Lung cancer presenting with central nervous system metastasis: Clinicopathological and molecular analysis of 171 cases.

A subset of lung carcinoma presents initially with brain metastasis. Precise subtyping is mandatory for optimized treatment of these advanced aggressive carcinomas. We herein analyzed surgical biopsies from 171 Patients (99 males and 72 females aged 48-96; mean, 72), who presented with brain metastasis of lung cancer. In addition to conventional subtyping, we applied an extended immunohistochemistry (IHC) panel and performed several molecular tests looking for potential therapeutic targets other than EGFR mutations. Non-small cell carcinoma (NSCLC) comprised 157 (91.8 %) of cases: 109 (63.7 %) adenocarcinomas, 27 (15.8 %) squamous cell (SCC), 18 (10.5 %) large cell undifferentiated, 1 (0.6 %) adenosquamous and 2 (1.2 %) unclassified carcinomas. Of the adenocarcinomas, 81.7 % were TTF1+. Notably, 45 % of those TTF1-negative cases expressed HepPar1. SMARCA4 and SMARCA2 loss was observed in 13/171 (7.6 %) and 32/163 (19.6 %) cases, respectively; mainly TTF1- (40.0 %) and HepPar1+ (38.1 %) adenocarcinomas were affected by SMARCA2/4 loss. Loss of at least one mismatch repair (MMR) protein was observed in 3/156 (1.9 %) cases (2 adenocarcinomas and 1 large cell neuroendocrine carcinoma/LCNEC). Limited available data on mutation testing showed a frequency of EGFR mutations of 4.3% and of KRAS mutations of 57%. HER2 expression (2+/3+) was found in 45/166 (27.1 %) of cases with amplification verified by CISH in 18/38 (47.4 % of immunopositive cases and 10.5 % of the whole cohort); all but one were adenocarcinomas. Other genetic abnormalities detected included EML4::ALK rearrangements in 3 (1.8 %; 2 TTF1+ adenocarcinomas and 1 LCNEC) and RET rearrangements in one SCNEC. Variable subsets of tumors revealed amplifications of several potentially therapeutically targetable genes including MYC (30.0 %), MET (10.1 %), HER2 (10 %), FGFR1 (9.6 %), FGFR3 (4.6 %), and FGFR2 (3.4 %). This study highlights a highly heterogeneous molecular background in lung cancer presenting with CNS metastases. These findings highlight the need for individualized tumor testing strategies looking for potential therapeutic targets for this aggressive disease.

Serrated epithelial colorectal polyps (hyperplastic polyps, sessile serrated adenomas) with perineurial stroma: Clinicopathological and molecular analysis of a new series.

Serrated colorectal fibroblastic polyps (FPs) are rare benign mucosal lesions composed of serrated epithelial crypts separated and distorted by intimately associated bland spindle cell proliferations with perineurial-like phenotype. We herein describe 21 new FPs affecting 10 females and 9 males aged 45 to 80 yrs. (mean, 62 yrs). Lesions originated in the sigmoid colon/rectosigmoid junction (n = 16), rectum (n = 2), and other parts of the colon (n = 3). Most patients had additional synchronous or metachronous polyps: classical adenomas (12 patients), sessile serrated adenoma/SSA (1 patient), hyperplastic polyps/HPs (7 patients), both HPs and adenomas (6 patients) and colorectal cancer (2 patients). Size of the lesions varied from 1 to 6 mm (mean: 3 mm). Histologically, all lesions were composed of serrated epithelial crypts that were separated and distorted by spindle cell stromal proliferations (consistently EMA+, claudin-1+ and GLUT-1+). The epithelial component displayed features of HPs (n = 17) and SSA (n = 4). Laser-microdissection-guided molecular testing was successful for 13 epithelial and 9 stromal components (9 paired samples). The BRAF V600E mutation was detected in 54% of the epithelial but in none of the stromal components. In conclusion, colorectal FPs represent genuine serrated epithelial polyps corresponding either to HP or (less frequently) SSA and should be better classified as such with a note on the presence of the stromal component. A more concise terminology reflecting their epithelial nature is needed to fulfill the requirements for colorectal cancer risk assessment and hence adopt appropriate follow-up strategies.

Comprehensive screening for mutations associated with colorectal cancer in unselected cases reveals penetrant and nonpenetrant mutations.

Germline mutation testing in patients with colorectal cancer (CRC) is offered only to a subset of patients with a clinical presentation or tumor histology suggestive of familial CRC syndromes, probably underestimating familial CRC predisposition. The aim of our study was to determine whether unbiased screening of newly diagnosed CRC cases with next generation sequencing (NGS) increases the overall detection rate of germline mutations. We analyzed 152 consecutive CRC patients for germline mutations in 18 CRC-associated genes using NGS. All patients were also evaluated for Bethesda criteria and all tumors were investigated for microsatellite instability, immunohistochemistry for mismatch repair proteins and the BRAF*V600E somatic mutation. NGS based sequencing identified 27 variants in 9 genes in 23 out of 152 patients studied (18%). Three of them were already reported as pathogenic and 12 were class 3 germline variants with an uncertain prediction of pathogenicity. Only 1 of these patients fulfilled Bethesda criteria and had a microsatellite instable tumor and an MLH1 germline mutation. The others would have been missed with current approaches: 2 with a MSH6 premature termination mutation and 12 uncertain, potentially pathogenic class 3 variants in APC, MLH1, MSH2, MSH6, MSH3 and MLH3. The higher NGS mutation detection rate compared with current testing strategies based on clinicopathological criteria is probably due to the large genetic heterogeneity and overlapping clinical presentation of the various CRC syndromes. It can also identify apparently nonpenetrant germline mutations complicating the clinical management of the patients and their families.

ypN0 nodal status after neoadjuvant chemoradiotherapy for rectal carcinoma is not associated with adverse prognosis as compared with pN0 after primary surgery.

BACKGROUND AND AIMS: Neoadjuvant chemoradiotherapy (nCRT) has been established as standard treatment for locally advanced rectal cancer (cT3/4 or cN+ cM0, distance from circumferential resection margin ≤1 mm) to enhance local disease control and minimize the rate of locoregional recurrence. As a consequence of downstaging, a significant proportion of patients show non-viable tumor deposits within regional lymph nodes that qualify as ypN0 according to the current TNM classification irrespective of the actual pre-treatment status. Accordingly, the prognostic relevance of ypN0 status as compared with true pN0 without preceding nCRT is not known. PATIENTS AND METHODS: We retrospectively analyzed 132 patients who underwent standard total mesorectal excision (TME) surgery after nCRT for rectal carcinoma and were classified as ypN0, and compared their prognoses with those of 341 patients with pN0 without nCRT. RESULTS: Re-evaluation of regional lymph nodes after nCRT showed no evidence of previous metastasis in 91 cases (ypN0(-)), sure but non-viable metastasis in 14 patients (ypN0(+)), and unsure status in 27 patients (ypN0(X)). The local recurrence rate, distant metastases, and disease-free, observed, and cancer-related survival were similar in all subgroups (p = 0.447, 0.695, 0.759, 0.655, and 0.354, respectively). CONCLUSION: Our results showed a similar outcome in patients with ypN0 and in the control group with pN0 regarding local recurrence rate, distant metastases, and disease-free, observed, and cancer-related survival. Validation of these results is necessary to clarify the questions regarding postoperative adjuvant chemotherapy for patients with ypN0(+).

ISL1 expression is not restricted to pancreatic well-differentiated neuroendocrine neoplasms, but is also commonly found in well and poorly differentiated neuroendocrine neoplasms of extrapancreatic origin.

The human insulin gene enhancer-binding protein islet-1 (ISL1) is a transcription factor involved in the differentiation of the neuroendocrine pancreatic cells. Recent studies identified ISL1 as a marker for pancreatic well-differentiated neuroendocrine neoplasms. However, little is known about ISL1 expression in pancreatic poorly differentiated and in extrapancreatic well and poorly differentiated neuroendocrine neoplasms. We studied the immunohistochemical expression of ISL1 in 124 neuroendocrine neoplasms. Among pancreatic neuroendocrine neoplasms, 12/13 with poor differentiation were negative, whereas 5/7 with good differentiation but a Ki67 >20% were positive. In extrapancreatic neuroendocrine neoplasms, strong positivity was found in Merkel cell carcinomas (25/25), pulmonary small cell neuroendocrine carcinomas (21/23), medullary thyroid carcinomas (9/9), paragangliomas/pheochromocytomas (6/6), adrenal neuroblastomas (8/8) and head and neck neuroendocrine carcinomas (4/5), whereas no or only weak staining was recorded in pulmonary carcinoids (3/15), olfactory neuroblastomas (1/4) and basaloid head and neck squamous cell carcinomas (0/15). ISL1 stained the neuroendocrine carcinoma component of 5/8 composite carcinomas and also normal neuroendocrine cells in the thyroid, adrenal medulla, stomach and colorectum. Poorly differentiated neuroendocrine neoplasms, regardless of their ISL1 expression, were usually TP53 positive. Our results show the almost ubiquitous expression of ISL1 in extrapancreatic poorly differentiated neuroendocrine neoplasms and neuroblastic malignancies and its common loss in pancreatic poorly differentiated neuroendocrine neoplasms. These findings modify the role of ISL1 as a marker for pancreatic neuroendocrine neoplasms and suggest that ISL1 has a broader involvement in differentiation and growth of neuroendocrine neoplasms than has so far been assumed.

Regulatory T cells and cytotoxic T cells close to the epithelial-stromal interface are associated with a favorable prognosis.

Cytotoxic T cells and regulatory T cells play a crucial role in the outcome of cancer patients. Besides the density of these cells, it was shown recently that the spatial distribution is equally important. Here, we specifically analyzed the spatial distribution of these T cell subtypes at the epithelial-stromal interface in a rectal cancer cohort and its relevance for prognosis. We studied a cohort of 191 patients with advanced rectal cancer treated by radiochemotherapy (RCT). Tissue microarrays were immunohistochemical double-stained by FoxP3+ and CD+. Cell densities were analyzed in the stromal and epithelial compartment. Additionally, an image analysis software calculated the distances of lymphocytes to the epithelial-stromal interface (ESI). CD8+ and FoxP3+ cell counts decreased clearly after RCT with the decrease of FoxP3+ being more pronounced than of CD8+ cells. In the invasive front, short distances of the ESI to CD8+ and to FoxP3+ cells were associated with improved overall survival. Cell counts in the stromal compartment had no influence on prognosis. No correlation between stromal and epithelial lymphocyte densities was observed. The distance of epithelial-stromal interface to CD8+ and FoxP3+ cells was more accurate in predicting prognosis in the stromal compartment of rectal cancer patients than mere cell counts and could thereby be means of better stratifying patients for therapy. This observation will have to be validated in future prospective studies with regard to other tumor entities and its implications for the responsiveness of tumors to new therapeutic modalities.

Loss of enhancer of zeste homologue 2 (EZH2) at tumor invasion front is correlated with higher aggressiveness in colorectal cancer cells.

PURPOSE: Enhancer of zeste homolog 2 (EZH2) is associated with epigenetic gene silencing and aggressiveness in many tumor types. However, the prognostic impact of high EZH2 expression is controversially discussed for colorectal cancer. For this reason, we immunohistochemically analyzed EZH2 expression in 105 specimens from colon cancer patients separately for tumor center and invasion front. METHODS: All sections from tissue microarrays were evaluated manually and digitally using Definiens Tissue Studio software (TSS). To mirror-image the EZH2 status at the tumor invasion front, we treated HCT116 colon cancer cells with the EZH2 inhibitor 3-Deazaneplanocin A (DZNep) and studied the growth of in ovo xenografts in the chorioallantoic membrane (CAM) assay. RESULTS: We showed a significant decrease in EZH2 expression and the repressive H3K27me3 code at the tumor invasion front as supported by the TSS-constructed heatmaps. Loss of EZH2 at tumor invasion front, but not in tumor center was correlated with unfavorable prognosis and more advanced tumor stages. The observed cell cycle arrest in vitro and in vivo was associated with higher tumor aggressiveness. Xenografts formed by DZNep-treated HCT116 cells showed loosely packed tumor masses, infiltrative growth into the CAM, and high vessel density. CONCLUSION: The differences in EZH2 expression between tumor center and invasion front as well as different scoring and cutoff values can most likely explain controversial literature data concerning the prognostic value of EZH2. Epigenetic therapies using EZH2 inhibitors have to be carefully evaluated for each specific tumor type, since alterations in cell differentiation might lead to unfavorable results.

Combination of growth pattern and tumor regression identifies a high-risk group in neoadjuvant treated rectal cancer patients.

OBJECTIVE: To analyze the prognostic effect of E-cadherin expression, the growth pattern of the tumor and the regression grade in a rectal cancer cohort treated with neoadjuvant radiochemotherapy (RCT). METHODS: A total of 223 patients with rectal cancer treated with neoadjuvant RCT followed by surgery were included. Altogether 88 biopsies prior to RCT and 213 tumor resections in an average of 55 days post-RCT were investigated. Protein expression of E-cadherin and tumor growth pattern (solid glandular vs single-cell pattern) was assessed by staining tissue microarrays. The regression grade at the invasion front was determined according to the Dworak scale. RESULTS: There was a significant decrease of E-cadherin expression (P = 0.002) and a significant increased single-cell growth (P < 0.001) at the invasion front in tumor samples after RCT compared with primary biopsies of the tumor. A low E-cadherin expression in the biopsy was related to a longer metastasis-free survival (P = 0.033) and tumor-specific survival (P = 0.030). Post-RCT single-cell growth at the tumor invasion front was a prognostic factor for longer tumor-specific survival (P = 0.021). A combination of growth pattern and the Dworak regression grade was an independent prognostic parameter for tumor-specific survival (P = 0.015). CONCLUSIONS: Loss of E-cadherin protein expression in the pretreatment biopsy of rectal cancer is associated with fewer metastases and improved survival. Furthermore, the growth pattern in the post-RCT resection specimen has a prognostic value for survival. A combination of growth pattern and tumor regression score (the RegPat score) showed the highest discriminatory power to identify high-risk patients.

Spatial distribution of FoxP3+ and CD8+ tumour infiltrating T cells reflects their functional activity.

BACKGROUND: Regulatory and cytotoxic T cells are key players in the host's anticancer immune response. We studied the spatial distribution of FoxP+ and CD8+ cells to identify potential interactions. METHODS: In 202 patients 103 pre-radiochemotherapy biopsies and 153 post-radiochemotherapy tumour specimens of advanced rectal cancer were available and an immunohistochemical double staining of FoxP3+ and CD8+ tumour-infiltrating lymphocytes was performed to investigate cell density and cell-to-cell distances. RESULTS: FoxP3+ cells decreased after radiochemotherapy by a factor of 3 while CD8+ cells remained nearly unchanged. High epithelial (p=0.033) and stromal (p=0.009) FoxP3+ cell density was associated with an improved overall survival. Cell-to-cell distances of randomly distributed cells were simulated and compared to observed cell-to-cell distances. Observed distances shorter than the simulated, random distances were hypothesized to represent FoxP3+ cells actively interacting with CD8+ cells. Epithelial short distances were associated with a favourable prognosis while the opposite was true for the stromal compartment. CONCLUSION: The analysis of cell-to-cell distances may offer a tool to predict outcome, maybe by identifying functionally active, interacting infiltrating inflammatory cells in different tumour compartments.

PD-L1 is upregulated by radiochemotherapy in rectal adenocarcinoma patients and associated with a favourable prognosis.

BACKGROUND: The influence of neoadjuvant radiochemotherapy (RCT) on programmed death-ligand 1 (PD-L1) expression, a predictive marker for programmed cell death protein 1 (PD-1) inhibitor therapy, was studied on tumour and inflammatory cells in rectal adenocarcinoma patients along with its prognostic value. METHODS: PD-L1 immunohistochemistry was performed on tissue microarrays of 103 pre-RCT biopsies and 159 post-RCT surgical specimens (central tumour, invasive front and normal tissue) of 199 patients. In 63 patients, both samples were available. Proportion and maximum intensity of PD-L1-positive (PD-L1+) cells were evaluated. RESULTS: RCT increased the proportion of PD-L1-expressing cancer cells from 2.1% to 7.8% in the central tumour (p < 0.001) or 9.3% in the invasive front (p < 0.001). Cancer cell PD-L1 on its own could not predict prognosis. High PD-L1 expression on pre-RCT inflammatory cells (maximum intensity: p = 0.048) and post-RCT invasive front inflammatory cells (p = 0.010) correlated with improved no evidence of disease survival. In multivariate analysis, the combination of low PD-L1 in cancer and inflammatory cells was an independent negative prognostic marker for overall survival (OS) pre-RCT (Cox's proportional hazard ratio 0.438, p = 0.045) and in the invasive front post-RCT (Cox's proportional hazard ratio 0.257, p = 0.030). CONCLUSION: Neoadjuvant RCT is associated with an increased PD-L1 expression in rectal adenocarcinoma patients, which should prompt clinical trials combining radiotherapy and PD-1/PD-L1 pathway blockade. Combined low PD-L1 expression on tumour and inflammatory cells is an independent negative prognostic marker for OS in RCT of rectal adenocarcinoma.

Inflammatory response in serrated precursor lesions of the colon classified according to WHO entities, clinical parameters and phenotype-genotype correlation.

Studies on traditional serrated adenoma (TSA) and sessile serrated adenoma with dysplasia (SSA-D) are rare due to the low frequency of these lesions, which are well defined by the latest WHO classification. However, introducing new morphological criteria such as intra-epithelial lymphocytes (IELs) might facilitate colorectal polyp diagnoses. Additionally, the phenotype-genotype correlation needs to be updated as the terminology has repeatedly changed. This study analysed 516 polyps, consisting of 118 classical adenomas (CAD), 116 hyperplastic polyps (HPP), 179 SSAs, 41 SSA-Ds, and 62 TSAs. The lesions were analysed in relation to the patients' clinical parameters including gender, age, localisation, and size. The inflammatory background of the polyps was quantified and BRAF and KRAS mutations as well as MLH1 and CDKN2A promoter methylation were assessed. In multivariate analyses, an increase in IELs was an independent and robust new criterion for the diagnosis of SSA-D (p < 0.001). Superficial erosions and acute neutrophil granulocytes led to reactive changes potentially resembling dysplasia. KRAS and BRAF mutations were associated with CAD/TSA and HPP/SSA, respectively. However, almost half of TSAs had a BRAF mutation and were KRAS wild type. CDKN2A seems to precede MLH1 hyper-methylation within the serrated carcinogenesis model. The genotyping of WHO-based entities - and especially SSA - has sharpened in comparison to previously published data. TSAs can be sub-grouped according to their mutation status. Of note, the higher number of IELs in SSA-D reflects their close relationship to colorectal cancers with micro-satellite instability. Therefore, IELs might represent a new diagnostic tool for SSA-D.

Expression of the LIM homeobox domain transcription factor ISL1 (Islet-1) is frequent in rhabdomyosarcoma but very limited in other soft tissue sarcoma types.

The transcription factor ISL1 (islet-1) has emerged as a useful marker for metastatic pancreatic well differentiated neuroendocrine neoplasms, but recent studies showed wider expression in poorly differentiated neuroendocrine carcinomas from different sites as well as poorly differentiated neuroblastoma. Expression of ISL1 in soft tissue sarcomas has not been studied before.We evaluated ISL1 expression in 249 soft tissue tumour specimens from 249 patients and 17 precursor cell lymphoblastic lymphomas (ALL). ISL1 was not detected in any of 63 liposarcomas of different subtypes, 55 leiomyosarcomas, 22 solitary fibrous tumours, 20 undifferentiated pleomorphic/spindle cell sarcomas, 13 small cell synovial sarcomas and 17 ALL cases. Variable nuclear expression was detected in rhabdomyosarcoma (15/25, 60%), rhabdomyoblastic areas of malignant müllerian mixed tumours (5/5), Ewing sarcoma (2/12, very weak) and monophasic fibrous synovial sarcoma (2/29). More extensive staining (moderate to strong) was restricted to rhabdomyosarcoma. Taken by histological subtype, ISL1 was expressed more frequently in alveolar (9/11, 82%) versus non-alveolar (6/14, 43%) rhabdomyosarcoma. ISL1 is commonly expressed in rhabdomyosarcoma, particularly the alveolar subtype and should be distinguished from poorly differentiated neuroendocrine and neuroblastic neoplasms. Awareness of this finding helps to avoid misinterpretation as neuroendocrine neoplasms that would result in inappropriate therapeutic and prognostic consequences.