Cases Of Primary Lung Adenocarcinoma Research Articles

Histology of Bronchiolar Tumor Spread Through Air Spaces.

The clinical significance of lung tumor spread through air spaces (STAS) has been extensively studied, and is recognized as a unique pattern of invasion. Previous studies of STAS have focused primarily on STAS in alveolar spaces, whereas STAS in the bronchiolar spaces (bronchiolar STAS) has been described in only a few case reports only. Here, we examined 306 cases of primary lung adenocarcinoma and found that bronchiolar STAS was present in 18%. Bronchiolar STAS was associated with an inferior prognosis, more advanced stage, and higher histologic grade. No significant difference in clinicopathological factors or prognosis was observed between cases with bronchiolar STAS and those with alveolar STAS alone. Notably, bronchiolar STAS often occurred simultaneously with alveolar STAS and endobronchial spread of adenocarcinoma, particularly when bronchiolar STAS was present outside the main tumor. We also identified cases where bronchiolar STAS and endobronchial spread of adenocarcinoma occurred simultaneously in the same bronchi or bronchioles located outside the main tumor, as well as cases with bronchiolar STAS adjacent to intrapulmonary metastatic nodules. Our results highlight the significant role of bronchiolar STAS in the aerogenous spread of adenocarcinoma cells. Bronchiolar STAS can be regarded as a histologic variant of alveolar STAS. This study also supports the idea that STAS is not a tissue processing artifact, but a true biological process with clinical implications, offering histologic evidence of aerogenous spread in lung adenocarcinoma.

α-Cyanoacrylate Rapid Medical Adhesive (Medical EC Glue) Localization of Pulmonary Nodules Guided by Computed Tomography before Thoracoscopic Surgery.

The study aims to conduct lung cancer screening by low-dose CT to identify the nature of the pulmonary nodule. The purpose of this study was to evaluate the role of preoperative medical EC glue localization of pulmonary nodules of uncertain nature by minimally invasive surgical resection. From December 2017 to December 2019, 18 patients (12 women, 6 men; median age: 54 years)with pulmonary nodules were located using medical EC glue under the guidance of preoperative CT and then resected under video thoracoscopy at Air Force Medical Center of PLA. The clinical characteristics were retrospectively collected to evaluate the effectiveness, safety and feasibility of the operation. The mean value of the maximum diameter of pulmonary nodules on CT images before the operation was 10.8 mm. The average depth was 10.3 mm (1.0-39.5 mm). Among 18 nodules, 8 were pure ground glass nodules, 3 were solid nodules, and 7 were partial solid nodules. The diagnosis rate of medical glue localization under the guidance of CT after the operation was 100%. Postoperative pathological diagnosis showed that there were 10 cases of primary lung adenocarcinoma, 1 case of invasive lung adenocarcinoma, 3 cases of adenocarcinoma in situ, 1 case of metastatic adenocarcinoma, and 3 cases of benign nodules. No obvious serious complications were found after localization. This study suggests that CT-guided percutaneous medical EC glue localization is a reliable, safe, feasible and practical method for undiagnosed pulmonary nodules and can significantly improve the rate of resection of small pulmonary nodules. Furthermore, it was considered to be more reasonable to remove pulmonary nodules and maximize the preservation of lung function.

EP-674 Metastases to the breast from a primary lung adenocarcinoma: A case report and literature review

Abstract Background Primary breast cancer is the commonest malignancy in adult females. However, metastases to the breast is a rare occurrence with a frequency of approximately 0.2% - 1.3%. Lung adenocarcinoma is the most common type of lung cancer and distant metastases to other organs carry a poor prognosis. A wide variety of malignancies can metastasise to the breast, melanoma being most common. On review of the literature there is a paucity of published cases of primary lung adenocarcinoma metastasizing to the breast. Case A 77-year-old lady present to the emergency department (ED) with a 5-day history of presyncope. There were no other associated symptoms and the no findings on clinical exam. Computed tomography (CT) of the thorax, abdomen & pelvis reported diffuse opacities in both lungs and a solitary, 3×2cm mass in the upper inner quadrant of the left breast. A core biopsy of the lesion was carried out. Immunohistochemistry did not support a diagnosis of breast carcinoma but strongly suggested a primary lung adenocarcinoma. After a multidisciplinary meeting (MDM) discussion the plan was to commence a course of chemotherapy. Conclusion Given its atypical presentation it can be challenging to distinguish between a primary breast cancer and metastases. Metastatic disease, although rare, should be considered in the differential diagnosis as the treatment and prognosis differ greatly from primary breast cancer. Immunohistochemistry is essential for identifying the primary origin of the tumour.

A Case of Primary Lung Adenocarcinoma due to Bilateral Adrenal Metastases with Addison's Disease as the Initial Symptom

A Case of Primary Lung Adenocarcinoma due to Bilateral Adrenal Metastases with Addison's Disease as the Initial Symptom

Atrial invasion from primary lung adenocarcinoma extension via the pulmonary vein.

Intravascular extension of lung adenocarcinoma is one of the four defined routes of metastasis to the heart but is rarely described in the literature. This is a rare case of primary lung adenocarcinoma with intravenous extension to the left atrium via the pulmonary vein. A 56-year-old female presented to the hospital with chest tightness and dyspnea. Chest computed tomography revealed a right hilar mass extending through the right superior pulmonary vein into the left atrium. Transthoracic echocardiography revealed a large, partially mobile left atrial mass occupying the entire atrial cavity and affecting mitral valve closure. Endobronchial ultrasound with transbronchial biopsy of the right middle lobe of the lung histologically showed a poorly differentiated adenocarcinoma compatible with the primary lung cancer. The patient was deemed a poor surgical candidate by cardiothoracic surgery due to the extent of metastasis and was started on chemoradiation. The patient's left atrial tumor mass started shrinking in size after starting the treatment. This unique case displaying intravascular extension of lung cancer to the left atrium has rarely been described in the literature.

Correlation of TTF-1 immunoexpression and EGFR mutation spectrum in non-small cell lung carcinoma.

BackgroundThyroid transcription factor (TTF-1) is a diagnostic marker expressed in 75%–85% of primary lung adenocarcinomas (ACs). Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene is the most common targetable driver alteration in lung AC. Previous studies have shown a positive correlation between TTF-1 and EGFR mutation status. We aimed to determine the predictive value of TTF-1 immunoexpression for underlying EGFR mutation status in a large Indian cohort. MethodsThis retrospective designed study was conducted with medical record data from 2011 to 2020. All cases of primary lung AC and non–small cell lung carcinoma not otherwise specified (NSCLC, NOS) with known TTF-1 expression diagnosed by immunohistochemistry using 8G7G3/1 antibodies and EGFR mutation status diagnosed by quantitative polymerase chain reaction were retrieved, reviewed, and theresults were analyzed. ResultsAmong 909 patient samples diagnosed as lung AC and NSCLC, NOS, TTF-1 was positive in 76.8% cases (698/909) and EGFR mutations were detected in 29.6% (269/909). A strong positive correlation was present between TTF-1 positivity and EGFR mutation status (odds ratio, 3.61; p < .001), with TTF-1 positivity showing high sensitivity (90%) and negative predictive value (87%) for EGFR mutation. TTF-1 immunoexpression did not show significant correlation with uncommon/dual EGFR mutations (odds ratio, 1.69; p = .098). EGFR–tyrosine kinase inhibitor therapy was significantly superior to chemotherapy among EGFR mutant cases irrespective of TTF-1 status; however, no significant differences among survival outcomes were observed. ConclusionsOur study confirms a strong positive correlation between TTF-1 expression and common EGFR mutations (exon 19 deletion and exon 21 L858R) in advanced lung AC with significantly high negative predictive value of TTF-1 for EGFR mutations.

Abstract 2218: MET alterations are enriched in lung adenocarcinoma brain metastases and define a distinct molecular and transcriptomic subtype

Abstract Lung cancer has the highest incidence of metastases to the brain, with up to 40% of non-small cell lung cancer (NSCLC) patients developing brain metastases (BM). There is a critical need to develop novel treatments to effectively prevent and treat NSCLC BM. MET is a receptor tyrosine kinase that upon binding hepatocyte growth factor (HGF), mediates proliferation, epithelial-mesenchymal transition (EMT), invasion, angiogenesis and metastasis. Recent studies have suggested that the MET pathway may be a significant determinant of metastatic potential to the brain. We evaluated 125 lung adenocarcinoma (LUAD) BM and 477 primary LUAD for MET amplification (amp) by FISH (MET/CEP &amp;gt; 2) as well as other molecular alterations using targeted next-generation sequencing in a subset of LUAD BM (N=74) and primary LUAD (N=171) samples, including 13 paired primary and brain sets. We identified a significant enrichment of MET amp in LUAD BM (19%) compared to primary LUAD (3%; p&amp;lt;0.00001) or liver metastases (4%, N=80; p=0.002). High MET amp (MET/CEP7 ratio &amp;gt;5) was present in 6.5% of BM compared to 1.3% of primary LUAD cases (p=0.0006). In matched samples, BM-specific MET amp was observed. Non-exon 14 skipping MET activating mutations were also significantly more frequent in LUAD BM (22%) compared to primary LUAD (12%; p=0.05), as well as TP53, KRAS, SMAD4, APC, RB1, RET, ABL1, ALK, and VHL variants (adj. p values &amp;lt;0.02). VHL and IDH1 mutations were significantly increased in MET altered compared to non-MET altered BM. In addition, KRAS Q61X variants were more common in LUAD BM compared to LUAD and specifically more common in MET amp BM. We also examined MET activation in paired tumors using an HGF-MET proximity binding, dual-antibody assay. MET expression was increased in the majority of BM compared to the paired LUAD, and there were brain-specific MET activation was observed. RNASeq analysis identified distinct gene signatures in MET amplified (N=11) versus non-MET amplified (N=24) LUAD BM, including upregulation of the EMT and glycolytic pathways. We validated the importance of the EMT transcription factor, TWIST1 in MET-driven NSCLC tumorigenesis preclinically and confirmed activation of the glycolytic pathway in MET amplified NSCLC. Finally, non-invasive strategies to detect brain-specific MET alterations will be needed to identify BM patients who can benefit from MET inhibitors. Therefore, we examined 277 metastatic NSCLC patients that underwent standard of care circulating tumor DNA testing with the Guardant360 platform, and found that both MET mutation and/or amp were more frequently detected in LUAD patients with BM (p=0.04). Together, we show that over a third of LUAD BM patients have MET alterations compared to primary LUAD and may be responsive to MET inhibitors. Further, our liquid biopsy approach may allow us to identify BM-specific alterations for patient selection in clinical trials. Citation Format: Timothy F. Burns, Sanja Dacic, Maria A. Velez, Ashwin Somasundaram, Saveri Bhattacharya, Anish Chakka, Zachary A. Yochum, Jingxiao Jin, Ethan Miller, Brenda F. Kurland, Riyue Bao, Danielle P. Normolle, Sameer Agnihotri, Uma R. Chandran, Laura P. Stabile. MET alterations are enriched in lung adenocarcinoma brain metastases and define a distinct molecular and transcriptomic subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2218.

Epidermal Growth Factor Receptor Gene Mutation Detection in Histology and Cytology Specimens of Primary Lung Adenocarcinoma: Immunohistochemistry Versus the Molecular Method.

Background:Epidermal growth factor receptor (EGFR) gene in lung adenocarcinoma is associated with good clinical response to EGFR-tyrosine kinase therapy. The two most common EGFR gene mutations, representing 80 to 90%, are the E746-A750 deletion in exon 19 and the L858R point mutation in exon 21. Materials and Methods:We have conducted the study to evaluate immunohistochemistry’s performance in detecting the E746-A750 deletion in exon 19 of the EGFR gene in primary lung adenocarcinoma cases. This study examined 133 cases of primary lung adenocarcinoma for three years duration. The selected cases were tested for EGFR gene mutations by real-time PCR by a reference laboratory. Most cases (124) were diagnosed by tissue biopsy, though nine used cell block cytology. We performed an immunohistochemistry test on 75 cases that contained adequate diagnostic material in the paraffin block. Results:The test result was scored as 0 to 3+, based on the staining intensity and percentage of positive tumor cells. We evaluated the immunohistochemistry test’s sensitivity and specificity compared to the EGFR gene mutations by real-time PCR. There was a significant association between gender, smoking status, and the EGFR gene mutations (P < 0.001). The overall sensitivity and specificity of the immunohistochemistry test were 40% and 100%, respectively. The positive predictive value and negative predictive values were 100% and 76.9%, each. Conclusions:The immunohistochemistry has high specificity but low sensitivity in the detection of E746-A750 deletion in exon 19 of the EGFR gene. The mutation-specific antibody used in this study was unable to detect other uncommon variants of exon 19 deletions. With high specificity value, immunohistochemistry may provide an adjunct to molecular testing for detecting the most common EGFR gene mutations in cases of a low cellularity sample, financially-limited situations, or in critically ill cases where urgent targeted therapy is needed.

Utility of liquid-based cytology on residual needle rinses collected from core needle biopsy for lung nodule diagnosis.

BackgroundCore needle biopsy (CNB) has become the most common tissue sampling modality for pathological diagnosis of peripheral lung nodules. However, approximately 10% of pulmonary CNB specimens cannot be unambiguously diagnosed, even with auxiliary techniques. This retrospective study investigated the diagnostic value of liquid‐based cytology on residual pulmonary CNB material collected from needle rinses.MethodsComputed tomography‐guided pulmonary CNB specimens and relevant cytology of CNB needle rinses (CNR) from July 2017 to June 2020 were reviewed. A total of 406 patients, each of whom underwent a CNB procedure, were included in the study.ResultsOf the 406 cases, a more serious diagnosis was rendered by CNR in 6.4% (n = 26) of cases. Furthermore, among these 26 cases, 13 malignancies were confirmed only from CNR. Of the remaining 13 patients with uncertain lesions identified from CNR, six were diagnosed with definite benign lesions from tissue samples, five were found to harbor malignant neoplasms through repeated CNB or follow‐up examination, and two had tuberculosis. The sensitivity (320/332, 96.4%) of combined CNR/CNB (both CNR and CNB) in distinguishing malignancies from benign lesions was higher than that of CNB alone (307/332, 92.5%). A total of 320 malignant neoplasms included 198 cases of primary lung adenocarcinoma and 71 cases of primary lung squamous cell carcinoma.ConclusionsCNR with higher nuclear and cytoplasmic resolution than CNB exhibited a high diagnostic efficacy for differentiating malignant from benign lesions in the lung. Moreover, combined CNR/CNB achieved optimal results in reducing the false‐negative rate and the subtyping of non‐small cell lung cancer.

P09.51 Lung Adenocarcinoma in Morocco

Lung cancer is the most common cancer and the leading cause of cancer death worldwide. Adenocarcinoma has become the most prevalent histological type. It represents about 60% of non-small cell lung cancers, and mainly related to changes in cigarette composition. This study aims to describe the epidemiological characteristics of lung adenocarcinoma in Rabat (capital of Morocco). This is a descriptive retrospective study of 162 cases of primary lung adenocarcinoma diagnosed and treated at Ibn Sina University Hospital Center in Rabat between January 2016 and December 2017. Of the 265 cases of primary lung cancer reported during the period of study, 162 (61.1%) were diagnosed with adenocarcinoma. Of these, 88.3% were men, with a male-female ratio of 7.5. The average age at diagnosis of lung cancer was 59.2 years (range 31-80 years). Nearly one-fifth (19.6%) of patients were aged less than 50 years, 54.2% were between 50 and 65 years and 26.2% were older than 65 years. According to the results, 86.7% of men and 15.8% of women were smokers. Immunohistochemistry was performed on 132 cases. All patients were considered positive for cytokeratin 7 (CK7) but negative for cytokeratin 20 (CK20) and 82.3% were positive for Thyroid Transcription Factor-1 (TTF-1). The majority of cancer cases (81.5%) were diagnosed with locally advanced or metastatic disease. The increasing incidence of lung adenocarcinoma is alarming. Most cases were diagnosed with metastatic disease and occurred among smokers,highlighting the need for smoking cessation and lung cancer early detection programs.

Primary lung adenocarcinoma in three adolescent patients affected by bone sarcomas

Pediatric primary lung carcinomas are extremely rare. Apart from known associations with congenital adenomatoid malformations, cases of primary lung adenocarcinomas after prolonged treatments of pediatric malignancy have been reported. We describe the morphological and molecular features of three cases of lung adenocarcinoma developed in adolescents aged 8 to 17years during progression of their bone osteosarcoma or Ewing sarcomas. The morphological features overlapped those of adult lung adenocarcinoma including in situ, minimally invasive, and invasive forms. EGFR gene mutations were found in all three cases by targeted next-generation sequencing. The two patients with Ewing sarcoma had no progression of their lung cancer and no further progression of the metastatic bone tumor after additional chemo- and radio-therapy. Conversely, the osteosarcoma patient refused further treatments after thoracic surgery for metastatic osteosarcoma and locally advanced adenocarcinoma and died 2years later of widespread distant metastases. Our results indicate that primary lung cancer might originate in pediatric patients during prolonged adjuvant therapies for primary bone neoplasm, and this possibility should be considered in the presence of suspected lung disease progression to correctly monitor the primary tumor evolution and define the appropriate therapeutic strategy at each time point. If appropriately treated, second primary lung cancer may not affect the patients' prognosis. The pathogenetic mechanisms of these rare lung adenocarcinomas are not clear, but the presence of EGFR mutations in all three cases indicates an oncogene addiction of the lung tumor, rather than a direct cancerogenic effect of the sarcoma-related treatment.

Primary lung adenocarcinoma with gross calcification and suspected bone formation.

Calcification in a lung tumor suggests that it is a benign tumor such as a hamartoma or a sclerosing lung cell tumor. In contrast, carcinoid, lung cancer, carcinosarcoma, and sarcoma rarely harbor calcification. Primary lung adenocarcinomas with gross calcification that is suggestive of bone formation are very rare. It is difficult to distinguish between calcification and bone formation purely on the basis of image definitive diagnosis of bone formation being difficult in the absence of a large surgical specimen. Lung cancers with bone formation are exceedingly rare: to the best of our knowledge, only 13 cases have been reported. Careful attention is needed when differentiating between benign and malignant tumors. Here, we report a case of primary lung adenocarcinoma with gross calcification that was suggestive of bone formation.

PD-L1 Expression of Lung Cancer Cells, Unlike Infiltrating Immune Cells, Is Stable and Unaffected by Therapy During Brain Metastasis

BackgroundApproximately 50% of brain metastases originate from non–small-cell lung cancer. The median survival of patients with brain metastases is 1 month without treatment. Novel immunotherapeutic strategies, such as those targeting the programmed death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) axis, are promising in patients with advanced systemic disease but are often preferentially administered to patients with tumors showing PD-L1 positivity. Patients and MethodsSurgically resected paired primary lung adenocarcinoma and brain metastasis samples of 61 patients were analyzed. We compared the paired samples regarding the amount of peritumoral and stromal mononuclear infiltration, PD-L1 expression of tumor and immune cells, and PD-1 expression of immune cells. We investigated the effect of radiotherapy, chemotherapy, and steroid therapy on PD-L1 expression in brain metastases. ResultsThere was significant positive correlation regarding the PD-L1 expression of tumor cells between the paired primary lung adenocarcinoma and brain metastatic samples with the use of different cutoff levels (1%, 5%, 50%). We found no impact of chemotherapy or steroid therapy on the changes of PD-L1 expression of tumor cells between the 2 sites. There is no or only limited concordance of the proportion of PD-1– or PD-L1–positive tumor-associated immune cells between the paired tumor samples, which suggests that brain metastases develop their own immune environment. ConclusionWe observed a strong correlation of PD-L1 positive tumor cells between primary lung adenocarcinoma cases and their corresponding brain metastases, which is not significantly influenced by chemotherapy or steroid therapy.

Abstract 3683: Reciprocal expressions of TFF-1 and TTF-1 in lung adenocarcinomas

Abstract The molecular targeted therapies against EGFR, ALK etc. have improved progression-free survival of patients with lung adenocarcinomas. However, targetable driver mutations have mainly been found in TTF-1-positive terminal respiratory unit (TRU)-type, but have yet been undiscovered in non-TRU type despite the advances in genomics technologies. In order to find the molecular candidate characterizing major subtype of non-TRU type adenocarcinomas, we compared TTF-1-positive/CDH1-positive adenocarcinoma cell lines (“TRU-type” cell lines) (n=11) and TTF-1-negative/CDH1-positive adenocarcinoma cell lines (“non-TRU-type” cell lines) (n=8), and found that TFF-1 (trefoil factor family-1), a gastric mucosal protective factor, was characteristically highly expressed in “non-TRU-type” cell lines. Immunohistochemical analysis of primary lung adenocarcinomas (n=70) revealed that TFF-1 expression was inversely correlated with TTF-1 expression (p=0.0116) and was very rare in primary lung adenocarcinoma cases (5.7%:4/70). So we focused on the non-TRU type adenocarcinomas on the basis of histologic pattern (acinar, solid or mucinous morphology predominant tumors without non-mucinous lepidic growth components) (n=43). Among them, TFF-1 was frequently expressed (62%:27/43), and significantly correlated with the expressions of MUC5AC, HNF4A and CK20 (p&amp;lt;0.0001, p=0.0001, and p=0.0007, respectively), and inversely correlated with TTF-1 expression (p=00039). Next generation sequencing analysis of the 43 cases revealed that KRAS gene mutations and Inactivating mutations and hypermethylation of the TTF-1 gene were frequent in TFF-1 positive cases. The analysis of publicly available data of 442 lung adenocarcinoma cases revealed that high expression of TFF-1 correlated with low expression of TTF-1 and worse prognosis. We confirmed that shRNA against TFF-1 inhibited the cell proliferation and the colony formation, and induced the apoptosis, in the TFF-1 positive “non-TRU-type” cell lines. These results indicate that TFF-1 is not only the marker but also can possibly be the molecular target of a subset of non-TRU-type lung adenocarcinomas. Citation Format: Daisuke Matsubara, Manabu Soda, Taichiro Yoshimoto, Yusuke Amano, Atsushi Kihara, Yuji Sakuma, Shunsuke Endo, Koichi Hagiwara, Masashi Fukayama, Hiroyuki Mano, Toshiro Niki. Reciprocal expressions of TFF-1 and TTF-1 in lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3683.

One Pulmonary Lesion, 2 Synchronous Malignancies.

Introduction. Mantle cell lymphoma (MCL) comprises approximately 3% to 10% of all non-Hodgkin lymphomas. Although there is an increased risk for secondary malignancies after treatment among non-Hodgkin lymphomas survivors, a synchronous diagnosis of primary lung cancer arising in conjunction with lymphoma at the same site has rarely been reported. We report an unusual case of primary lung adenocarcinoma with coexistent MCL within the same lung lesion. Case Presentation. A 55-year-old female with newly diagnosed stage IV-B MCL was referred for workup of a right upper lobe cavitary lesion detected during lymphoma staging. A whole-body positron-emission tomography-computed tomography scan revealed diffuse adenopathy but also identified a cavitary right upper lobe lesion atypical for lymphoma. Bronchoscopy was unremarkable with cytology (on lavage) negative for malignancy. At 2 months, a computed tomography scan of the chest showed a persistent lesion. A video-assisted thoracoscopic wedge resection was performed. Histopathological examination revealed a lepidic predominant, well-differentiated adenocarcinoma (stage T1a) and foci of lymphoid infiltrate within and adjacent to the adenocarcinoma consistent with lung involvement by MCL. Discussion. Synchronous presentation of primary lung adenocarcinoma and lymphoma at a single site is exceedingly rare. Nonresolving pulmonary lesions with features atypical for lymphoma should be viewed with caution and worked up comprehensively to rule out occult second malignancies, in order to guide a prompt diagnosis and appropriate treatment.

Anaplastic lymphoma kinase immunohistochemistry in lung adenocarcinomas: Evaluation of performance of standard manual method using D5F3 antibody.

Immunohistochemistry (IHC) with anaplastic lymphoma kinase (ALK) antibodies is considered as an economical screening method in lung adenocarcinomas. Automated Ventana D5F3-IHC is approved by US Food and Drug Administration for targeted therapy; however, the automated IHC apparatus are not widely used in most laboratories. We evaluated the performance of ALK IHC using the manual semiquantitative method to assess the concordance with Ventana ALK IHC assay. We tested 156 cases of primary lung adenocarcinomas for ALK protein expression by D5F3-IHC. The intensity of cytoplasmic staining was classified as 0 or 1+/2+/3+ (weak/medium/strong). Binary score of positive and negative was used for Ventana assay. A comparison analysis and clinicopathological features were recorded. ALK IHC was positive in 25 (16.02%) cases, of which 18 were men and mostly nonsmokers. The mean age for all patients was 55 years, and for ALK IHC-positive cases was 48 years. Nine of 25 (36%) ALK IHC-positive cases showed signet ring cell and mucinous morphology. On comparison, all, but one, cases positive by manual method showed positive results by automated assay. IHC negative cases by manual method were negative by Ventana assay. Manual IHC is equally effective in the detection of ALK-rearranged cases as automated methods. It can be easily integrated as a screening method into routine practice thus reducing the cost of automated systems. However, equivocal cases should be tested by approved methods.

P1.02-071 Detection of Multiple Low-Frequency Mutations by Molecular-Barcode Sequencing

Recent advantage of next-generation sequencing (NGS) in the field of cancer genome research has revealed extreme levels of genetic heterogeneity, suggesting the major roles of subclonal mutations in cancer relapse and in rapid emergence of acquired resistance. Unlike inherited mutations, somatic variants often occur at low allele frequencies that require sensitive methods for detection. Based on the results using another highly sensitive method, such as digital PCR, the study of cancer subclones requires to detect mutations that are present in <1% frequency, however, such a level of resolution cannot be obtained by conventional NGS approaches. In the current study, we performed molecular-barcode sequencing for primary lung adenocarcinoma cases in order to analyze mutations with <1% low frequency. Fresh frozen tumor samples from 58 primary lung adenocarcinoma patients whose tumors previously tested positive for EGFR by conventional method (the PNA-LNA PCR clamp method) were collected. All samples were obtained from surgically resected specimen. We used HaloPlex HS method, which is a high sensitivity amplicon-based targeted sequencing method incorporating molecular barcodes in the DNA library, allowing for the identification of duplicate reads to significantly improve the base calling accuracy even at low allelic frequencies compared to conventional NGS methods. We used a panel designed for 47 cancer-related genes including EGFR, and sequenced data was obtained by using Illumina Miseq Reagent v3 (600 Cycle). Normal tissue samples were also sequenced for threshold adjustment. Out of 58 samples, EGFR ‘major’ mutation (L858R, Exon19 deletion, G719A/S, T790M) profiles of 57 samples by molecular-barcode sequencing corresponded to those by clinical method (98.3%). The mean coverage of EGFR major mutation was 3078 (243-8285), and the minimal detectable frequency was 0.45%. Of note, we could detect the minor frequency of T790M mutation in addition to the major frequency of L858R mutation, at the allele frequency of 1.92% (20/1042) for T790M and 10.62% (155/1459) for L858R mutation, respectively. Minor genetic alterations, except major mutation, in EGFR were detected in 82.8% (48/58) cases, and the mean number was 2.3 (0-11). These results suggest the clinical applicability of this method. We could demonstrate good concordance rate between clinical examination and molecular barcode sequencing. Minor genetic alterations in EGFR were detected in 82.8% (48/58) cases. Further investigations are warranted to establish the confident detection of subclonal mutations with low frequency.

Immunohistochemistry with a novel mutation-specific monoclonal antibody as a screening tool for the EGFR L858R mutational status in primary lung adenocarcinoma.

Epidermal growth factor receptor (EGFR) mutation status is the best predictor of patient response to treatments with tyrosine kinase inhibitors in primary lung adenocarcinoma and is typically analyzed by DNA-based techniques, such as direct DNA sequencing and allele-specific PCR. Recently, however, two mutation-specific antibodies against delE746-A750 in exon 19 and L858R in exon 21 have opened the door for a more convenient and more efficient strategy to determine EGFR mutation status. To evaluate the clinical application of a new mutation-specific mouse monoclonal antibody for EGFR (L858R), we performed immunohistochemistry (IHC) studies with tumor samples from primary lung adenocarcinoma in retrospective and validation settings. A total of 215 cases of primary lung adenocarcinoma were examined and compared using a combination of DNA-based techniques (direct DNA sequencing and/or allele-specific PCR) and protein-based IHC. IHC staining was assessed on a 0 to 3+ score scale, and a cutoff value of 2+ was used as positive by IHC. In the retrospective setting, statistical analyses of the data showed that the sensitivity of IHC was 90.9% and the specificity was 96.8%. Findings from the validation study demonstrated that the sensitivity and specificity of IHC were 88.2% and 100%, respectively. IHC with the novel mutation-specific antibody could be used as a screening method to assess the EGFR L858R mutation status in primary lung adenocarcinoma.

High-resolution detection of recurrent aberrations in lung adenocarcinomas by array comparative genomic hybridization and expression analysis of selective genes by quantitative PCR.

Genomic abnormalities are the hallmark of cancers and may harbor potential candidate genes important for cancer development and progression. We performed array comparative genomic hybridization (array CGH) on 36 cases of primary lung adenocarcinoma (AD) using an array containing 2621 BAC or PAC clones spanning the genome at an average interval of 1 Mb. Array CGH identified the commonest aberrations consisting of DNA gains within 1p, 1q, 5p, 5q, 7p, 7q, 8q, 11q, 12p, 13q, 16p, 17q, 20q, and losses with 6q, 9p, 10q and 18q. High-level copy gains involved mainly 7p21-p15 and 20q13.3. Dual color fluorescence in situ hybridization (FISH) was performed on a selective locus for validation of array CGH results. Genomic aberrations were compared with different clinicopathological features and a trend of higher number of aberrations in tumors with aggressive phenotypes and current tobacco exposure was identified. According to array CGH data, 23 candidate genes were selected for quantitative PCR (qPCR) analysis. The concordance observed between the genomic and expression changes in most of the genes suggested that they could be candidate cancer-related genes that contributed to the development of lung AD.

Sirt1 is a tumor promoter in lung adenocarcinoma.

Sirtuin 1 (Sirt1) is a nicotinamide adenine dinucleotide-dependent class III histone deacetylase. It reportedly can repress cellular apoptosis and senescence to affect DNA repair, stress response and aging. Notably, previous data have indicated that Sirt1 is both a tumor promoter and a tumor suppressor in tumorigenesis. However, Sirt1 expression in primary lung adenocarcinoma remains unknown. Immunohistochemical staining was performed to investigate Sirt1 expression in cancer cells in 125 consecutive resected cases of primary lung adenocarcinoma. Sirt1 expression was found to be increased in 26 (20.8%) of the 125 cases, which correlated significantly with five clinicopathological factors: Ki67 index, hypoxia-inducible factor 1 (HIF1) molecule expression, tumor-node-metastasis (TNM) classification, pulmonary vein invasion and lymphatic duct invasion. In the Sirt1-positive expression group, Sirt1 expression correlated with a higher Ki67 index and higher TNM classification, particularly for lymph node invasion and metastasis, and with a higher number of pulmonary vein invasion and lymphatic duct invasion. Additionally, a negative correlation was identified between HIF1-positive expression and Sirt1-negative expression. These results indicate that Sirt1 overexpression plays a promotional role in tumorigenesis and is closely associated with invasion and metastasis and, thus, it may be associated with prognosis.