Endobronchial ultrasound-guided transbronchial fine needle aspiration of mediastinal lymphadenopathy: Diagnostic performance and clinical implications of the World Health Organization reporting system.

INTRODUCTION: Lymph node fine-needle aspiration cytology (LN-FNAC) is a common, rapid, minimally invasive and cost-effective diagnostic method. For mediastinal lymph nodes, endobronchial ultrasound (EBUS) guided LN-FNAC is a first-line investigation and has an indispensable role in the diagnosis and staging of patients with suspected lung cancer. Recently, a new WHO system has been proposed for classification of LN-FNAC heralding five different diagnostic categories; insufficient, benign, atypical, suspicious for malignancy and malignant. The aim of this study was to evaluate the diagnostic accuracy and risk of malignancy (ROM) of these categories in EBUS-guided LN-FNAC from mediastinal lymph nodes. METHOD: We evaluated 2110 consecutive mediastinal lymph nodes during this one-year retrospective study. Corresponding radiological images and histologic material were used as ground truth to calculate accuracy, sensitivity, specificity and ROM. RESULTS: The WHO system showed an overall accuracy of 93.7% with a sensitivity of 83.0% and a specificity of 97.5%. The positive predictive value was 92.3% and the negative predictive value 94.2%. The overall ROM for each category in the WHO classification system was 12.8% for the inadequate, 2.4% for the benign, 47.4% for the atypical, 81.0% for the suspicious for malignancy and 93.6% for the malignant category. CONCLUSION: The results of the present study indicate that the new WHO system entails a high diagnostic accuracy regarding EBUS-guided LN-FNAC assessment of mediastinal lymph nodes and supports its integration into clinical practice. Application of the WHO system standardizes risk assessment thus facilitating communication between cytopathologists and clinicians and minimizes the need for histopathological analysis.

PD-L1 Testing in Cytological Non-Small Cell Lung Cancer Specimens: A Comparison with Biopsies and Review of the Literature.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) expression is used for treatment prediction in non-small cell lung cancer (NSCLC). While cytology may be the only available material in the routine clinical setting, testing in clinical trials has mainly been based on biopsies. METHODS: We included 2 retrospective cohorts of paired, concurrently sampled, cytological specimens and biopsies. Also, the literature on PD-L1 in paired cytological/histological samples was reviewed. Focus was on the cutoff levels ≥1 and ≥50% positive tumor cells. RESULTS: Using a 3-tier scale, PD-L1 was concordant in 40/47 (85%) and 66/97 (68%) of the paired NSCLC cases in the 2 cohorts, with kappa 0.77 and 0.49, respectively. In the former cohort, all discordant cases had lower score in cytology. In both cohorts, concordance was lower in samples from different sites (e.g., biopsy from primary tumor and cytology from pleural effusion). Based on 25 published studies including about 1,700 paired cytology/histology cases, the median (range) concordance was 81-85% (62-100%) at cutoff 1% for a positive PD-L1 staining and 89% (67-100%) at cutoff 50%. CONCLUSIONS: The overall concordance of PD-L1 between cytology and biopsies is rather good but with significant variation between laboratories, which calls for local quality assurance.

Clinical Utility of Targeted Sequencing in Lung Cancer: Experience From an Autonomous Swedish Health Care Center.

OBJECTIVES: Mutation analysis by massive parallel sequencing (MPS) is routinely performed in the clinical management of lung cancer in Sweden. We describe the clinical and mutational profiles of lung cancer patients subjected to the first 1.5 years of treatment predictive MPS testing in an autonomous regional health care region. METHODS: Tumors from all patients with lung cancer who had an MPS test from January 2015 to June 2016 in the Skåne health care region in Sweden (1.3 million citizens) were included. Six hundred eleven tumors from 599 patients were profiled using targeted sequencing with a 26-gene exon-focused panel. Data on disease patterns and characteristics of the patients subjected to testing were assembled, and correlations between mutational profiles and clinical features were analyzed. RESULTS: MPS with the 26-gene panel revealed alterations in 92% of the 611 lung tumors, with the most frequent mutations detected in the nontargetable genes TP53 (62%) and KRAS (37%). Neither KRAS nor TP53 mutations were associated with disease pattern, chemotherapy response, progression-free survival, or overall survival in advanced-stage disease treated with platinum-based doublet chemotherapy as a first-line treatment. Among targetable genes, EGFR driver mutations were detected in 10% of the tumors, and BRAF p.V600 variants in 2.3%. For the 71 never smokers (12%), targetable alterations (EGFR mutations, BRAF p.V600, MET exon 14 skipping, or ALK/ROS1 rearrangement) were detected in 59% of the tumors. CONCLUSION: Although the increasing importance of MPS as a predictor of response to targeted therapies is indisputable, its role in prognostics or as a predictor of clinical course in nontargetable advanced stage lung cancer requires further investigation.

Clinical framework for next generation sequencing based analysis of treatment predictive mutations and multiplexed gene fusion detection in non-small cell lung cancer.

Precision medicine requires accurate multi-gene clinical diagnostics. We describe the implementation of an Illumina TruSight Tumor (TST) clinical NGS diagnostic framework and parallel validation of a NanoString RNA-based ALK, RET, and ROS1 gene fusion assay for combined analysis of treatment predictive alterations in non-small cell lung cancer (NSCLC) in a regional healthcare region of Sweden (Scandinavia). The TST panel was clinically validated in 81 tumors (99% hotspot mutation concordance), after which 533 consecutive NSCLCs were collected during one-year of routine clinical analysis in the healthcare region (~90% advanced stage patients). The NanoString assay was evaluated in 169 of 533 cases. In the 533-sample cohort 79% had 1-2 variants, 12% >2 variants and 9% no detected variants. Ten gene fusions (five ALK, three RET, two ROS1) were detected in 135 successfully analyzed cases (80% analysis success rate). No ALK or ROS1 FISH fusion positive case was missed by the NanoString assay. Stratification of the 533-sample cohort based on actionable alterations in 11 oncogenes revealed that 66% of adenocarcinomas, 13% of squamous carcinoma (SqCC) and 56% of NSCLC not otherwise specified harbored ≥1 alteration. In adenocarcinoma, 10.6% of patients (50.3% if including KRAS) could potentially be eligible for emerging therapeutics, in addition to the 15.3% of patients eligible for standard EGFR or ALK inhibitors. For squamous carcinoma corresponding proportions were 4.4% (11.1% with KRAS) vs 2.2%. In conclusion, multiplexed NGS and gene fusion analyses are feasible in NSCLC for clinical diagnostics, identifying notable proportions of patients potentially eligible for emerging molecular therapeutics.

The expression pattern of matrix-producing tumor stroma is of prognostic importance in breast cancer.

BACKGROUND: There are several indications that the composition of the tumor stroma can contribute to the malignancy of a tumor. Here we utilized expression data sets to identify metagenes that may serve as surrogate marker for the extent of matrix production and vascularization of a tumor and to characterize prognostic molecular components of the stroma. METHODS: TCGA data sets from six cancer forms, two breast cancer microarray sets and one mRNA data set of xenografted tumors were downloaded. Using the mean correlation as distance measure compact clusters with genes representing extracellular matrix production (ECM metagene) and vascularization (endothelial metagene) were defined. Explorative Cox modeling was used to identify prognostic stromal gene sets. RESULTS: Clustering of stromal genes in six cancer data sets resulted in metagenes, each containing three genes, representing matrix production and vascularization. The ECM metagene was associated with poor prognosis in renal clear cell carcinoma and in lung adenocarcinoma but not in other cancers investigated. Explorative Cox modeling using gene pairs identified gene sets that in multivariate models were prognostic in breast cancer. This was validated in two microarray sets. Two notable genes are TCF4 and P4HA3 which were included in the sets associated with positive and negative prognosis, respectively. Data from laser-microdissected tumors, a xenografted tumor data set and from correlation analyses demonstrate the stroma specificity of the genes. CONCLUSIONS: It is possible to construct ECM and endothelial metagenes common for several cancer forms. The molecular composition of matrix-producing cells, rather than the extent of matrix production seem to be important for breast cancer prognosis.

Mutational and gene fusion analyses of primary large cell and large cell neuroendocrine lung cancer.

Large cell carcinoma with or without neuroendocrine features (LCNEC and LC, respectively) constitutes 3-9% of non-small cell lung cancer but is poorly characterized at the molecular level. Herein we analyzed 41 LC and 32 LCNEC (including 15 previously reported cases) tumors using massive parallel sequencing for mutations in 26 cancer-related genes and gene fusions in ALK, RET, and ROS1. LC patients were additionally subdivided into three immunohistochemistry groups based on positive expression of TTF-1/Napsin A (adenocarcinoma-like, n = 24; 59%), CK5/P40 (squamous-like, n = 5; 12%), or no marker expression (marker-negative, n = 12; 29%). Most common alterations were TP53 (83%), KRAS (22%), MET (12%) mutations in LCs, and TP53 (88%), STK11 (16%), and PTEN (13%) mutations in LCNECs. In general, LCs showed more oncogene mutations compared to LCNECs. Immunomarker stratification of LC revealed oncogene mutations in 63% of adenocarcinoma-like cases, but only in 17% of marker-negative cases. Moreover, marker-negative LCs were associated with inferior overall survival compared with adenocarcinoma-like tumors (p = 0.007). No ALK, RET or ROS1 fusions were detected in LCs or LCNECs. Together, our molecular analyses support that LC and LCNEC tumors follow different tumorigenic paths and that LC may be stratified into molecular subgroups with potential implications for diagnosis, prognostics, and therapy decisions.

Sex differences in the prognostic significance of KRAS codons 12 and 13, and BRAF mutations in colorectal cancer: a cohort study.

BACKGROUND: Activating KRAS and BRAF mutations predict unresponsiveness to EGFR-targeting therapies in colorectal cancer (CRC), but their prognostic value needs further validation. In this study, we investigated the impact of KRAS codons 12 and 13, and BRAF mutations on survival from CRC, overall and stratified by sex, in a large prospective cohort study. METHODS: KRAS codons 12 and 13, and BRAF mutations were analysed by pyrosequencing of tumours from 525 and 524 incident CRC cases in The Malmö Diet and Cancer Study. Associations with cancer-specific survival (CSS) were explored by Cox proportional hazards regression, unadjusted and adjusted for age, TNM stage, differentiation grade, vascular invasion and microsatellite instability (MSI) status. RESULTS: KRAS and BRAF mutations were mutually exclusive. KRAS mutations were found in 191/ 525 (36.4%) cases, 82.2% of these mutations were in codon 12, 17.3% were in codon 13, and 0.5% cases had mutations in both codons. BRAF mutations were found in 78/524 (14.9%) cases. Overall, mutation in KRAS codon 13, but not codon 12, was associated with a significantly reduced CSS in unadjusted, but not in adjusted analysis, and BRAF mutation did not significantly affect survival. However, in microsatellite stable (MSS), but not in MSI tumours, an adverse prognostic impact of BRAF mutation was observed in unadjusted, but not in adjusted analysis. While KRAS mutation status was not significantly associated with sex, BRAF mutations were more common in women. BRAF mutation was not prognostic in women; but in men, BRAF mutation was associated with a significantly reduced CSS in overall adjusted analysis (HR = 3.50; 95% CI = 1.41-8.70), but not in unadjusted analysis. In men with MSS tumours, BRAF mutation was an independent factor of poor prognosis (HR = 4.91; 95% CI = 1.99-12.12). KRAS codon 13 mutation was associated with a significantly reduced CSS in women, but not in men in unadjusted, but not in adjusted analysis. CONCLUSIONS: Results from this cohort study demonstrate sex-related differences in the prognostic value of BRAF mutations in colorectal cancer, being particularly evident in men. These findings are novel and merit further validation.

Associations of beta-catenin alterations and MSI screening status with expression of key cell cycle regulating proteins and survival from colorectal cancer.

BACKGROUND: Despite their pivotal roles in colorectal carcinogenesis, the interrelationship and prognostic significance of beta-catenin alterations and microsatellite instability (MSI) in colorectal cancer (CRC) needs to be further clarified. In this paper, we studied the associations between beta-catenin overexpression and MSI status with survival from CRC, and with expression of p21, p27, cyclin D1 and p53, in a large, prospective cohort study. METHODS: Immunohistochemical MSI-screening status and expression of p21, p27 and p53 was assessed in tissue microarrays with tumours from 557 cases of incident CRC in the Malmö Diet and Cancer Study. Chi Square and Spearman's correlation tests were used to explore the associations between beta-catenin expression, MSI status, clinicopathological characteristics and investigative parameters. Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the relationship between beta-catenin overexpression, MSI status and cancer specific survival (CSS). RESULTS: Positive MSI screening status was significantly associated with older age, female sex, proximal tumour location, non-metastatic disease, and poor differentiation, and inversely associated with beta-catenin overexpression. Beta-catenin overexpression was significantly associated with distal tumour location, low T-stage and well-differentiated tumours. Patients with MSI tumours had a significantly prolonged CSS in the whole cohort, and in stage III-IV disease, also in multivariable analysis, but not in stage I-II disease. Beta-catenin overexpression was associated with a favourable prognosis in the full cohort and in patients with stage III-IV disease. Neither MSI nor beta-catenin status were predictive for response to adjuvant chemotherapy in curatively treated stage III patients. P53 and p27 expression was positively associated with beta-catenin overexpression and inversely associated with MSI. Cyclin D1 expression was positively associated with MSI and beta-catenin overexpression, and p21 expression was positively associated with MSI but not beta-catenin overexpression. CONCLUSIONS: Findings from this large, prospective cohort study demonstrate that MSI screening status in colorectal cancer is an independent prognostic factor, but not in localized disease, and does not predict response to adjuvant chemotherapy. Beta-catenin overexpression was also associated with favourable outcome but not a treatment predictive factor. Associations of MSI and beta-catenin alterations with other investigative and clinicopathological factors were in line with the expected. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/8778585058652609.

Molecular correlates and prognostic significance of SATB1 expression in colorectal cancer.

BACKGROUND: Special AT-rich sequence-binding protein 1 (SATB1) is a global gene regulator that has been reported to confer malignant behavior and associate with poor prognosis in several cancer forms. SATB1 expression has been demonstrated to correlate with unfavourable tumour characteristics in rectal cancer, but its association with clinical outcome in colorectal cancer (CRC) remains unclear. In this study, we examined the prognostic impact of SATB1 expression in CRC, and its association with important molecular characteristics; i.e. beta-catenin overexpression, microsatellite instability (MSI) screening status, and SATB2 expression. METHODS: Immunohistochemical expression of SATB1 and beta-catenin was assessed in tissue microarrays with tumours from 529 incident CRC cases in the prospective population-based Malmö Diet and Cancer Study, previously analysed for SATB2 expression and MSI screening status. Spearmans Rho and Chi-Square tests were used to explore correlations between SATB1 expression, clinicopathological and investigative parameters. Kaplan Meier analysis and Cox proportional hazards modelling were used to explore the impact of SATB1 expression on cancer specific survival (CSS) and overall survival (OS). RESULTS: SATB1 was expressed in 222 (42%) CRC cases and negative, or sparsely expressed, in adjacent colorectal mucosa (n = 16). SATB1 expression was significantly associated with microsatellite stable tumours (p < 0.001), beta-catenin overexpression (p < 0.001) and SATB2 expression (p < 0.001). While not prognostic in the full cohort, SATB1 expression was significantly associated with poor prognosis in SATB2 negative tumours (HR = 2.63; 95% CI 1.46-4.71; p(interaction) = 0.011 for CSS and HR = 2.31; 95% CI 1.32-4.04; p(interaction) = 0.015 for OS), remaining significant in multivariable analysis. CONCLUSIONS: The results of this study demonstrate that SATB1 expression in CRC is significantly associated with beta-catenin overexpression, microsatellite stability and SATB2 expression. Furthermore, SATB1 expression is a factor of poor prognosis in SATB2 negative tumours. Altogether, these data indicate an important role for SATB1 in colorectal carcinogenesis and suggest prognostically antagonistic effects of SATB1 and SATB2. The mechanistic basis for these observations warrants further study. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1922643082772076.