In the United States, lung adenocarcinoma is the most common form of primary lung cancer and is classified as non-small cell lung cancer (NSCLC). It is closely associated with previous smoking history. Although incidence and mortality rates have declined, it remains the leading cause of cancer-related deaths in the country Lung adenocarcinoma, originating from mucosal glands, makes up around 40% of all lung cancers. It is particularly prevalent among non-smokers and typically arises in the outer parts of the lungs. This type of cancer is often detected in areas with existing scars or chronic inflammation. (1). The primary factors strongly linked to lung cancer in individuals who have never smoked include exposure to acknowledged and suspected carcinogens like radon, second-hand tobacco smoke, and various indoor air pollutants. (2). Initially, lung adenocarcinomas were classified by architectural subtypes and predominant histological patterns, correlating with prognosis(3,4A non-official grading system that divides assessments into three distinct levels emerged, categorizing tumors into low, intermediate, and high grades(5). Cribriform and fused gland patterns, referred to as complex glandular patterns (CGPs), fall under the acinar subtype. In 2015, the WHO classified the cribriform pattern as a high-grade form of the acinar subtype. (5). In 2020, the IASLC introduced a new grading system considering both predominant and highest-grade patterns, with tumors containing ≥ 20% high-grade patterns classified as poorly differentiated, including cribriform glandular patterns with solid and micro papillary patterns(6,7Following the introduction of lung adenocarcinoma (LUAD) growth patterns as outlined by the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) in 2011, these criteria were later incorporated into the World Health Organization (WHO) classification. (8) Numerous investigations have been conducted to assess their prognostic significance in resected specimens (9, 10, 11, and 12).Accurate grading of tumors is crucial for prognosis determination and treatment planning (13). Biopsy and resection specimens are key for tumor grading, but their discordance presents challenges in clinical decisions. Biopsies, usually the first diagnostic step, may not fully capture the tumor's histology due to sampling limitations. In contrast, resection specimens provide a more comprehensive view of the tumor's architecture. (14,15Several studies suggest that the total number of involved lymph nodes or lymph node stations may provide a more precise indication of prognosis in certain cancers, such as lung adenocarcinoma. (16, 17, 18)

The aim of our study is to examine the consistency of tumor grading between biopsy and resection specimens in lung adenocarcinoma and to assess its prognostic significance in relation to lymph node metastasis.

This research was conducted at Pathology Department of Shaukat Khanum Memorial Cancer Hospital and Research Center in Lahore, Pakistan, after approval from the Institutional Review Board (IRB). We retrospectively retrieved 24 in-house patients diagnosed with lung adenocarcinoma from December 2010 to December 2020 from hospital archives. Our study included in house cases of lung adenocarcinoma who underwent complete resection here and for which complete core biopsy and corresponding resection specimen were available with us, follow-up status duration of 125 months was available in hospital database, excluding those with other histological subtypes, loss of follow-up, unavailable results of molecular studies and scanty biopsies.

Two consultant pathologists, each with significant experience in the relevant field, conducted a histological slide review to assess the tumor grade in initial biopsies. Biopsy specimens were categorized into lepidic, acinar, papillary, micro papillary and solid types, using 5% increments to quantify the proportions of the observed growth patterns (fig 1-6). This classification was based on the dominant growth pattern present in the tumors. All patients underwent complete tumor resection along with lymph node dissection. Data for this study were retrieved from the hospital database, which included the results of molecular studies conducted on these patients. In evaluating the resection specimens, the histological tumor grade was assigned based on the grading system recommended by the International Association for the Study of Lung Cancer (IASLC) (19). This system classifies tumors into three grades: Grade 1 for lepidic predominant tumors, Grade 2 for acinar or papillary predominant tumors, both of which show no or less than 20% high-grade patterns. Grade 3 includes any tumor with 20% or more high-grade patterns, such as solid, micro papillary, or complex glandular structures.

The pathological stage (pT) and nodal stage (pN) were assessed using the tumor protocols for lung cancer provided by the College of American Pathologists (CAP) (20). Additionally, advanced research has revealed a variety of molecular markers with predictive and prognostic significance in lung cancer (21, 22). Molecular studies were conducted on these patients, and data were collected from archives.

Data Analysis:

Data was entered and analyzed in SPSS 27. Association between grades on resection and categorical variables like gender, tumor staging and prognostic marker was assessed by using Chi Square test. Age differences among three grades on resection were observed by Friedman test. Concordance between Grades on biopsy and resection was assessed through Cohen kappa statistic. Kaplan Meier was used for calculate overall, metastasis free and recurrence free survival among different grades on resection. The p≤0.05 was considered significant.

Our study comprised of 24 patients out of which 18(75.0%) were males and 6(25.0%) were females. The average age of the patients was 61.46±12 years. On biopsy, there were 3(12.5%) cases with Grade 1, 14(58.14%) cases with Grade 2, and 7(29.2%) cases with Grade 3, while on resection grade distribution was 1(4.2%), 9(37.5%) and 14(58.3%) for Grade 1, Grade 2 and Grade 3, respectively. Upon analyzing the biopsy and resection grades, it was observed that among the three cases initially diagnosed as Grade 1 tumors on biopsy, 1(33.3%) remained Grade 1, 1(33.3%) progressed to Grade 2 and 1(33.3%) advanced to Grade 3. Among the 14(58.14%) cases identified as Grade 2 tumors on biopsy, 8(57.1%) retained Grade 2, while 6(42.9%) transitioned to Grade 3. All the cases diagnosed as Grade 3 tumors on biopsy remained Grade 3 upon resection.

Hence, a moderate to substantial level of concordance between tumor grades observed on core biopsies and resection specimens (Kappa=0.450, p= 0.002). The PT1 stage predominantly consisted of Grade 2 tumors 3(75.0%) on resection with a minority 1(25%) being Grade 3. PT2 cases were largely 9(64.0%) in Grade 3. PT 4 cases were entirely Grade 3. This pattern revealed that the higher PT stages tend to exhibit higher-grade tumors (p=0.335). For Nx (nodal stage not assessed),

1(50%) case had Grade 1 tumors, 1(50%) had Grade 2. In N0 (no regional lymph node metastasis), 7(43.8%) cases had Grade 2, and 9(64.3%) cases had Grade 3. N1 (metastasis to peribronchial/hilar lymph nodes) had 3(75.0%) cases with Grade 3. for N2 (metastasis to mediastinal lymph nodes), 2(100%) cases had Grade 3 tumors. This provided an association between lymph node involvement and tumor severity on resection (p=0.129). Out of 24 patients, EGFR was performed on 10(41.7%) patients and all they were negative (p=0.504). ALK testing was conducted on 7(29.2%) patients, resulting in 6(25%) patients negative for ALK mutation and 1(4.2%) patients positive who had Grade 3 tumor (p=0.839). BRAF testing was conducted on 2(8.3%) patients, both yielding negative results. Among these patients, 1(50%) patient displayed Grade 2 tumors, and 1(50%) patient had Grade 3 tumor (p=0.901). (Table 1)

Among the 24 cases examined, 11(45.8%) patients died. The 4(44.4%) patients with the Grade 2 and 7(50%) with Grade 3 tumor observed death... The estimated mean survival time for patients with Grade 2 was estimated to be approximately 81.42±19.64 (Months). For the patients with Grade 3, the estimated mean survival time was estimated to be approximately 73.69±14.48 (Months). Patients with grade 3 tumors exhibited a lower estimated mean survival time compared to those with Grade 2 tumor however, no statistically significant difference was observed (p=0.916). The overall 5-years survival rate for Grade 2 tumor was estimated to be approximately 46.70% (95% C.I: 9.46%-83.940%). Among Grade 3 patients, the 5-year survival rate was 42.0% (95% C.I: 13.97%-70.03%). (Figure 7)

Out of 24 patients 11(45.8%) were metastatic. The metastasis free estimated mean survival time for Grade 2 and Grade 3 was approximately 96.09±19.67 (Months) and 60.12±14.32 (Months), respectively with no significant difference (p=0.267). The overall 5-years metastasis free survival rate for Grade 2 and grade 3 tumor was estimated to be approximately 64.8%(95% C.I: 32.46%-97.14%) and 37.30% (95% C.I: 8.49%-66.11%), respectively. (Figure 8)

Recurrence was observed in 2(8.3%) patients within the study population, and both of them had Grade 3 tumor. The recurrence free estimated mean survival time for patients with Grade 3 was approximately 105.889±15.041 (Months) The 5-year recurrence free survival rate for Grade 3 tumor was projected to be approximately 77.80% (95% C.I: 50.56%-100.0%) (Figure 9)

Identifying high-grade adenocarcinoma in patients with early-stage lung adenocarcinoma prior to surgery is crucial. However, there are a limited number of studies comparing the accuracy of adenocarcinoma subtyping between small biopsy samples and surgical resection specimens (23, 24, and 25). Among the few available studies, Matsuzawa et al. (2019) reported a 66% concordance in predominant subtypes between 326 resected specimens and their corresponding biopsy samples. Importantly, their study did not explore the concordance related to the presence or absence of high-grade adenocarcinoma another study by Yang Huang et al. in 2017 demonstrated that in 58.6% of cases, there was agreement in the adenocarcinoma subtypes between the predominant patterns observed in resection specimens and those identified in biopsy sections from CTNB/R-EBUS. Our recent study showed that notably, cases initially diagnosed as Grade 1 tumors on biopsy exhibit a 100% concordance, as the lone case maintains Grade 1 upon

resection. Conversely, Grade 2 tumors on biopsy demonstrate an 88.9% concordance, with the majority retaining their Grade 2 status on resection, while 42.9% progress to Grade 3. All Grade 3 tumors on biopsy show a 100% concordance, persisting as Grade 3 upon resection.

Distinct histologic subtypes in lung adenocarcinoma exhibit varied biological invasiveness, with micro papillary and solid predominant subtypes showing heightened aggressiveness and susceptibility to lymph node metastasis. This underscores the influence of histologic subtype on lymph node involvement. Additionally, the absence of lepidic predominant subtype in lymph nodes aligns with the better prognosis associated with this subtype compared to others. (26,27,28,29,30).

Previous study conducted by Y.YU et al in 2016 showed that, the percentages of lymph node involvement varied among histologic subtypes, with the highest rates found in solid predominant and micro papillary predominant subtypes at 47.6% and 66.7%, respectively and study by long Xu et al in 2022 showed that the patients with grade 3 LUAD had a significantly higher proportion of advanced clinical T stage (P<0.001), mediastinal nodal metastasis (P<0.001), vascular permeation invasion (VPI) (P<0.001), and spread through air spaces (STAS) (P<0.001) compared to those with grade 1 and grade 2 LUAD. Our present study shows that for those with no regional lymph node metastasis (N0), none had Grade 1, 77.8% had Grade 2, and 64.3% had Grade 3. In instances of metastasis to peribronchial/hilar lymph nodes (N1), none had Grade 1, 11.1% had Grade 2, and 21.4% had Grade 3. For metastasis to mediastinal lymph nodes (N2), none had Grade 1 or Grade 2, and both cases (14.3%) had Grade 3 tumors.

Molecular testing in lung cancer primarily targets adenocarcinomas and poorly differentiated non-small cell carcinomas, while routine testing is not conducted for squamous cell carcinoma, small cell carcinoma, or low-grade neuroendocrine tumors (31). Research shows a higher frequency of K-ras, EGFR, and BRAF mutations in adenocarcinomas with a micro papillary growth pattern. (32,33,34). Previous studies conducted by Rosane et al in 2009, Arne Warth et al in 2014 and Weijing Cai et al in 2015 showed that Lung adenocarcinomas with a predominant micro papillary growth pattern exhibit a higher occurrence of K-ras, EGFR, and BRAF mutations compared to conventional adenocarcinoma. In the present study, EGFR testing (10 patients) yielded all negative results, mostly associated with Grade 3 tumors. ALK testing (7 patients) showed predominantly negative results (6 cases), correlated with Grade 3 tumors. Limited BRAF data (2 patients) hindered definitive conclusions, but negative BRAF results were associated with Grade 3 tumors, emphasizing the significance of EGFR, ALK, and BRAF as potential markers influencing lung adenocarcinoma grades.

Study limitations involve a small, in-house patient sample, excluding out-house patients, and an inability to follow all cases. Interobserver variability and differences in grading criteria among pathologists may impact the concordance of tumor grade in biopsy and resection specimens.

The exclusion criteria included multiple lung nodules, adenocarcinoma in situ or minimally invasive adenocarcinoma, clinical stage II-III according to the TNM classification, a history of previous malignancies or preoperative treatment, and patients who were treated outside the hospital.

In summary, our study encompassed 24 patients, predominantly males (75%) with a mean age of 42 to 70 years, and a median follow-up duration of 125 months. Notably, our findings revealed variations in tumor grades upon resection, with specific transitions noted from biopsy to resection, especially in Grade 2 cases.

Furthermore, the analysis of prognostic markers (ALK, BRAF, and EGFR) highlighted the influence of these markers on tumor grade and clinical outcomes. EGFR negativity was associated with Grade 3 tumors, while ALK-negative cases predominantly exhibited Grade 3 tumors. BRAF-negative results were also linked to Grade 3 tumors. The survival analysis demonstrated the impact of tumor grade on recurrence-free survival, metastasis-free survival, and overall survival, emphasizing the clinical significance of grading in predicting disease outcomes. These insights underscore the importance of comprehensive molecular and pathological evaluations for a more nuanced understanding of lung adenocarcinoma prognosis and tailored therapeutic approaches.

Acknowledgements

I would like to express my sincere gratitude to Dr Sajid Mushtaq and Dr Maryam Hameed, whose guidance and support were invaluable throughout this research. Special thanks to of Shaukat Khanum Memorial Cancer Hospital and Research Center in Lahore, Pakistan, for providing the necessary resources and facilities for conducting this study. I also extend my appreciation to my colleagues and fellow researchers for their insightful discussions and collaboration. Lastly, I would like to acknowledge my family and friends for their unwavering encouragement and support during this journey.

1. Li C, Lu H. Adenosquamous carcinoma of the lung. OncoTargets and therapy. 2018 Aug 14:4829-35.
2. Samet JM, Avila-Tang E, Boffetta P, Hannan LM, Olivo-Marston S, Thun MJ, Rudin CM. Lung cancer in never smokers: clinical epidemiology and environmental risk factors. Clinical Cancer
3. //Research. 2009 Sep 15; 15(18):5626-45.
4. Travis WD, Brambilla E, Müller-Hermelink HK, Harris CC. Tumours of the lung, pleura, thymus and heart: World Health Organization Classification of Tumors. Pathology and genetics, Lyon. 2004.
5. Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG, Powell CA, Riely GJ, Van Schil PE, Garg K. International association for the study of lung cancer/american thoracic society/European respiratory society international multidisciplinary classification of lung adenocarcinoma. Journal of thoracic oncology. 2011 Feb 1; 6(2):244-85.
6. Travis WD, Brambilla E, Nicholson AG, Yatabe Y, Austin JH, Beasley MB, Chirieac LR, Dacic S, Duhig E, Flieder DB, Geisinger K. The 2015 World Health Organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification. Journal of thoracic oncology. 2015 Sep 1; 10(9):1243-60.
7. Moreira AL, Ocampo PS, Xia Y, Zhong H, Russell PA, Minami Y, Cooper WA, Yoshida A, Bubendorf L, Papotti M, Pelosi G. A grading system for invasive pulmonary adenocarcinoma: a proposal from the International Association for the Study of Lung Cancer Pathology Committee. Journal of Thoracic Oncology. 2020 Oct 1; 15(10):1599-610.
8. Willner J, Narula N, Moreira AL. Updates on lung adenocarcinoma: invasive size, grading and STAS. Histopathology. 2024 Jan; 84(1):6-17.
9. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. Introduction to the 2015 World Health Organization classification of tumors of the lung, pleura, thymus, and heart. Journal of Thoracic Oncology. 2015 Sep 1; 10(9):1240-2.
10. Yoshiya T, Mimae T, Tsutani Y, Tsubokawa N, Sasada S, Miyata Y, Kushitani K, Takeshima Y, Murakami S, Ito H, Nakayama H. Prognostic role of subtype classification in small-sized pathologic N0 invasive lung adenocarcinoma. The Annals of Thoracic Surgery. 2016 Nov 1;102(5):1668-73.
11. Yoshizawa A, Motoi N, Riely GJ, Sima CS, Gerald WL, Kris MG, Park BJ, Rusch VW, Travis WD. Impact of proposed IASLC/ATS/ERS classification of lung adenocarcinoma: prognostic subgroups and implications for further revision of staging based on analysis of 514 stage I cases. Modern pathology. 2011 May 1; 24(5):653-64.

12. Wolf JL, Trandafir TE, Akram F, Andrinopoulou ER, Maat AW, Mustafa DA, Kros JM, Stubbs AP, Dingemans AC, von der Thüsen JH. The value of prognostic and predictive parameters in early-stage lung adenocarcinomas: A comparison between biopsies and resections. Lung Cancer. 2023 Feb 1; 176:112-20.
13. Warth A, Muley T, Meister M, Stenzinger A, Thomas M, Schirmacher P, Schnabel PA, Budczies J, Hoffmann H, Weichert W. The novel histologic International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification system of lung adenocarcinoma is a stage-independent predictor of survival. Journal of clinical oncology. 2012 May 1;30(13):1438-46.
14. Sica G, Yoshizawa A, Sima CS, Azzoli CG, Downey RJ, Rusch VW, Travis WD, Moreira AL. A grading system of lung adenocarcinomas based on histologic pattern is predictive of disease recurrence in stage I tumors. The American journal of surgical pathology. 2010 Aug 1;34(8):1155-62.
15. Huang KY, Ko PZ, Yao CW, Hsu CN, Fang HY, Tu CY, Chen HJ. Inaccuracy of lung adenocarcinoma subtyping using preoperative biopsy specimens. The Journal of thoracic and cardiovascular surgery. 2017 Jul 1; 154(1):332-9.
16. Wolf JL, Trandafir TE, Akram F, Andrinopoulou ER, Maat AW, Mustafa DA, Kros JM, Stubbs AP, Dingemans AC, von der Thüsen JH. The value of prognostic and predictive parameters in early-stage lung adenocarcinomas: A comparison between biopsies and resections. Lung Cancer. 2023 Feb 1; 176:112-20.
17. Griff S, Taber S, Bauer TT, Pfannschmidt J. Prognostic significance of the pattern of pathological N1 lymph node metastases for non-small cell lung cancer. Journal of thoracic disease. 2019 Aug;11(8):3449.
18. Lee JG, Lee CY, Park IK, Kim DJ, Park SY, Kim KD, Chung KY. Number of metastatic lymph nodes in resected non–small cell lung cancer predicts patient survival. The Annals of thoracic surgery. 2008 Jan 1;85(1):211-5.
19. Wei S, Asamura H, Kawachi R, Sakurai H, Watanabe SI. Which is the better prognostic factor for resected non-small cell lung cancer: the number of metastatic lymph nodes or the currently used nodal stage classification?. Journal of Thoracic Oncology. 2011 Feb 1;6(2):310-8.
20. Moreira AL, Ocampo PS, Xia Y, Zhongsss H, Russell PA, Minami Y, Cooper WA, Yoshida A, Bubendorf L, Papotti M, Pelosi G. A grading system for invasive pulmonary adenocarcinoma: a proposal from the International Association for the Study of Lung Cancer Pathology Committee. Journal of Thoracic Oncology. 2020 Oct 1;15(10):1599-610.
21. Cancer Protocol Templates | College of American Pathologists (cap.org) Lung_4.3.0.1.REL_CAPCP.pdf
22. Puderecki M, Szumiło J, Marzec-Kotarska B. Novel prognostic molecular markers in lung cancer. Oncology Letters. 2020 Jul 1;20(1):9-18.
23. Iyengar P, Tsao MS. Clinical relevance of molecular markers in lung cancer. Surgical oncology. 2002 Dec 1;11(4):167-79.
24. Trejo Bittar HE, Incharoen P, Althouse AD, Dacic S. Accuracy of the IASLC/ATS/ERS histological subtyping of stage I lung adenocarcinoma on intraoperative frozen sections. Modern Pathology. 2015 Aug;28(8):1058-63.
25. Matsuzawa R, Kirita K, Kuwata T, Umemura S, Matsumoto S, Fujii S, Yoh K, Kojima M, Niho S, Ohmatsu H, Ochiai A. Factors influencing the concordance of histological subtype diagnosis from biopsy and resected specimens of lung adenocarcinoma. Lung Cancer. 2016 Apr 1;94:1-6.
26. Yeh YC, Nitadori JI, Kadota K, Yoshizawa A, Rekhtman N, Moreira AL, Sima CS, Rusch VW, Adusumilli PS, Travis WD. Using frozen section to identify histological patterns in stage I lung adenocarcinoma of≤ 3 cm: accuracy and interobserver agreement. Histopathology. 2015 Jun;66(7):922-38.
27. Lee MC, Kadota K, Buitrago D, Jones DR, Adusumilli PS. Implementing the new IASLC/ATS/ERS classification of lung adenocarcinomas: results from international and Chinese cohorts. Journal of Thoracic Disease. 2014 Oct;6(Suppl 5):S568.
28. Sakurai H, Asamura H, Miyaoka E, Yoshino I, Fujii Y, Nakanishi Y, Eguchi K, Mori M, Sawabata N, Okumura M, Yokoi K. Differences in the prognosis of resected lung adenocarcinoma according to the histological subtype: a retrospective analysis of Japanese lung cancer registry data. European Journal of Cardio-Thoracic Surgery. 2014 Jan 1;45(1):100-7.
29. Park JK, Kim JJ, Moon SW, Lee KY. Lymph node involvement according to lung adenocarcinoma subtypes: lymph node involvement is influenced by lung adenocarcinoma subtypes. Journal of thoracic disease. 2017 Oct;9(10):3903.
30. Yu Y, Jian H, Shen L, Zhu L, Lu S. Lymph node involvement influenced by lung adenocarcinoma subtypes in tumor size≤ 3 cm disease: a study of 2268 cases. European Journal of Surgical Oncology (EJSO). 2016 Nov 1;42(11):1714-9.
31. Xu L, Su H, Hou L, Wang F, Xie H, She Y, Gao J, Zhao S, Dai C, Xie D, Zhu Y. The IASLC Proposed Grading System Accurately Predicts Prognosis and Mediastinal Nodal Metastasis in Patients With Clinical Stage I Lung Adenocarcinoma. The American Journal of Surgical Pathology. 2022 Dec 29;46(12):1633-41.
32. Warth A, Penzel R, Lindenmaier H, Brandt R, Stenzinger A, Herpel E, Goeppert B, Thomas M, Herth FJ, Dienemann H, Schnabel PA. EGFR, KRAS, BRAF and ALK gene alterations in lung adenocarcinomas: patient outcome, interplay with morphology and immunophenotype. European Respiratory Journal. 2014 Mar 1;43(3):872-83.
33. De Oliveira Duarte Achcar R, Nikiforova MN, Yousem SA. Micropapillary lung adenocarcinoma: EGFR, K-ras, and BRAF mutational profile. American journal of clinical pathology. 2009 May 1;131(5):694-700.
34. Cai W, Lin D, Wu C, Li X, Zhao C, Zheng L, Chuai S, Fei K, Zhou C, Hirsch FR. Intratumoral heterogeneity of ALK-rearranged and ALK/EGFR coaltered lung adenocarcinoma. Journal of clinical oncology. 2015 Nov 11;33(32):3701.
35. Warth A, Penzel R, Lindenmaier H, Brandt R, Stenzinger A, Herpel E, Goeppert B, Thomas M, Herth FJ, Dienemann H, Schnabel PA. EGFR, KRAS, BRAF and ALK gene alterations in lung adenocarcinomas: patient outcome, interplay with morphology and immunophenotype. European Respiratory Journal. 2014 Mar 1;43(3):872-83.