A study of multinucleated giant cells in esophageal cancer.

OBJECTIVES: To evaluate the occurrence, abundance, distribution, nature and clinical significance of multinucleated giant cell (MGC) in esophageal cancer. MATERIALS AND METHODS: MGCs were examined with conventional pathology, immunohistochemistry and immunofluorescence in 107 esophageal cancer tissues. The findings were correlated to pathological diagnosis and clinical behavior of the cancers. RESULTS: MGCs were identified in 31.7% (34/107) of the cases. MGCs were positive for CD11c, CD11b, CD32, CD16, HLA-DR and MMP9, and negative for CD163, CD206 and CD64 giving a molecular profile of proinflammatory M1 but not immunosuppressive M2. MGCs were significantly related to decreased lymph node metastasis (p = 0.011), low pTNM stage (p = 0.044), favorable survival (p = 0.04), squamous cell cancer type rather than other histopathological subtypes (p = 0.020) and associated to better differentiation (p = 0.063). CONCLUSIONS: MGCs belong to M1 macrophage and perform phagocytosis and scavenging of cancer cells that would benefit patients' survival and could serve as a prognostic marker.

Targeted sequencing of circulating cell-free DNA in stage II-III resectable oesophageal squamous cell carcinoma patients.

BACKGROUND: The aim of this study was to investigate the potential of cell-free DNA (cfDNA) as a disease biomarker in oesophageal squamous cell carcinoma (ESCC) that can be used for treatment response evaluation and early detection of tumour recurrence. METHODS: Matched tumour tissue, pre- and post-surgery plasma and WBCs obtained from 17 ESCC patients were sequenced using a panel of 483 cancer-related genes. RESULTS: Somatic mutations were detected in 14 of 17 tumour tissues. Putative harmful mutations were observed in genes involved in well-known cancer-related pathways, including PI3K-Akt/mTOR signalling, Proteoglycans in cancer, FoxO signalling, Jak-STAT signalling, Chemokine signalling and Focal adhesion. Forty-six somatic mutations were found in pre-surgery cfDNA in 8 of 12 patients, with mutant allele frequencies (MAF) ranging from 0.24 to 4.91%. Three of the 8 patients with detectable circulating tumour DNA (ctDNA) had stage IIA disease, whereas the others had stage IIB-IIIB disease. Post-surgery cfDNA somatic mutations were detected in only 2 of 14 patients, with mutant allele frequencies of 0.28 and 0.36%. All other somatic mutations were undetectable in post-surgery cfDNA, even in samples collected within 3-4 h after surgery. CONCLUSION: Our study shows that somatic mutations can be detected in pre-surgery cfDNA in stage IIA to IIIB patients, and at a lower frequency in post-surgery cfDNA. This indicates that cfDNA could potentially be used to monitor disease load, even in low disease-stage patients.

Mesenchymal cystic hamartoma of the lung: A case report.

RATIONALE: Mesenchymal cystic pulmonary hamartoma is a rare type of hamartoma that has been reported in all cases in the literature. Most patients were reported to have spontaneous pneumothorax and were treated by surgery, and finally confirmed to be caused by rupture of the cystic hamartoma. Here, we report a case of mesenchymal cystic pulmonary hamartoma detected using computed tomography (CT) during a health check-up without obvious symptoms. PATIENT CONCERNS: A 60-year-old woman was detected using CT during her health check-up. She was a non-smoker and had no symptoms or history of specific diseases. DIAGNOSIS: The final pathological examination confirmed that the lesion was a mesenchymal cystic hamartoma of the lung. INTERVENTIONS: A uniportal video-assisted thoracic surgery wedge resection was performed for biopsy. OUTCOMES: The patient recovered smoothly and was discharged on postoperative day 3. LESSONS: For cystic pulmonary hamartoma, it is usually difficult to make a correct diagnosis using CT imaging. A chest magnetic resonance imaging examination may be helpful for differentiation diagnosis before video-assisted thoracic surgery biopsy.

Metastatic adenocarcinoma to the breast from the lung simulates primary breast carcinoma-a clinicopathologic study.

BACKGROUND: Rare extra-mammary metastases of adenocarcinoma to the breast closely mimic primary invasive breast carcinoma (PBC), and specifically without an aware of clinical history, pose a difficult diagnostic issue. METHODS: With the aim to improve differential diagnosis of lung adenocarcinoma metastasis and primary breast carcinoma in the breast, we retrieved 41 breast metastases from lung adenocarcinoma, seven of which were from the archived pathologic files of Cancer Hospital, Chinese Academy of Medical Science (CHCAMS) between 2001 and 2019, and the other 34 cases were collected from the published literatures. Clinicopathological features were collected and analyzed for differential diagnosis of primary lung malignancy, triple negative breast pathology and breast lesions without ipsilateral axillary lymphadenopathy or with contralateral axillary lymphadenopathy. Supplementary breast (GCDFP-15, or GATA-3) and lung-lineage (TTF-1) immunostaining plus genetic alternation analysis were also recorded and analyzed. RESULTS: Among the 41 cases, there were 37 females and four males, with a median age of 63 (range, 40-81) years at diagnosis of the breast lesion. Twenty-four cases (58.5%, 24/41) were detected metachronously to the counterpart of the lung. Strikingly, 13 cases (31.7%, 13/41) were initially misdiagnosed as primary breast cancer, and differential diagnostic factors were compared and analyzed between the correct and misdiagnosed cases, among which a documentation of lung cancer history showed significant difference. Pathologist initially misinterpreted six cases (46.2%, 6/13) as PBC on needle biopsy of breast mass with an unknown lung cancer history. The clinical diagnosis was considered two cases (15.4%, 2/13) to be either a primary breast tumor with lung and pleural metastasis or two synchronous primary tumors. Three cases (23.1%, 3/13) were initially misinterpreted as PBC by breast ultrasonography. TTF-1 immunostaining was found to be critical for a correct diagnosis of metastatic lesion (84.6%, 11/13) from the initially misdiagnosed cases as PBC. CONCLUSIONS: Metastatic lung adenocarcinoma to the breast, although rare, should be considered in the differential diagnosis of primary breast carcinoma, especially when the breast lesion exhibits as a "triple-negative invasive carcinoma". A documented lung cancer history combined with the clinicoradiological assessment and pathological evaluation are essential to make a correct differential diagnosis. TTF-1 immunostaining is crucial in approaching the diagnosis.

YAP1 protein expression has variant prognostic significance in small cell lung cancer (SCLC) stratified by histological subtypes.

BACKGROUND: Recently, expression of YAP1, a nuclear effector of an inactivated HIPPO pathway, has been identified as one of four molecular subtypes of SCLC. However, the clinicopathological relevance and prognostic significance of YAP1 expression in SCLC stratified by histological subtypes has not been systematically reported to date. METHODS: Tumor sections and corresponding formalin-fixed paraffin-embedded (FFPE) samples of 297 SCLC patients were retrieved from the pathological specimen repository and were subsequently reviewed by pathologists. Forty-six C-SCLCs (combined SCLCs) (15.5%) and 251P-SCLCs (pure SCLCs) (84.5%) were identified respectively. YAP1 expression was examined by immunohistochemistry (IHC) and assessed semi-quantitatively on tumor tissue array (TMA). Propensity score was used to match C-SCLCs and P-SCLCs in a ratio of 1 to 2 to balance age, gender, tumor stage and treatment methods. Finally, 46C-SCLCs and 92P-SCLCs were included for prognostic analysis. RESULTS: The positive rate of YAP1 expression was significantly higher in C-SCLCs than P-SCLCs before matching (52.2% vs 29.1%, P = 0.004). After matching by propensity score, the prescribed clinical parameters were well balanced between P-SCLCs and C-SCLCs. Expression of YAP1 was associated worse overall survival (OS) (5- year OS%, 39.0% vs. 74.9%, P = 0.013) and was an independent risk factor for OS (HR = 2.93, 95% CI: 1.01-8.51; P = 0.048) exclusively in C-SCLC. Univariate survival analysis in subgroups of different clinical variables also confirmed the prognostic impact of YAP1 was most significant in C-SCLC. But for P-SCLCs, expression of YAP1 showed no prognostic impact. CONCLUSIONS: Expression of YAP1 in small cell components of C-SCLC was significantly higher than that in P-SCLC. Besides, it served as an unfavorable predictor for OS in C-SCLC but not in P-SCLC, which suggested different entities of small cell components with variant YAP1 expression and potential different targetable oncogenic pathway between C-SCLC and P-SCLC.

EGFR mutation is positively correlated with C-Met protein expression: a study of 446 resected lung adenocarcinoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutation and mesenchymal-epithelial transition factor (C-Met) amplification are known factors for primary resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in advanced primary lung adenocarcinoma. However, little is known about the relationship between high expression of C-Met protein and primary EGFR mutation. This research aims to investigate the correlation between EGFR mutation and C-Met protein expression in resected primary lung adenocarcinoma. METHODS: Four hundred and forty-six surgically resected lung adenocarcinoma between 2013-2015 were collected for EGFR mutation analysis by real-time PCR (RT-PCR) and C-Met protein expression by immunohistochemistry (IHC). The relationship between the two biomarkers and clinicopathological features were analyzed. RESULTS: The positive rate of EGFR mutation and C-Met protein expression were 66.4% (296/446) and 96.4% (430/446). EGFR mutation was significantly higher in female, mild to moderate differentiation, lepidic, acinar and papillary histological subtypes (P<0.05). C-Met expression was more prominent in female than male (201 vs. 123, 45.07% vs. 27.57%). EGFR mutation was found positively correlated with C-Met protein expression (P<0.05). CONCLUSIONS: EGFR mutation and C-Met protein expression are prone to have a female predominance, and are positively correlated with each other in surgically resected lung adenocarcinoma specimens. This finding may be beneficial in explaining some of the resistance mechanisms of EGFR-mutated cases, which is worth further study.

Clinicopathological features and prognostic analysis of 247 small cell lung cancer with limited-stage after surgery.

The objective of this study was to analyze the clinical and pathological characteristics of patients with small cell lung cancer (SCLC) after curative surgery and to explore prognostic factors for disease-free survival (DFS) and overall survival (OS). Clinical data of 247 patients were collected, and clinicopathological features were retrieved, including gender, age, smoking history, tumor location, and distant metastasis. Histopathological features were also reviewed by three pathologists, including primary tumor (T), lymph node metastasis (N), pleural invasion, bronchial invasion, nerve invasion, spread through air spaces (STAS), tumor thrombosis, major cell shape (round Vs. spindle), tumor necrosis, stromal fibrosis, and tumor-infiltrating lymphocytes (TILs). Immunohistochemical staining of neuroendocrine markers (CD56, synapsin, chromogranin A) was also reviewed. All patients were followed up for recurrence, distant metastasis, and survival. Kaplan-Meier curves and log-rank tests were applied for survival analysis. The median DFS was 98 months, and the 1-year, 3-year, and 5-year DFS rates were 70.9%, 54.4%, and 52.2%, respectively. The median OS was not reached, and the 1-year, 3-year, and 5-year survival rates were 94.2%, 72.3%, and 65.4%, respectively. Univariate analysis revealed clinicopathological features with DFS (gender, smoking history, primary tumor, regional lymph node metastasis, major cell shape, and TILs) and OS (age, primary tumor, regional lymph node metastasis, distant metastasis, nerve invasion, major cell shape, and TILs). Multivariate analysis revealed DFS-related factors (smoking history, regional lymph node metastasis and major cell shape) and OS-related factors (age, primary tumor, distant metastasis in the brain, liver, bone, nerve invasion, and TILs). Age more than 65 years, smoking, advanced stage (T and N), distant metastasis, nerve invasion, major cell shape as spindle and TILs >30% were negatively correlated with survival. Neuroendocrine immunostaining markers showed no correlation with survival. Of interest, spindle cell type and TILs >30% are revealed as independent negative prognostic factors, and further molecular mechanisms need to be explored.

Clinicopathological features and prognostic implications of ASCL1 expression in surgically resected small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is one of the most aggressive lung cancers. Treatment of SCLC has remained unchanged during the past decades. Preclinical studies have revealed ASCL1 as a transcription regulator in the neuroendocrine (NE) differentiation and carcinogenesis of SCLC. However, there are few studies on correlation of ASCL1 expression and clinicopathological factors in resected SCLCs. Here, we aimed to analyze the ASCL1 expression of SCLC and investigate its associations with clinicopathological factors and survival. METHODS: A total of 247 surgically resected pure SCLC specimens were included in this retrospective study, all of which were processed using tissue microarrays for immunohistochemistry analysis of ASCL1. A total of 48 of 247 cases were tested by NanoString for mRNA expression analysis on 50 SCLC related genes. Statistical analysis was performed using R studio and SPSS software. RESULTS: NE scores of 48 pure SCLC specimens were calculated by analyzing 50 preselected genes. A significant correlation between NE score with both ASCL1 mRNA expression and ASCL1 protein expression were observed. For the entire cohort of 247 patients, ASCL1 was highly expressed in 42.5% of pure SCLC patients according to IHC results. Significant differences were observed between ASCL1 high and low expression groups in variables including staging, lymph node metastasis, nerve invasion and overall survival. CONCLUSIONS: In limited staged pure SCLC, ASCL1 expression was positively correlated with NE signature, pTNM stage, nerve invasion and OS. ASCL1 may therefore serve as a potential biomarker to predict prognosis as well as in the selection of patients for therapies targeting ASCL1-regulated downstream molecules.

Clinical Value of EGFR Copy Number Gain Determined by Amplicon-Based Targeted Next Generation Sequencing in Patients with EGFR-Mutated NSCLC.

BACKGROUND: The clinical relevance of epidermal growth factor receptor (EGFR) copy number gain in patients with EGFR mutated advanced non-small cell lung cancer on first-line tyrosine kinase inhibitor treatment has not been fully elucidated. OBJECTIVE: We aimed to estimate EGFR copy number gain using amplicon-based next generation sequencing data and explored its prognostic value. PATIENTS AND METHODS: Next generation sequencing data were obtained for 1566 patients with non-small cell lung cancer. EGFR copy number gain was defined based on an increase in EGFR read counts relative to internal reference amplicons and normal controls in combination with a modified z-score ≥ 3.5. Clinical follow-up data were available for 60 patients treated with first-line EGFR-tyrosine kinase inhibitors. RESULTS: Specificity and sensitivity of next generation sequencing-based EGFR copy number estimations were above 90%. EGFR copy number gain was observed in 27.9% of EGFR mutant cases and in 7.4% of EGFR wild-type cases. EGFR gain was not associated with progression-free survival but showed a significant effect on overall survival with an adjusted hazard ratio of 3.14 (95% confidence interval 1.46-6.78, p = 0.003). Besides EGFR copy number gain, osimertinib in second or subsequent lines of treatment and the presence of T790M at relapse revealed significant effects in a multivariate analysis with adjusted hazard ratio of 0.43 (95% confidence interval 0.20-0.91, p = 0.028) and 0.24 (95% confidence interval 0.1-0.59, p = 0.001), respectively. CONCLUSIONS: Pre-treatment EGFR copy number gain determined by amplicon-based next generation sequencing data predicts worse overall survival in EGFR-mutated patients treated with first-line EGFR-tyrosine kinase inhibitors. T790M at relapse and subsequent treatment with osimertinib predict longer overall survival.

Comparative study of clinicopathological characteristics and prognosis between combined and pure small cell lung cancer (SCLC) after surgical resection.

BACKGROUND: Histologically, SCLC are classified as pure (P-SCLC) and combined subtypes (C-SCLC). Currently, few studies compare the clinicopathological characteristics and explore the treatment strategies applied to them. METHODS: Between July 2005 and April 2016, the clinical records of 297 postoperative patients with pathologically confirmed SCLC were retrospectively analyzed. Kaplan-Meier method and Cox regression model were separately used for stratified univariate and multivariate survival analysis. RESULTS: A total of 46 cases (15.5%) of C-SCLCs and 251 cases (85.5%) of pure SCLCs (P-SCLCs) were included in this study. The average age of C-SCLCs was a little higher than that of P-SCLCs (59.65 ± 8.72 vs. 56.56 ± 10.12; P = 0.053). More patients had a history of smoking in C-SCLC (78.3% vs. 63.3%; P = 0.074). The five-year overall survival (OS) rate for P-SCLCs and C-SCLCs was 65.1% and 56.7%, respectively (P = 0.683). For P-SCLC, stage and an intervention of prophylactic cranial irradiation (PCI) were independent factors that affected OS. In C-SCLCs cases, performing sublobectomy was an independent risk factor for poor prognosis. CONCLUSIONS: We identified no significant difference in clinical characteristics and outcome between C-SCLCs and P-SCLCs. However, the factors affecting the prognosis of the two subtypes were slightly inconsistent. For C-SCLCs, the extent of resection had a greater impact on survival, and lobectomy combined with systemic lymph node dissection should therefore be performed as extensively as possible. In addition, PCI was beneficial in improving the SCLC OS rate. KEY POINTS: This study demonstrated the prognosis of C-SCLCs did not significantly differ from that of P-SCLCs, but was more susceptible to the extent of resection. Patients with C-SCLC who underwent limited resection had a significantly increased risk of shorter OS. This study highlighted the importance of performing lobectomy for resectable C-SCLC patients. This study also proved the benefit of PCI in improving the OS rate for both P-SCLC and C-SCLC patients.

Different pathologic responses to neoadjuvant anti-PD-1 in primary squamous lung cancer and regional lymph nodes.

Neoadjuvant immunotherapy provides a unique opportunity for understanding therapeutic responses. We analyzed pathologic responses in surgical specimens obtained from 31 squamous non-small cell lung cancer (NSCLC) patients receiving neoadjuvant anti-PD-1 treatment. Fifteen (48.4%) patients achieved pathologic complete response (pCR) or major pathologic response (MPR). Among them, seven (46.7%) were assessed as radiological partial response and eight (53.3%) as stable disease. Among 20 patients with pathologically identified tumor beds in lymph nodes (LNs), 10 and six patients achieved pCR/MPR in primary tumors and paired LNs, respectively. pCR was achieved in 6/19 N1 nodes and 1/7 N2 nodes. Residual viable tumor (RVT) cells in 8/9 MPR specimens had 100% immune-activated phenotype, while a median of 80% of RVT cells in pathologic nonresponse specimens presented immune-excluded/desert phenotype. These findings demonstrated that assessment of pathologic responses in both primary tumor and LNs may be important as a surrogate for assessing neoadjuvant immunotherapeutic efficacy.

An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients.

The number of genomic aberrations known to be relevant in making therapeutic decisions for non-small cell lung cancer patients has increased in the past decade. Multiple molecular tests are required to reliably establish the presence of these aberrations, which is challenging because available tissue specimens are generally small. To optimize diagnostic testing, we developed a transcriptome-based next-generation sequencing (NGS) assay based on single primed enrichment technology. We interrogated 11 cell lines, two patient-derived frozen biopsies, nine pleural effusion, and 29 formalin-fixed paraffin-embedded (FFPE) samples. All clinical samples were selected based on previously identified mutations at the DNA level in EGFR, KRAS, ALK, PIK3CA, BRAF, AKT1, MET, NRAS, or ROS1 at the DNA level, or fusion genes at the chromosome level, or by aberrant protein expression of ALK, ROS1, RET, and NTRK1. A successful analysis is dependent on the number of unique reads and the RNA quality, as indicated by the DV200 value. In 27 out of 51 samples with >50 K unique reads and a DV200 >30, all 19 single nucleotide variants (SNVs)/small insertions and deletions (INDELs), three MET exon 14 skipping events, and 13 fusion gene transcripts were detected at the RNA level, giving a test accuracy of 100%. In summary, this lung-cancer-specific all-in-one transcriptome-based assay for the simultaneous detection of mutations and fusion genes is highly sensitive.

Mutations in EMT-Related Genes in ALK Positive Crizotinib Resistant Non-Small Cell Lung Cancers.

Crizotinib is an effective drug for patients with anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC), but upon treatment, the tumors inevitably become crizotinib resistant in time. The resistance mechanisms are only partly understood. In this study, we aim to identify gene mutations associated with resistance in ALKpositive advanced non-squamous NSCLC treated with crizotinib. Four ALK positive patients with progressive disease following crizotinib treatment were identified with paired pre- and post-crizotinib tumor tissue from our previously published cohort. Somatic variants in these samples were detected by whole exome sequencing. In one of the four patients, an ALK-resistance associated mutation was identified. In the other three patients, no ALK-resistance associated mutations were present. In these patients we identified 89 relevant somatic mutations in 74 genes that were specific to the resistant tumors. These genes were enriched in 15 pathways. Four pathways, were related to epithelial-mesenchymal transition (EMT): proteoglycans in cancer, HIF-1 signaling, FoxO signaling pathway, and ECM-receptor interaction. Analysis of other EMT-related pathways revealed three additional genes with mutations specific to the crizotinib-resistant tumor samples. The enrichment of mutations in genes associated with EMT-related pathways indicates that loss of epithelial differentiation may represent a relevant resistance mechanism for crizotinib.

The involvement of sirtuins during optic nerve injury of rats.

Sirtuins, comprised of seven members, protect cells from injury, possibly through different roles. In this study, we used two young rat optic nerve injury models to analyze the changes in Sirts 1-7 at different time points to better understand the role of sirtuins during optic nerve injury. Twelve-week-old adult male F344 rats (total n=42) were divided randomly into two groups. One group was subjected to optic nerve cut (ON-cut) and the other group was subjected to a peripheral nerve-optic nerve graft (PN-ON graft) on the left eye. At 1 and 3 days and 1, 2, and 4 weeks, rats were euthanized and retinas of both eyes were removed. Total RNA was extracted and first-strand cDNA was synthesized. Sirts 1-7 and housekeeping ß-actin quantitative real-time PCR were performed. The quantitative real-time PCR profile showed that sirtuin mRNAs in both groups increased following optic nerve injury with and without peripheral nerve grafting. Sirt1 mRNA increased rapidly, reaching its peak at 3 days after surgery. Sirts 2-7 showed an increasing trend and remained high through 4 weeks after surgery. Sirts 4 and 6 were the only Sirts that increased in number in the PN-graft group at 4 weeks after surgery, where neuronal survival should be higher. Our data indicate that Sirt1 and Sirts 2-7 may play different or complementary roles in optic nerve injury and that Sirts 4 and 6 may play a greater role than the remaining Sirts in axon regeneration.