (8.3%) AEM patient and two (5.4%) KRAS-mutated MA patients died of disease, and one (8.3%) AEM patient and six (24.0%) KRAS-mutated MA patients experienced tumour recurrence. However, there was no significant difference in patient prognosis between AEMs and KRAS-mutated MAs. Lymphovascular invasion was more frequent in AEMs, but frequency of lymph node metastasis was not different between two groups. Grossly, both AEMs and KRAS-mutated MAs had gelatinous cut surface (Fig. 2A, arrows) and peripheral mucin pool (Fig. 2B, arrowheads) as usual MAs. Histologically, all cases had abundant extracellular mucin and columnar mucus cells with basally located nuclei. The most predominant growth pattern was an acinar pattern in both AEMs and KRASmutated MAs. Only in two patients with AEMs were cribriform pattern and tubulopapillary pattern predominant patterns, respectively. With regard to ALK and KRAS status, AEMs had several distinguishing histological features from KRAS-mutated MAs. At least one of the histological features associated with ALK rearrangement—solid signet ring cells, cribriform pattern, tubulopapillary pattern—were identified in all AEMs. Particularly, tubulopapillary pattern was found in 91.7% of AEMs whereas only two KRAS-mutated MAs had focal cribriform and tubulopapillary pattern, respectively (Fig. 2C). TILs and peritumoural muciphages were more predominant in KRAS-mutated MAs (Fig. 2D). Peritumoural leukocytic infiltration was significantly more prominent in KRAS-mutated MAs, and intracytoplasmic leukocytic
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infiltration was observed in 36.0% of KRAS-mutated MAs whereas no AEMs had intracytoplasmic leukocytic infiltration. Cytologically, AEMs demonstrated less irregular nuclear membrane compared to the KRAS-mutated MAs (Fig. 2E). Nucleoli were more easily visible in AEMs, displayed as a single prominent nucleolus (Fig. 2F). Apical snouts were present in 83.3% of AEMs (Fig. 2G). Although two cases (8.0%) of KRAS-mutated MA had apical snouts focally, most of KRAS-mutated MAs had smooth apical cytoplasmic borders (Fig. 2H). Comparison of clinicopathological parameters and detailed histological features based on ALK and KRAS status are summarised in Tables 1 and 2.
DISCUSSION There have been efforts to correlate the cytomorphology with molecular alteration in lung adenocarcinoma, since prediction and detection of specific mutations are critical to treatment and prognosis of patients.11 With regard to mutations, tumours with EGFR mutation have been associated with females, non-smokers, and definitive adenocarcinoma histology (acinar/papillary/lepidic pattern).11 KRAS-mutated tumour has been associated with history of smoking, solid histology, and nuclear pleomorphism.11,12 ALK-rearranged tumours have been associated with a younger age than EGFR- or KRAS-mutated tumours and show higher pathological stage and frequent nodal metastasis.12,13 Since ALK rearrangement is a target of crizotinib, detection of ALK rearrangement is essential for companion diagnosis of lung adenocarcinoma. ALK-rearranged adenocarcinoma shows several distinctive features implying the molecular alteration, including any type of mucus cell, solid signet ring
Grossly, gelatinous cut surface (A, arrows) and peripheral mucin pool (B, arrowheads) are found in both ALK-rearranged adenocarcinoma with extensive mucin production (AEM) (A) and KRAS-mutated mucinous adenocarcinoma (MA) (B). AEM has frequent psammomatous calcifications and floating tubulopapillary clusters of tumour cells (C). Mucinous background with relatively scant peritumoural inflammation is characteristic (C). KRAS-mutated MA has dense tumour infiltrating leukocytes and peritumoural muciphages (D). AEM demonstrates monotonous round nuclei with prominent nucleolus (E). Apical snouts are evident in high-power view (E). Nuclei of KRAS-mutated MA are more irregular and raisinoid without identifiable nucleoli (F). Apical snouts are a specific finding of AEM (G), whereas KRAS-mutated MA shows relatively smooth cytoplasmic border without any snouts (H).
Fig. 2
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Pathology (2016), 48(4), June
Table 1 Clinicopathological parameters of ALK-rearranged adenocarcinomas with extensive mucin production and and KRAS-mutated mucinous adenocarcinomas
Male gender (%) Age, mean years ± SD Follow up period, months Smoking history (%) None Ex-smoker Current smoker Tumour size, cm Lymphovascular invasion (%) Lymph node metastasis (%) Pathological stage (%) IA IB IIA IIB IIIA IIIB IV Death (%) Recurrence (%)
AEM (n=12)
KRAS-mutated MA (n=25)
p value
2 (40) 56.8 ± 12.4 12.2 ± 13.7
8 (32) 64.8 ± 11.9 6.7 ± 5.5
1.000 0.044 0.385 0.670
7 (58.3) 2 (16.7) 3 (25.0) 3.3 ± 1.2 7 (58.3) 5 (55.6)
18 (72.0) 4 (16.0) 3 (12.0) 3.7 ± 1.9 5 (20.0) 4 (16.0)
3 2 2 1 4 0 0 1 1
6 8 3 2 5 0 1 2 6
(25.0) (16.7) (16.7) (8.3) (33.3) (8.3) (8.3)
0.603 0.102 0.116 0.908
(24.0) (32.0) (12.0) (8.0) (20.0) (4.0) (5.4) (24.0)
1.000 0.389
AEM, ALK-rearranged adenocarcinoma with extensive mucin production; MA, mucinous adenocarcinoma.
Table 2 Comparison of histological features of ALK-rearranged adenocarcinomas with extensive mucin production and KRAS-mutated mucinous adenocarcinomas AEM (n=12)
KRAS-mutated MA (n=25)
Predominant growth pattern Acinar 10 (83.3) Cribriform 1 (2.7) Tubulopapillary 1 (2.7) Presence of known histological patterns associated ALK-rearrangement (%) Solid-signet rings 3 (25.0) Cribriform 5 (41.7) Tubulopapillary 11 (91.7) Tumour-infiltrating leukocytes (%) Peritumoural 5 (41.7) Intracytoplasmic 0 (0.0) Peritumoural muciphages (%) 8 (66.7) Psammona bodies (%) 3 (60.0) Nuclear membrane irregularity (%) Absent/Minimal 3 (25.0) Moderate 9 (75.0) Marked 0 (0.0) Prominent nucleoli (%) Absent/Minimal 0 (0.0) Moderate 6 (50.0) Marked 6 (50.0) Apical snouts (%) 10 (83.3)
p value 0.099
25 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (4.0) 1 (4.0)
0.028 0.009 <0.001
21 (84.0) 9 (36.0) 24 (96.0) 1 (4.0)
0.018 0.018 0.030 0.009 0.004
3 (12) 9 (36) 13 (52) <0.001 18 (72.0) 7 (28.0) 0 (0.0) 2 (8.0)
<0.001
AEM, ALK-rearranged adenocarcinoma with extensive mucin production; MA, mucinous adenocarcinoma.
cells, cribriform and tubulopapillary patterns, monotonous nuclei without significant pleomorphism, psammomatous calcification, and extracellular mucin.2,4,13 However, the histological features cannot be fully correlated with the molecular alterations, and ALK rearrangement has been rarely reported in MAs that mostly demonstrate histology of MA rather than ALK-specific histology.5 Therefore, AEMs can be confused with MAs, and previously reported ALKrearranged MAs would have been AEMs that mimicked MAs. In the present study, all AEMs had at least focal known ALK-specific histological features, whereas only two KRASmutated MAs had those features. Particularly, floating
tubulopapillary pattern was present in all AEMs except for one case, which seemed to be highly related to ALK rearrangement. KRAS is the most frequently mutated gene in MAs, and Sugano et al. showed that pulmonary MAs expressed HNF4a and lacked TTF-1 expression, and suggested that this immunoprofile was strongly correlated with mucinous histology along with positive KRAS mutation and negative EGFR and ALK mutation.14 According to Song et al., KRASmutated adenocarcinomas had severe nuclear pleomorphism, macronucleoli, and a neutrophilic background.15 Rekhtman et al. reported that KRAS mutation was associated with
ALK-REARRANGED ADENOCARCINOMA WITH EXTENSIVE MUCIN PRODUCTION
TILs and solid pattern in lung adenocarcinoma.9 Although Rekhtman et al. included only seven cases of KRAS-mutated MAs and focused on the solid pattern predominant adenocarcinomas, frequent peritumoural and intracytoplasmic leukocytic infiltration in KRAS-mutated MAs in the current study seemed to be related to the KRAS mutation, and partly corroborated previous studies.9,15 Furthermore, frequent TILs in KRAS-mutated tumours, in both mucinous and nonmucinous adenocarcinoma, might be targets of therapeutic immunomodulatory agents, such as PD1 and PD-L1.16 Cytologically, KRAS-mutated MAs exhibited irregular nuclear membranes that were contrasted with relatively monotonous and round nuclei of AEMs. However, macronucleoli were not significant in KRAS-mutated MAs in the present study, although Song et al. documented macronucleoli as a characteristic feature of KRAS-mutated adenocarcinomas.15 Rather, AEMs demonstrated prominent single nucleolus. This discordance could result from different materials and methods between the two studies. Song et al. used lymph node aspiration specimens and reviewed cytology slides.15 Since cells of cytology specimen are entirely prepared they may provide more visible nucleoli than cells from sectioned formalin fixed, paraffin embedded tissue. Additionally, most of the lymph node aspiration cases were in an advanced stage, whereas most of our KRAS-mutated MAs were relatively earlystage resected cases with rare lymph node metastasis. Besides, ALK-rearranged adenocarcinoma has been reported to have nuclear clearing like papillary thyroid carcinoma, and this nuclear feature would have been retained in AEMs, which may make the nucleoli more visible compared with those of KRASmutated MAs.4 Interestingly, apical snouts were observed in 83.3% of AEMs in the present study; these have not been introduced as an ALK-specific histological feature so far. Apical snouts are documented in breast lesions, including apocrine and columnar cell lesions.17,18 In contrast to AEMs, most of the KRAS-mutated MAs showed smooth apical cytoplasmic borders devoid of any snouts, except for two cases. Thus, we suggested that apical snouts would be one of the ALK-specific histological features in cases with extensive mucin that can mimic MAs.
CONCLUSIONS We compared clinicopathological features of AEMs with KRAS-mutated MAs and correlated the histology and molecular alterations. Since companion diagnosis with integrated histological and molecular findings is essential for appropriate treatment and construction of data for comprehensive analysis in the future, we suggest that AEMs should be distinguished from MAs. Although AEMs are rare and only a small number of patients could be included in the current study, we found that AEMs occurred in patients of younger age and exhibited distinct histological features compared to the KRAS-mutated MAs. It appears to be critical to not overlook any ALK-specific histology, especially floating tubulopapillary pattern and apical snouts in extensive mucin producing adenocarcionomas, because they strongly suggest the possibility of ALK rearrangement. Further study with a larger number of patients is required to clarify the clinicopathological features of AEMs.
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Acknowledgement: The authors are deeply indebted to Dr Hyo Sup Shim in the Department of Pathology, Yonsei University College of Medicine, for his comments that greatly improved the manuscript. Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose. Address for correspondence: Prof Joungho Han, Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, South Korea. E-mail: hanjho@ skku.edu
References 1. Lee SE, Lee B, Hong M, et al. Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma. Mod Pathol 2015; 28: 468–79. 2. Yoshida A, Tsuta K, Nakamura H, et al. Comprehensive histologic analysis of ALK-rearranged lung carcinomas. Am J Surg Pathol 2011; 35: 1226–34. 3. Rodig SJ, Mino-Kenudson M, Dacic S, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res 2009; 15: 5216–23. 4. Ha SY, Ahn J, Roh MS, et al. Cytologic features of ALK-positive pulmonary adenocarcinoma. Korean J Pathol 2013; 47: 252–7. 5. Shim HS, Kenudson M, Zheng Z, et al. Unique genetic and survival characteristics of invasive mucinous adenocarcinoma of the lung. J Thorac Oncol 2015; 10: 1156–62. 6. Finberg KE, Sequist LV, Joshi VA, et al. Mucinous differentiation correlates with absence of EGFR mutation and presence of KRAS mutation in lung adenocarcinomas with bronchioloalveolar features. J Mol Diagn 2007; 9: 320–6. 7. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010; 363: 1693–703. 8. Travis WD, Brambilla E, Noguchi M, et al. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 2011; 6: 244–85. 9. Rekhtman N, Ang DC, Riely GJ, et al. KRAS mutations are associated with solid growth pattern and tumor-infiltrating leukocytes in lung adenocarcinoma. Mod Pathol 2013; 26: 1307–19. 10. Yi ES, Boland JM, Maleszewski JJ, et al. Correlation of IHC and FISH for ALK gene rearrangement in non-small cell lung carcinoma: IHC score algorithm for FISH. J Thorac Oncol 2011; 6: 459–65. 11. Li Z, Dacic S, Pantanowitz L, et al. Correlation of cytomorphology and molecular findings in EGFR+, KRAS+, and ALK+ lung carcinomas. Am J Clin Pathol 2014; 141: 420–8. 12. Wang J, Dong Y, Cai Y, et al. Clinicopathologic characteristics of ALK rearrangements in primary lung adenocarcinoma with identified EGFR and KRAS status. J Cancer Res Clin Oncol 2014; 140: 453 – 60. 13. Kim H, Jang SJ, Chung DH, et al. A comprehensive comparative analysis of the histomorphological features of ALK-rearranged lung adenocarcinoma based on driver oncogene mutations: frequent expression of epithelial-mesenchymal transition markers than other genotype. PLoS One 2013; 8: e76999. 14. Sugano M, Nagasaka T, Sasaki E, et al. HNF4alpha as a marker for invasive mucinous adenocarcinoma of the lung. Am J Surg Pathol 2013; 37: 211–8. 15. Song DH, Lee B, Shin Y, et al. Cytomorphological identification of advanced pulmonary adenocarcinoma harboring KRAS mutation in lymph node fine-needle aspiration specimens: Comparative investigation of adenocarcinoma with KRAS and EGFR mutations. Diagn Cytopathol 2015; 43: 539–44. 16. De Palma M, Lewis CE. Macrophage regulation of tumor responses to anticancer therapies. Cancer Cell 2013; 23: 277–86. 17. Tanaka K, Imoto S, Wada N, et al. Invasive apocrine carcinoma of the breast: clinicopathologic features of 57 patients. Breast J 2008; 14: 164–8. 18. Guerra-Wallace MM, Christensen WN, White Jr RL. A retrospective study of columnar alteration with prominent apical snouts and secretions and the association with cancer. Am J Surg 2004; 188: 395–8.
A N ATO M I C A L PAT H O L O G Y
ALK-rearranged adenocarcinoma with extensive mucin production can mimic mucinous adenocarcinoma: clinicopathological analysis and comprehensive histological comparison with KRAS-mutated mucinous adenocarcinoma YOON JIN CHA1,2, JOUNGHO HAN1, SOO HYUN HWANG1, TAE BUM LEE1, HOJOONG KIM3 AND JEA ILL ZO4 1
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 2Department of Pathology, Yonsei University College of Medicine, 3Division of Pulmonary and Critical Care Medicine, and 4Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
Summary We aimed to investigate clinicopathological features and histology of ALK-rearranged adenocarcinomas with extensive mucin production (AEM) that mimic mucinous adenocarcinoma (MA). Retrospectively, 12 cases of AEM and 25 cases of MA harbouring KRAS mutation were retrieved. The clinicopathological profile and detailed histological features were analysed and compared based on the ALK and KRAS status. AEMs occurred in younger patients (p = 0.044) and were characterised by floating tubulopapillary pattern (p < 0.001), prominent nucleolus (p < 0.001), and apical cytoplasmic snouts (p < 0.001). In contrast, KRAS-mutated MAs lacked ALK-specific histological patterns (p < 0.05). Instead, tumour-infiltrating leukocytes (p = 0.018) and smooth cytoplasmic borders (p < 0.001) with vesicular nuclei (p = 0.004) were prominent in KRAS-mutated MAs. AEMs demonstrated characteristic tubulopapillary pattern and apical snouts, which were distinguishing features from MAs with KRAS mutation. Apical snouts can be a useful histological surrogate for ALK rearrangement in the pulmonary adenocarcinomas showing extensive mucin that mimic MA. Key words: Adenocarcinoma of lung; anaplastic lymphoma kinase; adenocarcinoma; mucinous; histology. Received 21 December 2015, revised 29 January, accepted 8 February 2016 Available online 22 April 2016
INTRODUCTION Anaplastic lymphoma kinase (ALK) gene rearrangement in pulmonary adenocarcinomas was first reported by Soda et al. in 2007.1 ALK rearrangement accounts for about 5–6% of pulmonary adenocarcinomas and correlates with non- or light smoking and young age.2,3 Histologically, extra/intracytoplasmic mucin, cribriform, tubulopapillary, and solid
signet-ring cell patterns are the most well-known indicators of ALK rearrangement.2–4 ALK-rearranged adenocarcinomas can rarely present with extensive mucin that mimic mucinous adenocarcinomas (MAs). Shim et al. reported unexpected ALK rearrangement in approximately 3% of MAs, which would have been ALK-rearranged adenocarcinomas with extensive mucin production (AEM).5 Histologically, pulmonary MA displays columnar cells with basally located nuclei and intracytoplasmic mucin and mucin pool. KRAS mutations are the most frequent driver mutation in pulmonary MA, followed by NRG1 rearrangement.5,6 KRAS mutation in lung cancer has been considered undruggable, in spite of many efforts to improve progression-free survival. Thus, it is important to suspect the possibility of AEM in tumour with extensive mucin production, because patients with ALKrearranged adenocarcinoma respond to treatment with the ALK inhibitor crizotinib.7 In this study, we compared the clinicopathological features and detailed histological features of AEMs and KRASmutated MAs, and aimed to describe the distinguishing features of AEMs.
MATERIALS AND METHODS This retrospective study was approved by the Institutional Review Board of the Samsung Medical Center. Case selection Among 1777 cases of surgical resection of primary lung adenocarcinomas at Samsung Medical Center from 2009 to 2014, ALK-rearranged adenocarcinomas accounted for 76 cases (4.3%), and MAs accounted for 109 cases (6.1%). KRAS mutation was present in 41 of the 109 MAs (37.6%). Among the 76 ALK-rearranged adenocarcinomas, 12 cases were classified as AEM, based on the mucinous histological features (goblet or columnar tumour cells) and extensive mucin production, and were selected for analysis. For comparison, 25 available cases of KRAS-mutated MA were selected. Clinicopathological analysis The following clinicopathological parameters were recorded: age, gender, smoking status (never, ex-, and current smoker), pack-year smoking history (defined as packs of cigarettes per day multiplied by years of smoking), tumour size, lymphovascular invasion, lymph node metastasis, pathological
Print ISSN 0031-3025/Online ISSN 1465-3931 © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved. DOI: http://dx.doi.org/10.1016/j.pathol.2016.02.016
326
CHA et al.
stage (American Joint Committee on Cancer seventh edition), death, and recurrence. Histological analysis For each case, whole tumour sections were re-reviewed by two pathologists (Han and Cha). Using a modification of the histological scoring system proposed by IASLC/ATS/ERS8 and described in previous studies,2,9 predominant histological pattern and histological features of ALK rearrangement (cribriform, solid signet-ring cell, tubulopapillary patterns) previously described,2,4 and presence of psammomatous calcification were recorded. Tubulopapillary pattern was defined as ring-shaped, floating micropapillary structures having dilated central luminal space without central fibrovascular cores, that sometimes coalesced to each other4 (Fig. 1A). Cytological features including nuclear membrane irregularity and nucleolar prominancy were checked and categorised into marked, moderate, or absent/ minimal, according to the previously described criteria.9 Tumour-infiltrating leukocytes (TILs) and peritumoural muciphages were evaluated according to the criteria used in the previous study:9 predominant = prominent at low power (×4 objective) or easily noticeable at low power; not predominant = none or inconspicuous at low power. In addition, the presence of apical snouts, which were defined as accumulation of secreted granules in the apical portion of mucin-containing tumour cells, was evaluated (Fig. 1B, arrowheads). ALK immunohistochemistry and fluorescent in situ hybridisation Immunohistochemical staining for ALK [NCL-ALK (clone 5A4), 1:40; Novocastra, UK] was performed using a biotin-avidin peroxidase complex on a BOND-MAX autostainer (Leica, Germany) after retrieval with ER2 solution. As described in previous studies, diffuse strong ALK-positivity (3+) can expect ALK rearrangement.10 Faint cytoplasmic (1+) staining was considered
Pathology (2016), 48(4), June
negative for ALK rearrangement. Cases with moderate, smooth cytoplasmic staining (2+) were further analysed by fluorescent in situ hybridisation using an ALK break-apart probe (Vysis LSI ALK Dual Color, break-apart rearrangement probe; Abbott Molecular, USA) to confirm the ALK rearrangement. Only diffuse, strong ALK-expressing (3+) cases were selected (Fig. 1C,D). Detection of EGFR and KRAS mutations The mutational analyses of EGFR (exons 18–21) and KRAS (exons 2, 3) were performed using directional sequencing of polymerase chain reaction (PCR) fragments amplified with genomic DNA from paraffin embedded tissue. PCR was performed in a 20 mL volume containing 100 ng of template DNA, 10x PCR buffer; 0.25 mMdNTPs, 10 pmol primers, and 1.25 U Taq DNA polymerase (iNtRON, Korea). PCR products were electrophoresed on 2% agarose gels and purified with a QIAquick PCR purification kit (Qiagen, Germany). Bidirectional sequencing was performed using the BigDye Terminator v 1.1 kit (Applied Biosystems, USA) on an ABI 3130xl genetic analyser (Applied Biosystems). Statistical analysis The comparison of clinicopathological and histological parameters based on ALK and KRAS status were assessed using the Chi-square (for categorical parameters) and Mann-Whitney U (for continuous parameters) tests. Statistical analyses were performed using SPSS 19.0 (IBM, USA), and p value less than 0.05 was considered statistically significant.
RESULTS Clinically, the mean age of patients with AEM was 56.8 years, which was approximately 10 years younger than
Representative pictures of floating tubulopapillary pattern (A) and apical snouts (B, arrowheads). Note diffuse strong cytoplasmic ALK expression in the attenuated cytoplasm due to the prominent intracytoplasmic mucin (C). Apical snouts are also positive for ALK immunohistochemical staining (D).
Fig. 1
ALK-REARRANGED ADENOCARCINOMA WITH EXTENSIVE MUCIN PRODUCTION
patients with KRAS-mutated MA. Tumour size, smoking history, and pathological stage showed no significant difference between AEMs and KRAS-mutated MAs. There was no gender predilection in both groups. One (8.3%) AEM patient and two (5.4%) KRAS-mutated MA patients died of disease, and one (8.3%) AEM patient and six (24.0%) KRAS-mutated MA patients experienced tumour recurrence. However, there was no significant difference in patient prognosis between AEMs and KRAS-mutated MAs. Lymphovascular invasion was more frequent in AEMs, but frequency of lymph node metastasis was not different between two groups. Grossly, both AEMs and KRAS-mutated MAs had gelatinous cut surface (Fig. 2A, arrows) and peripheral mucin pool (Fig. 2B, arrowheads) as usual MAs. Histologically, all cases had abundant extracellular mucin and columnar mucus cells with basally located nuclei. The most predominant growth pattern was an acinar pattern in both AEMs and KRASmutated MAs. Only in two patients with AEMs were cribriform pattern and tubulopapillary pattern predominant patterns, respectively. With regard to ALK and KRAS status, AEMs had several distinguishing histological features from KRAS-mutated MAs. At least one of the histological features associated with ALK rearrangement—solid signet ring cells, cribriform pattern, tubulopapillary pattern—were identified in all AEMs. Particularly, tubulopapillary pattern was found in 91.7% of AEMs whereas only two KRAS-mutated MAs had focal cribriform and tubulopapillary pattern, respectively (Fig. 2C). TILs and peritumoural muciphages were more predominant in KRAS-mutated MAs (Fig. 2D). Peritumoural leukocytic infiltration was significantly more prominent in KRAS-mutated MAs, and intracytoplasmic leukocytic
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infiltration was observed in 36.0% of KRAS-mutated MAs whereas no AEMs had intracytoplasmic leukocytic infiltration. Cytologically, AEMs demonstrated less irregular nuclear membrane compared to the KRAS-mutated MAs (Fig. 2E). Nucleoli were more easily visible in AEMs, displayed as a single prominent nucleolus (Fig. 2F). Apical snouts were present in 83.3% of AEMs (Fig. 2G). Although two cases (8.0%) of KRAS-mutated MA had apical snouts focally, most of KRAS-mutated MAs had smooth apical cytoplasmic borders (Fig. 2H). Comparison of clinicopathological parameters and detailed histological features based on ALK and KRAS status are summarised in Tables 1 and 2.
DISCUSSION There have been efforts to correlate the cytomorphology with molecular alteration in lung adenocarcinoma, since prediction and detection of specific mutations are critical to treatment and prognosis of patients.11 With regard to mutations, tumours with EGFR mutation have been associated with females, non-smokers, and definitive adenocarcinoma histology (acinar/papillary/lepidic pattern).11 KRAS-mutated tumour has been associated with history of smoking, solid histology, and nuclear pleomorphism.11,12 ALK-rearranged tumours have been associated with a younger age than EGFR- or KRAS-mutated tumours and show higher pathological stage and frequent nodal metastasis.12,13 Since ALK rearrangement is a target of crizotinib, detection of ALK rearrangement is essential for companion diagnosis of lung adenocarcinoma. ALK-rearranged adenocarcinoma shows several distinctive features implying the molecular alteration, including any type of mucus cell, solid signet ring
Grossly, gelatinous cut surface (A, arrows) and peripheral mucin pool (B, arrowheads) are found in both ALK-rearranged adenocarcinoma with extensive mucin production (AEM) (A) and KRAS-mutated mucinous adenocarcinoma (MA) (B). AEM has frequent psammomatous calcifications and floating tubulopapillary clusters of tumour cells (C). Mucinous background with relatively scant peritumoural inflammation is characteristic (C). KRAS-mutated MA has dense tumour infiltrating leukocytes and peritumoural muciphages (D). AEM demonstrates monotonous round nuclei with prominent nucleolus (E). Apical snouts are evident in high-power view (E). Nuclei of KRAS-mutated MA are more irregular and raisinoid without identifiable nucleoli (F). Apical snouts are a specific finding of AEM (G), whereas KRAS-mutated MA shows relatively smooth cytoplasmic border without any snouts (H).
Fig. 2
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CHA et al.
Pathology (2016), 48(4), June
Table 1 Clinicopathological parameters of ALK-rearranged adenocarcinomas with extensive mucin production and and KRAS-mutated mucinous adenocarcinomas
Male gender (%) Age, mean years ± SD Follow up period, months Smoking history (%) None Ex-smoker Current smoker Tumour size, cm Lymphovascular invasion (%) Lymph node metastasis (%) Pathological stage (%) IA IB IIA IIB IIIA IIIB IV Death (%) Recurrence (%)
AEM (n=12)
KRAS-mutated MA (n=25)
p value
2 (40) 56.8 ± 12.4 12.2 ± 13.7
8 (32) 64.8 ± 11.9 6.7 ± 5.5
1.000 0.044 0.385 0.670
7 (58.3) 2 (16.7) 3 (25.0) 3.3 ± 1.2 7 (58.3) 5 (55.6)
18 (72.0) 4 (16.0) 3 (12.0) 3.7 ± 1.9 5 (20.0) 4 (16.0)
3 2 2 1 4 0 0 1 1
6 8 3 2 5 0 1 2 6
(25.0) (16.7) (16.7) (8.3) (33.3) (8.3) (8.3)
0.603 0.102 0.116 0.908
(24.0) (32.0) (12.0) (8.0) (20.0) (4.0) (5.4) (24.0)
1.000 0.389
AEM, ALK-rearranged adenocarcinoma with extensive mucin production; MA, mucinous adenocarcinoma.
Table 2 Comparison of histological features of ALK-rearranged adenocarcinomas with extensive mucin production and KRAS-mutated mucinous adenocarcinomas AEM (n=12)
KRAS-mutated MA (n=25)
Predominant growth pattern Acinar 10 (83.3) Cribriform 1 (2.7) Tubulopapillary 1 (2.7) Presence of known histological patterns associated ALK-rearrangement (%) Solid-signet rings 3 (25.0) Cribriform 5 (41.7) Tubulopapillary 11 (91.7) Tumour-infiltrating leukocytes (%) Peritumoural 5 (41.7) Intracytoplasmic 0 (0.0) Peritumoural muciphages (%) 8 (66.7) Psammona bodies (%) 3 (60.0) Nuclear membrane irregularity (%) Absent/Minimal 3 (25.0) Moderate 9 (75.0) Marked 0 (0.0) Prominent nucleoli (%) Absent/Minimal 0 (0.0) Moderate 6 (50.0) Marked 6 (50.0) Apical snouts (%) 10 (83.3)
p value 0.099
25 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (4.0) 1 (4.0)
0.028 0.009 <0.001
21 (84.0) 9 (36.0) 24 (96.0) 1 (4.0)
0.018 0.018 0.030 0.009 0.004
3 (12) 9 (36) 13 (52) <0.001 18 (72.0) 7 (28.0) 0 (0.0) 2 (8.0)
<0.001
AEM, ALK-rearranged adenocarcinoma with extensive mucin production; MA, mucinous adenocarcinoma.
cells, cribriform and tubulopapillary patterns, monotonous nuclei without significant pleomorphism, psammomatous calcification, and extracellular mucin.2,4,13 However, the histological features cannot be fully correlated with the molecular alterations, and ALK rearrangement has been rarely reported in MAs that mostly demonstrate histology of MA rather than ALK-specific histology.5 Therefore, AEMs can be confused with MAs, and previously reported ALKrearranged MAs would have been AEMs that mimicked MAs. In the present study, all AEMs had at least focal known ALK-specific histological features, whereas only two KRASmutated MAs had those features. Particularly, floating
tubulopapillary pattern was present in all AEMs except for one case, which seemed to be highly related to ALK rearrangement. KRAS is the most frequently mutated gene in MAs, and Sugano et al. showed that pulmonary MAs expressed HNF4a and lacked TTF-1 expression, and suggested that this immunoprofile was strongly correlated with mucinous histology along with positive KRAS mutation and negative EGFR and ALK mutation.14 According to Song et al., KRASmutated adenocarcinomas had severe nuclear pleomorphism, macronucleoli, and a neutrophilic background.15 Rekhtman et al. reported that KRAS mutation was associated with
ALK-REARRANGED ADENOCARCINOMA WITH EXTENSIVE MUCIN PRODUCTION
TILs and solid pattern in lung adenocarcinoma.9 Although Rekhtman et al. included only seven cases of KRAS-mutated MAs and focused on the solid pattern predominant adenocarcinomas, frequent peritumoural and intracytoplasmic leukocytic infiltration in KRAS-mutated MAs in the current study seemed to be related to the KRAS mutation, and partly corroborated previous studies.9,15 Furthermore, frequent TILs in KRAS-mutated tumours, in both mucinous and nonmucinous adenocarcinoma, might be targets of therapeutic immunomodulatory agents, such as PD1 and PD-L1.16 Cytologically, KRAS-mutated MAs exhibited irregular nuclear membranes that were contrasted with relatively monotonous and round nuclei of AEMs. However, macronucleoli were not significant in KRAS-mutated MAs in the present study, although Song et al. documented macronucleoli as a characteristic feature of KRAS-mutated adenocarcinomas.15 Rather, AEMs demonstrated prominent single nucleolus. This discordance could result from different materials and methods between the two studies. Song et al. used lymph node aspiration specimens and reviewed cytology slides.15 Since cells of cytology specimen are entirely prepared they may provide more visible nucleoli than cells from sectioned formalin fixed, paraffin embedded tissue. Additionally, most of the lymph node aspiration cases were in an advanced stage, whereas most of our KRAS-mutated MAs were relatively earlystage resected cases with rare lymph node metastasis. Besides, ALK-rearranged adenocarcinoma has been reported to have nuclear clearing like papillary thyroid carcinoma, and this nuclear feature would have been retained in AEMs, which may make the nucleoli more visible compared with those of KRASmutated MAs.4 Interestingly, apical snouts were observed in 83.3% of AEMs in the present study; these have not been introduced as an ALK-specific histological feature so far. Apical snouts are documented in breast lesions, including apocrine and columnar cell lesions.17,18 In contrast to AEMs, most of the KRAS-mutated MAs showed smooth apical cytoplasmic borders devoid of any snouts, except for two cases. Thus, we suggested that apical snouts would be one of the ALK-specific histological features in cases with extensive mucin that can mimic MAs.
CONCLUSIONS We compared clinicopathological features of AEMs with KRAS-mutated MAs and correlated the histology and molecular alterations. Since companion diagnosis with integrated histological and molecular findings is essential for appropriate treatment and construction of data for comprehensive analysis in the future, we suggest that AEMs should be distinguished from MAs. Although AEMs are rare and only a small number of patients could be included in the current study, we found that AEMs occurred in patients of younger age and exhibited distinct histological features compared to the KRAS-mutated MAs. It appears to be critical to not overlook any ALK-specific histology, especially floating tubulopapillary pattern and apical snouts in extensive mucin producing adenocarcionomas, because they strongly suggest the possibility of ALK rearrangement. Further study with a larger number of patients is required to clarify the clinicopathological features of AEMs.
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Acknowledgement: The authors are deeply indebted to Dr Hyo Sup Shim in the Department of Pathology, Yonsei University College of Medicine, for his comments that greatly improved the manuscript. Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose. Address for correspondence: Prof Joungho Han, Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, 135-710, South Korea. E-mail: hanjho@ skku.edu
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