Natural history and clinical characteristics of multiple pulmonary nodules with ground glass opacity

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1 Editor's Choice ORIGINAL ARTICLE Natural history and clinical characteristics of multiple pulmonary nodules with ground glass opacity YUKI SATO, 1 DAICHI FUJIMOTO, 1 TAKESHI MORIMOTO, 2,3 KEIICHIRO UEHARA, 4 KAZUMA NAGATA, 1 ICHIRO SAKANOUE, 5 HIROSHI HAMAKAWA, 5 YUTAKA TAKAHASHI, 5 YUKIHIRO IMAI 4 AND KEISUKE TOMII 1 1 Department of Respiratory Medicine, 2 Department of Clinical Research Center, 4 Department of Clinical Pathology, 5 Department of Thoracic Surgery, Kobe City Medical Center General Hospital, Kobe and 3 Department of Clinical Epidemiology, Hyogo College of Medicine, Nishinomiya, Japan ABSTRACT Background and objective: Ground glass nodules (GGNs) are frequently encountered in the lungs. We report the natural history and characteristics of multiple GGNs, and propose a management plan for patients with multiple GGNs. Methods: We retrospectively analysed patients with GGNs that met the following criteria: (i) GGN diameter of 3 cm or less, (ii) ground glass opacity proportion of 50% or more and (iii) observation without treatment for 6 months. We evaluated size changes in computed tomography images. Two end points, incidence of growth at 36 months and time to growth were analysed using logistic regression models and Cox proportional hazards model. Results: Between April 2008 and December 2014, 187 patients fulfilled the inclusion criteria (78 (42%) had multiple lesions). Among the multiple-ggn patients, the median observation period was 45.5 months, 25 patients (32%) experienced GGN progression at 36 months and 4 patients (5.1%) after 36 months. Between the multiple and single GGNs, there were no significant differences in growth incidence at 36 months (P = 0.1), after 36 months (P = 0.6) or in time to growth (P = 0.3). Among patients with multiple GGNs who experienced one GGN growth, 41% of patients experienced residual GGN growth afterwards. Conclusion: Patients with multiple GGNs showed a tendency to growth within the first 36 months, and a significant proportion of patients experienced multiple GGN progression. We suggest that the optimal observation period for patients with multiple GGNs is 36 months, but careful observation is needed after a lesion begins to grow. Key words: adenocarcinoma, follow-up, ground glass nodule, multiple, natural history. Correspondence: Daichi Fujimoto, Department of Respiratory Medicine, Kobe City Medical Center General Hospital, Minatojima-Minamimachi, Chuo-ku, Kobe , Japan. daichi@kcho.jp Received 16 February 2017; invited to revise 21 March 2017; revised 13 April 2017; accepted 14 April 2017 (Associate Editor: David Barnes). SUMMARY AT A GLANCE We describe the natural history of patients with multiple ground glass nodules (GGNs). In most GGNs, growth occurs within the first 36 months. Residual GGN growth after initial growth was common in multiple-ggn patients. A past history of lung cancer and size >10 mm are risk factors for growth. Abbreviations: AAH, atypical adenomatous hyperplasia; AIS, adenocarcinoma in situ; EGFR, epidermal growth factor receptor gene; G12x, other codon of 12 KRAS mutant; GGN, ground glass nodule; GGO, ground glass opacity; HR, hazard ratio; HRCT, high-resolution computed tomography; HU, Hounsfield unit; KRAS, v-ki-ras2 Kirsten rat sarcoma viral oncogene homologue; LLL, left lower lobe; LUL, left upper lobe; MIA, minimally invasive adenocarcinoma; OR, odds ratio; PCR, polymerase chain reaction; RLL, right lower lobe; RML, right middle lobe; RUL, right upper lobe. INTRODUCTION Pulmonary nodules with ground glass opacity (GGO) have been frequently observed since the clinical introduction of HRCT. 1 A ground glass nodule (GGN) is defined as a hazy opacity that does not obscure the view of underlying bronchial structures or pulmonary vessels on HRCT. 2 GGN is observed in a wide variety of clinical conditions, for example, focal interstitial fibrosis, inflammation, haemorrhage and adenocarcinoma. 3,4 Previous reports demonstrated that most of the persistent GGNs are preinvasive adenocarcinoma, 20 30% of the nodules grow to invasive adenocarcinoma due to multistep progression, and most of these lesions grow within 36 months GGNs frequently appear as multiple nodules. Previous genetic research on multiple GGNs indicates that these tumours are mostly multifocal, independent cancers When GGN lesions are scattered in different lobes of the lung in patients with multifocal GGNs, it can be difficult to resect all; therefore, observation is a standard procedure for multiple GGNs without any invasive features. doi: /resp.13089

2 1616 Y Sato et al. The natural history of multiple GGNs and the difference between single and multiple lesions have not been fully examined, and it has also not been determined how patients with multiple GGNs should be managed. Existing guidelines are based on previous reports, which included only small numbers of multiple GGNs. 11,15 19 The objective of this study was to understand the natural history and characteristics of multiple GGNs, and to identify areas where it might be possible to improve the management of these patients. METHODS Patients We retrospectively collected patients from a departmental database with pulmonary nodules who met the following criteria: (i) tumour diameter of 3 cm or less, (ii) GGO proportion of 50% or more and (iii) observation without treatment for 6 months. 10,16 All patients were regularly evaluated by HRCT scans; the intervals were at the discretion of the attending physician. Generally, during the first year, follow-up visits were conducted every 3 6 months and after 1 year they were conducted every 6 12 months. Patients who reported never having smoked were defined as never-smokers, those who had smoked within 1 year of the diagnosis were categorized as current smokers and the rest were considered to be former smokers. This study was reviewed and approved by the Institutional Review Board of Kobe City Medical Center General Hospital (zn160804). All tumour specimens enrolled in the genetic analysis were obtained with informed consent (or formal waiver of consent) with approval by our Ethics Committee. The informed consent was waived from individual participants enrolled in the retrospective study part, as all HRCT imaging was performed during routine medical care. Radiological analysis HRCT scans were performed with various scanners but mainly with Light Speed VCT or Optima CT 660 and analysed by the AW server (GE Yokogawa Medical System, Hino, Japan). Image data were reconstructed with a thickness of mm, and obtained using a lung window setting with a level of 650 Hounsfield units (HU) and a width of 1500 HU. All radiological images were evaluated by two independent specialists (pulmonologist and radiologist). A GGN was defined as an area with a slight, homogeneous increase in density that did not obscure the underlying vascular markings. 20 For this study, we performed central reviews of HRCT scans, and the size of each lesion was recorded by measuring the largest diameter in the axial dimension using a calliper tool in the software on the lung window. In cases of multiple GGNs, we only recorded the diameters of the largest lesions. In this study, we discriminate part-solid GGN from pure GGN. 11,21 We scored the ratio of the solid portion to the pure GGO portion by visually estimating the proportion of the GGN component in each tumour without measuring the diameter, as previously reported. 22 According to the previous study, GGN progression was concluded when one of the following points was recognized: (i) a gross increase in the greatest dimension by at least 2 mm from the initial HRCT scan, (ii) a gross increase in the size of the solid part by at least 2 mm or (iii) a new solid part of any size. 23 Patients who underwent surgical resection without experiencing GGN progression were censored. Pathological and genetic analysis We retrospectively analysed these specimens according to the World Health Organization (WHO) classification of lung adenocarcinoma. 24 We isolated tumour DNA and analysed the epidermal growth factor receptor gene (EGFR) mutation status at exons using the peptide nucleic acid-locked nucleic acid PCR clamp method, as described previously. 25 For EGFR mutationnegative patients, we investigated v-ki-ras2 Kirsten rat sarcoma viral oncogene homologue (KRAS) mutation using both the multiplex kit and direct sequencing methods, as these mutations are mutually exclusive. 26,27 All pathological analyses were evaluated by two experienced pathologists who were unaware of the patients conditions. Statistical analysis The follow-up time period was defined from the date of the initial HRCT to the date of the latest HRCT. The time to GGN growth was defined from the date of the initial HRCT to the date of the HRCT when progressive GGN growth was determined. Continuous variables were analysed using the Student s t-test. Dichotomous variables were analysed using Fisher s exact test. To clarify the factors that may affect GGN growth incidence, logistic regression models were applied to estimate ORs and 95% CIs on all clinically important factors (age, sex, smoking status, past history of lung cancer, GGN pattern, GGN size and GGN multiplicity), followed by multivariate models with all factors included simultaneously. The Kaplan Meier method was used to estimate the time to GGN growth, and groups were compared using the log-rank statistic. Cox proportional hazards models were fitted to determine the associations between patient characteristics and time to GGN progression in both univariate and multivariate analyses. The results are expressed as hazard ratios (HRs) with 95% CI. A P-value of <0.05 indicated statistical significance. We conducted the statistical analyses using JMP 11 software (SAS Institute, Cary, NC, USA). RESULTS Patient characteristics Between April 2008 and December 2014, 193 patients met the inclusion criteria. We excluded patients with transient GGNs (n = 6). In total, 187 patients with GGN were included in the study and 78 (42%) had multiple nodules (Fig. 1). Patient characteristics and comparison between multiple and single GGNs are summarized in Table 1. A total of 299 GGN lesions were detected in patients with multiple GGNs. Among them, the median

3 Natural history of multiple GGNs 1617 P < 0.001) was independently associated with shorter time to multiple GGN progression (Table 3). Treatment modalities and their outcomes Treatment modalities and outcomes for multiple and single GGNs are shown in Table 1. A total of 14 multiple-ggn patients and 18 single-ggn patients underwent surgical resection, and all of the nodules were determined to be adenocarcinoma or preinvasive adenocarcinoma. Three out of 14 multiple-ggn patients were resected without experiencing GGN growth; physician s discretion: n = 1; patient s request: n = 1; and simultaneous resection of concurrent cancer: n =1. The post-operative stages were pt1an0m0: n = 10; pt1bn0m0: n = 3; and pt2an0m0: n = 1, and no recurrence was observed during the follow-up period. Patients with multiple GGNs were more likely to undergo radiation therapy (P = 0.045). Figure 1 Flow chart for the study patients. A total of 187 patients were included in this study. Of those, 78 (42%) patients had multiple ground glass nodule (GGN) lesions and 14 patients underwent surgical resection. GGO, ground glass opacity. number of GGN lesions per patient was 3 (range: 2 22 GGNs), the median diameter was 13.1 mm and the median observation period was 45.5 months; 22 patients (28%) had a past history of lung cancer. Comparison of clinical profiles of patients with multiple or single GGNs showed that the multiple-ggn patient group included a significantly higher percentage of patients with a past history of lung cancer (P = 0.004). Other characteristics did not differ significantly between the two groups. Analysis of progression rate at 36 months and after 36 months, and clinical factors Table 1 indicates that the progression rates of patients with multiple or single GGNs at 36 months were 32% and 22%, respectively (P = 0.1), and those of patients who experienced no progression over 36 months were 5.1% and 8.3%, respectively (P = 0.6). In patients with multiple GGNs, GGN size 10 mm (OR: 43.6, 95% CI: , P < 0.001) and past history of lung cancer (OR: 5.22, 95% CI: , P = 0.015) independently predicted a higher growth rate at 36 months (Table 2). In patients with single GGNs, the same analysis showed partly solid pattern (OR: 3.69, 95% CI: , P = 0.02) and GGN size 10 mm (OR: 4.06, 95% CI: , P = 0.02) as independent predictors of higher growth rate at 36 months (Table S1, Supplementary Information). Analysis of time to GGN progression Kaplan Meier curves of time to GGN progression in patients with multiple or single GGNs are shown in Figure 2A. The time to progression did not differ significantly (P = 0.3) between the two groups. Cox s multivariate regression analysis of the influence of clinical characteristics on time to GGN progression indicated that a GGN size 10 mm (HR: 23.3, 95% CI: , Respirology (2017) 22, Pathological and genetic analysis of resected multiple and single GGNs Table 4 indicates pathological and genetic characteristics of resected GGNs. Patients whose specimens were unavailable were excluded. No apparent difference in the proportions of invasive adenocarcinoma and predominant subtype was observed between multiple and single GGNs. Among multiple-ggn patients, there were nine (69%) patients with EGFR mutations (two patients with del19 and seven patients with L858R point mutation), whereas no patients with KRAS mutations were found. Among single-ggn patients, there were eight (47%) patients with EGFR mutation (two patients with del19 and six patients with L858R point mutation). There were five patients with KRAS mutations among single- GGN patients (two patients with G12C, one patient with G12D, G12C + A146T double mutation, and G12x (other codon of 12 KRAS mutant)). Between multiple- GGN and single-ggn patients, there was no significant difference in the frequency of EGFR mutations (P = 0.3), whereas the frequency of KRAS mutations appeared to be relatively low among patients with multiple GGNs (P = 0.053). Subsequent change after first progression in patients with multiple GGNs During the total follow-up period, 29 patients with multiple GGNs experienced progression. Of those, 12 (41%) patients experienced remaining GGN progression after the first progression (Table S2, Supplementary Information). The time to the second progression was 18.9 months. The Kaplan Meier curve of time to second GGN progression is shown in Figure 2B. DISCUSSION To the best of our knowledge, this is the first report to show the natural history and characteristics of multiple GGNs. Importantly, most of the multiple-ggn patients experienced a new GGN growth within 36 months, and 2017 Asian Pacific Society of Respirology

4 1618 Y Sato et al. Table 1 Clinical, radiological characteristics and differences by GGN multiplicity Patient characteristics Multiple GGNs (n = 78) Single GGN (n = 109) Total (n = 187) P Follow-up period (months) Median Range GGN number Total Median (range) 3 (2 22) 1 (1 1) 1 (1 22) Age (years) Mean (SD) 66.5 (10.9) 64.8 (12.1) 65.5 (11.6) 0.3 Sex Male 24 (31) 45 (41) 69 (37) 0.2 Smoking status Never 55 (71) 70 (64) 125 (67) 0.4 Current or former 23 (29) 39 (36) 62 (33) Past history of lung cancer Yes 22 (28) 12 (11) 34 (18) Adenocarcinoma Squamous cell carcinoma No 56 (72) 97 (89) 153 (82) Past history of extrapulmonary cancer Yes 35 (45) 40 (37) 75 (40) 0.3 GGN pattern Part-solid GGN 27 (35) 26 (24) 53 (28) 0.1 GGN size (mm) Mean (SD) 13.1 (6.8) 11.5 (5.5) 12.2 (6.1) 0.08 Occupying lobe RUL 22 (28) 33 (30) 55 (29) 1.0 RML 5 (6) 8 (7) 13 (7) RLL 16 (21) 18 (17) 34 (18) LUL 19 (24) 28 (26) 47 (25) LLL 16 (21) 22 (20) 38 (20) Growth incidence At 36 months 25 (32) 24 (22) 49 (26) 0.1 After 36 months 4 (5.1) 9 (8.3) 13 (7.0) 0.6 Treatment Surgical resection 14 (18) 18 (17) 32 (17) 0.8 Radiation therapy 9 (12) 4 (4) 13 (7) No treatment 55 (71) 87 (80) 142 (76) 0.2 Comparison between patients with or without history of lung cancer. In case of multiple GGNs, the lobe of the largest GGN is indicated. Comparison between patients with right-side or left-side GGN. Comparison between patients who did or did not receive surgery. Comparison between patients who did or did not receive radiation therapy. Comparison between patients who received or did not receive any treatment. GGN, ground glass nodule; LLL, left lower lobe; LUL, left upper lobe; RLL, right lower lobe; RML, right middle lobe; RUL, right upper lobe; SD, standard deviation. a second GGN progression was often detected after one progression. In our analysis, 42% of patients had multiple GGNs, and one-third of patients with multiple GGNs experienced progression. The proportion of multiple GGNs in this study is slightly higher than that in previous reports. 10,28 In our institute, pulmonologists perform HRCT of the whole lung in all patients; therefore, small GGN lesions are more likely to be detected than in the previous studies. To propose an appropriate management strategy for patients with multiple GGNs, we analysed the natural history of multiple GGNs in comparison to single GGNs. In the existing guidelines, it is recommended to do follow-up HRCT with 3 12-month intervals for at least 2 3 years for patients with GGN lesions Our results indicated that there was no significant difference in the time to progression between multiple and single GGNs, and neither lymph node metastasis nor post-operative recurrence was observed in multiple- GGN patients. Furthermore, we demonstrated that there was no significant difference in growth incidence at 36 months between multiple and single GGNs, and that the growth rate of multiple GGNs after 36 months

5 Natural history of multiple GGNs 1619 Table 2 Univariate and multivariate analyses of factors affecting the GGN growth incidence at 36 months in multiple- GGN patients Characteristics Growth incidence Multivariate analysis Univariate analysis P OR (95% CI) P Age (years) (35) ( ) 0.9 <65 8 (28) Reference Sex Male 11 (46) ( ) 0.2 Female 14 (26) Reference Smoking status Never 14 (25) ( ) 0.7 Current or former 11 (48) Reference Past history of lung cancer Yes 12 (55) ( ) No 13 (23) Reference GGN pattern Part-solid GGN 14 (52) ( ) 0.5 Pure GGN 11 (22) Reference GGN size (mm) (50) < ( ) <0.001 <10 1 (3) Reference CI, confidence interval; GGN, ground glass nodule; OR, odds ratio. Figure 2 (A) Kaplan Meier curves of time to ground glass nodule (GGN) progression. The curve for multiple-ggn patients is shown as a bold line (, multiple GGN (n = 78);, single GGN (n = 109); P = 0.3). (B) Kaplan Meier curve of time to second GGN progression in patients with multiple GGN (n = 29). was 5.1%. Previously, Kobayashi et al. suggested that a 3-year observation period is optimal for patients with GGN lesions, as they detected no GGN growth after 36 months. 10 Another study suggested that the frequency of subsequent growth of GGNs that had been stable during the initial 3 years was 6.7%. 11 From these findings, we suggest that HRCT scans with 3 12-month intervals are appropriate and, importantly, 36 months is the optimal observation period for multiple GGNs. The risk factors for patients with multiple GGNs were slightly different from those for patients with single GGNs. We determined that a GGN size 10 mm and a past history of lung cancer were risk factors for progression at 36 months for patients with multiple GGNs, Respirology (2017) 22, whereas a partly solid pattern and a GGN size 10 mm were independent risk factors for patients with single GGNs. A partly solid pattern, size and lung cancer history have been reported to be risk factors in the previous studies, which included both multiple and single GGNs. 6,7,15,28 We have to consider a past history of lung cancer as an independent risk factor for growth for patients with multiple GGNs. Surprisingly, our study revealed that 41% of patients who had experienced one GGN growth experienced subsequent growth of the remaining GGNs. These results were consistent with the above-mentioned finding that the multiple-ggn patient group comprised a significantly higher percentage of patients with a past 2017 Asian Pacific Society of Respirology

6 1620 Y Sato et al. Table 3 Univariate and multivariate analyses of factors affecting the time to GGN growth in multiple-ggn patients Characteristics Patients Univariate Multivariate analysis analysis P HR (95% CI) P Age (years) (63) ( ) 0.7 <65 29 (37) Reference Sex Male 24 (31) ( ) 0.4 Female 54 (69) Reference Smoking status Never 55 (71) ( ) 0.4 Current or 23 (29) Reference former Past history of lung cancer Yes 22 (28) ( ) 0.2 No 56 (72) Reference GGN pattern Part-solid 27 (35) ( ) 0.2 GGN Pure GGN 51 (65) Reference GGN size (mm) (62) < ( ) <0.001 <10 30 (38) Reference CI, confidence interval; GGN, ground glass nodule; HR, hazard ratio. history of lung cancer, and that these were at high risk for GGN growth. Considering the high percentage of patients with a second GGN growth, we suggest that one should pay careful attention to patients with multiple GGNs and a lung cancer history. Further studies are needed to establish appropriate intervals and duration of follow-up periods after the first growth for patients with multiple GGNs. Interestingly, genetic analyses revealed that the frequency of KRAS mutations in multiple GGNs was low, compared with the frequency in single GGNs, although it was not statistically significant. Previous studies reported that driver mutations are common in GGN lesions, and that GGNs with KRAS mutations rarely progress. 9,29 We speculate that patients with multiple GGNs might have different genetic profiles than those with single GGNs. Further large-scale research is warranted to elucidate the pathogenesis and genetic profiles of multiple GGNs. This study has some limitations. First, we recognize the retrospective, single-centre design of this study and the limited number of Japanese patients in the study group as important limitations. Second, we were unable to diagnose all GGNs, so some of them may be non-malignant lesions. Third, measurement errors should be considered. A previous report suggested that a diameter increase of >1.72 mm is necessary to identify true growth. 23 Therefore, we defined growth as a more than 2-mm increase in the size of a GGN lesion in this analysis. Fourth, there were no clear-cut criteria for GGN treatment in our institute. We basically Table 4 Comparison of histological and genetic characteristics between multiple and single GGNs with surgical resection Patient characteristics Multiple GGNs (n = 13) Single GGN (n = 17) Total (n = 30) P WHO classification AAH/AIS/MIA 2 (15) 3 (18) 5 (20) 1.0 Invasive 11 (85) 14 (82) 25 (80) adenocarcinoma Lepidic Papillary Acinar Mucinous Mutation status EGFR mutation 9 (69) 8 (47) 17 (57) 0.3 del L858R KRAS mutation 0 (0) 5 (29) 5 (17) G12C G12D G12C + A146T G12x Wild type 4 (31) 4 (24) 8 (27) 0.7 Two patients were excluded because their samples were unavailable. Comparison between patients with preinvasive lesions and invasive adenocarcinoma. Comparison between patients with lepidic type and other subtype. Comparison between patients with and without EGFR mutations. Comparison between patients with and without KRAS mutations. Comparison between patients with and without any mutations. AAH, atypical adenomatous hyperplasia; AIS, adenocarcinoma in situ; EGFR, epidermal growth factor receptor gene; G12x, other codon of 12 KRAS mutant; GGN, ground glass nodule; KRAS, v-ki-ras2 Kirsten rat sarcoma viral oncogene homologue; MIA, minimally invasive adenocarcinoma; WHO, World Health Organization. performed treatment when the lesions began to grow in size or the solid component began to increase; however, a number of clinical factors influenced the timing of pulmonary resection. In conclusion, we suggest that the optimal observation period for multiple GGNs is 36 months; however, we should pay careful attention to patients with multiple GGNs who experience just one GGN progression. Acknowledgements The authors would like to thank Munehiro Ito, Shunsuke Teraoka, Atsushi Nakagawa and Kojiro Otsuka for data acquisition, and Keiko Sakuragawa for her administrative assistance. Disclosure statement This study was supported by internal funding of Kobe City Medical Center General Hospital.

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Lung Cancer 2009; 64: Sakamoto H, Shimizu J, Horio Y, Ueda R, Takahashi T, Mitsudomi T, Yatabe Y. Disproportionate representation of KRAS gene mutation in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutation from preinvasive to invasive adenocarcinomas. J. Pathol. 2007; 212: Supplementary Information Additional supplementary information can be accessed via the html version of this article at the publisher s website. Table S1 Univariate and multivariate analyses of factors affecting ground glass nodule (GGN) growth incidence at 36 months in single-ggn patients. Table S2 Disease course after first growth in multiple-ground glass nodule patients. Respirology (2017) 22, Asian Pacific Society of Respirology