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Prognostic Value of Cyclin A2 and B1 Expression in Lung Carcinoids

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Prognostic Value of Cyclin A2 and B1 Expression in Lung Carcinoids Pathology (August 2019) 51(5), pp. 481–486 ANATOMICAL PATHOLOGY Prognostic value of cyclin A2 and B1 expression in lung carcinoids 1 1 2 1 LUKA BRCIC ,MARTIN HEIDINGER ,ANITA ZENKO SEVER ,MARTIN ZACHARIAS , 3 4 4 5 MARKO JAKOPOVIC ,MELANIE FEDIUK ,ALFRED MAIER ,FRANZ QUEHENBERGER , 6 1 SVEN SEIWERTH ,HELMUT POPPER 1Medical University of Graz, Diagnostic and Research Institute of Pathology, Graz, Austria; 2University Hospital Centre Zagreb, Clinical Department of Pathology and Cytology, Zagreb, Croatia; 3University of Zagreb School of Medicine, Department for Respiratory Diseases Jordanovac, University Hospital Centre Zagreb, Zagreb, Croatia; 4Medical University of Graz, Department of Surgery, Division of Thoracic and Hyperbaric Surgery, Graz, Austria; 5Medical University of Graz, Institute for Medical Informatics, Statistics and Documentation, Graz, Austria; 6University of Zagreb School of Medicine, Institute of Pathology, Zagreb, Croatia Summary INTRODUCTION Carcinoid classification in thelungisstillbasedon Cell cycle progression is regulated by the interaction of morphological criteria. Although there are many studies cyclin, cyclin dependent kinases (CDKs) and CDK in- investigating the role of Ki-67 proliferation index in the hibitors.1 There are more than 20 different CDKs and cyclins, classification of lung neuroendocrine tumours, it is still but only some cyclin-CDK complexes are important in pro- not used in routine diagnostics. Interestingly, cyclins, gression and control of the cell cycle.2 Therefore, it is not which have a crucial role in controlling the cell cycle, surprising that their molecular changes and changes in their have not been thoroughly studied in lung neuroendocrine expression have been detected in many tumours.3,4 However, tumours. The aim of our study was to investigate the different cyclins are differently expressed and regulated in correlation of cyclin A2 and B1 expression with prog- different cell cycle phases. nosis, Ki-67 proliferation index, and carcinoid After binding of cyclin A2 to CDK2, DNA replication is morphology. A cohort of 134 resected typical and atyp- induced, and the cell passes through S phase. Furthermore, ical carcinoids was stained with antibodies against Ki-67, the cyclin A2-CDK2 complex is essential for the progression cyclin A2 and B1. The positive nuclear reaction was of G2 phase into mitosis.5 Cyclin A is localised in the nucleus assessed in hot spot areas and expressed as the per- during S phase, controlling DNA synthesis.6 In G2 phase it centage of tumour cells. Univariate analyses found the moves to centrosomes where it regulates cyclin B-CDK1 highest relative hazard between low and high cyclin A2 complex activation.7 Cyclin B binds to CDK1 at the end of S expression both with respect to overall survival [hazard phase and the complex initiates mitosis (M phase); thereafter ratio (HR)=16; 95% confidence interval (CI) 4.8–51; the complex is degraded.8 Since the role of the cyclin B- p=0.0000054], and relapse (HR=8; 95% CI 3.1–21; CDK1 complex in mitosis is crucial, it is not unexpected to p=0.00002). In multivariate analysis for overall survival see its dysregulation in many tumours. Several studies cyclin A2 (HR=10; 95% CI 2.5–>100; p=0.0082) and B1 demonstrated high cyclin B1 expression in different carci- (HR=6.5; 95% CI 1.5–35; p=0.02) remained significant nomas, which was associated with aggressiveness of tumour, – when adjusted for other risk factors, whereas Ki-67 was and might serve as a prognostic marker.9 11 It can be present no longer significant (HR=0.64; 95% CI 0.003–5.5; in the cytoplasm and in the nucleus, the latter associated with p=0.65). This suggests that Ki-67 is closer to conven- poorer prognosis (as demonstrated in breast and oesophageal tional risk factors for survival than cyclin A2 and B1. carcinomas).12,13 On the other hand, Zheng et al. showed that Furthermore, the analysis revealed 4 mitoses per 2 mm2 high expression of cyclin B1 is present in ovarian tumours as a more powerful prognostic cut-off than currently with low malignant potential, but not in ovarian carci- accepted 2 mitoses. We have clearly demonstrated that nomas.14 Cyclin D1 accumulates in the nucleus in G1 phase, application of cyclin A2 and cyclin B1 might bring addi- and after dimerisation with CDK4 and CDK6 regulates tional value regarding the overall and progression-free transition from G1 to S phase. Independently of CDK it also survival of patients with carcinoids of the lung. regulates cell differentiation, growth and proliferation. Increased expression of cyclin D1 is present in different – Key words: Carcinoid; lung; cyclin; Ki-67; mitosis. carcinomas for which it is also a poor prognostic marker.15 17 Received 24 November 2018, revised 18 February, accepted 22 March 2019 Igarashi et al. demonstrated a different expression of cyclin Available online 20 June 2019 B1 in typical (TC) versus atypical carcinoids (AC), while no Print ISSN 0031-3025/Online ISSN 1465-3931 © 2019 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved. DOI: https://doi.org/10.1016/j.pathol.2019.03.011 482 BRCIC et al. Pathology (2019), 51(5), August significant difference in cyclin D1 was observed.11 However, The fixation process was more or less consistent throughout the investi- they did not look closely into this, and the role of cyclins A2 gated period, due to the routinely tested pH of formalin. and B1 in pulmonary carcinoids until now has not been Immunohistochemistry evaluated. Ki-67, cyclin A2 and cyclin B1 immunostaining was performed on formalin- In the 2015 World Health Organization (WHO) classifi- fixed, paraffin embedded, 3–4 mm thick sections. Ki-67 antibody [clone MIB- cation, TC and AC, together with large cell neuroendocrine 1 (GA62661), ready to use, with EnVision FLEX Kit, both from Dako, carcinoma and small cell carcinoma, are grouped into Denmark] after low pH antigen retrieval was stained on Omnis (Dako). 18 neuroendocrine tumours (NET) of the lung. TC represents Polyclonal cyclin A2 antibody (ab80792; Abcam, United Kingdom) was the low-grade end of this spectrum and AC is a tumour of stained on Dako Autostainer, after antigen retrieval in waterbath (40 min at intermediate malignancy. Both represent 1–2% of all pri- 98C), using EnVision detection system (Dako). Monoclonal antibody for mary lung malignancies; however, they are the most cyclin B1 (clone Y106, ab32053, dilution 1:200; Abcam) was used on common primary lung malignancies in children and late Ventana Benchmark XT with Ultraview DAB detection Kit (Ventana, USA). adolescents.19 Distinction between TC and AC is still based Slides were counterstained with haematoxylin, dehydrated and mounted. on morphology alone: tumours with neuroendocrine Positive reaction was assessed as the percentage of tumour cells showing nuclear expression of Ki-67, cyclin A2, and cyclin B1. Counting was morphology (i.e., rosettes, trabeculae, nests), <2 mitoses per 2 fi performed in the hot spot areas (areas of highest nuclear staining) after 2mm, and absence of tumour necrosis are classi ed as TC, scanning the whole slide on the low power magnification, by counting of – 2 while tumours with 2 10 mitoses per 2 mm , and/or pres- 2000 tumour cells, as previously described.23 ence of tumour necrosis, should be classified as AC.18 Differentiation between TC and AC is important not only Statistical analysis because AC more often have distant metastases and worse The significance level for statistical tests was 0.05. Homogeneity of two by prognosis, but also because therapy protocols might differ. two tables was tested by Fisher’s exact test. Spearman’s rank correlation Ki-67 as a proliferation marker was introduced in the last quantified associations. Risk factors for overall survival and recurrence were – WHO classification as a tool to distinguish carcinoids (with analysed by Kaplan Meier estimation and proportional hazards regression. proliferation index <20%) from high-grade NET [small cell Observation time was limited to 10 years as no deaths occurred afterwards. Median time of follow-up was calculated by the Kaplan–Meier method with lung cancer (SCLC) and large cell neuroendocrine carci- 24 18 the roles of censoring and event reversed. For immunohistological staining, noma (LCNEC)] where proliferation index is >40%. This optimal cut-off values were calculated that minimised p value of the log rank is in line with the European Neuroendocrine Tumour So- tests for survival. Age was dichotomised at the median, T stage at one and N ciety expert consensus and recommendations, where Ki-67 at zero. All risk factors were coded as zero or one. Therefore, the regression was found to be useful in small biopsies as a tool for coefficients in Cox regression are the logarithm of the hazard ratios of two separating carcinoids from high-grade NET.19 Different groups. As in the multivariate Cox model, many of the risk factors were studies analysed Ki-67 expression in carcinoids, applying a strongly correlated, and the estimates had to be calculated by the elastic net 25 range of cut-off values (2.5–7.0%); but most of them were method that penalises overfitting. R 3.4.4 (www.r-project.org) and the not able to clearly separate TC and AC.20,21 packages glmnet 2.0.16 and selectiveInference 1.2.4 were used for As previously demonstrated, there is some inter-observer calculations. variability in carcinoid classification using only morpho- logical criteria.22 Furthermore, in small biopsies it is diffi- RESULTS cult to assess all histological criteria, since the area of the Clinicopathological characteristics of the study cohort whole tumour sample is often smaller than 2 mm2. Even Our study cohort consisted of 97 TCs and 37 ACs. There with the help of Ki-67, proper prognostic stratification is not were 72 female and 62 male patients, median age of 56 years always possible.
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