Nora Sahnane

Colorectal poorly differentiated neuroendocrine carcinomas and mixed adenoneuroendocrine carcinomas: insights into the diagnostic immunophenotype, assessment of methylation profile, and search for prognostic markers.

Colorectal poorly differentiated neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are well-recognized entities generally known to be associated with biological aggressiveness and poor patient survival. However, a few published papers have highlighted the existence of a subgroup of tumors with a better survival than expected; however, to date, there are no established parameters that usefully identify this category. In the present study we have investigated the morphologic features, the CpG methylator phenotype (CIMP), microsatellite instability (MSI), and the immunohistochemical profile, including the expression of transcription factors (TTF1, ASH1, CDX2, and PAX5), stem cell markers (CD117 and CD34), and cytokeratins 7 and 20, in a series of 39 carcinomas (27 NECs and 12 MANECs) to better characterize such neoplasms and to search for prognostic indicators. No different patient survival was observed between NECs and MANECs. Neoplasms showed a heterogenous spectrum of morphologic and immunohistochemical features; however, only large-cell subtype, significant peritumoral lymphoid reaction, CD117 immunoreactivity, vascular invasion, and MSI/CIMP+ status were significantly correlated with prognosis on univariable analysis. Furthermore, vascular invasion and CD117 immunoreactivity were independent prognostic markers on multivariable analysis. In addition to these prognostic features, neoplasms showed different expression of transcription factors, stem cell markers, and cytokeratins that should be considered for diagnostic purposes and, especially, for discriminating among possible differential diagnoses.

Androgen receptor is frequently expressed in HER2-positive, ER/PR-negative breast cancers

The estrogen receptor (ER)/progesterone receptor (PR)-negative breast carcinomas (BCs) encompass three molecular subtypes: one with human epidermal growth factor receptor 2 (HER) overexpression, one normal like, and the triple negative. The androgen receptor (AR) is expressed in 70–90% of invasive BCs. The aim of our study is to detect the expression of AR in a series of ER/PR-negative BCs to ascertain if there is clinical significance in relation to BC molecular subtypes. A immunohistochemical study for all receptors and cytokeratin expression was performed in 232 cases of ER/PR-negative BCs. According to cytokeratin expression, BCs were classified into two groups: luminal-type BCs (44.2%) and basal-like-type BCs (55.8%). According to the expression of HER2, 59.3% were triple-negative BCs (when ER, PR, and HER2 were negative) and 40.7% were HER2-positive BCs. AR expression was observed in 128 tumors (56.6%). One hundred and ten cases (48.8%) had >10% and 18 (7.8%) had <10% of positively stained cells. AR immunoreactivity was found in 31.2% basal-like BCs, while in the luminal group 71.1% of cases were positive, showing highly significant correlation (p < 10−8). Regarding HER2 status, 76.7% of HER2-positive BC cases were AR positive compared with only 30.4% of triple-negative BC types, showing a strong statistically significant correlation. In conclusion, we show that AR is frequently expressed in ER/PR-negative BCs and that expression of HER2 and AR is highly correlated (p < 0.005). Our results point out the role of AR and HER2 in the pathogenesis of BCs and suggest the potential role of AR in clinical management of ER/PR-negative BCs.

Microsatellite unstable gastrointestinal neuroendocrine carcinomas: a new clinicopathologic entity

Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are heterogeneous neoplasms characterized by poor outcome. Microsatellite instability (MSI) has recently been found in colorectal NECs showing a better prognosis than expected. However, the frequency of MSI in a large series of GEP-NEC/MANECs is still unknown. In this work, we investigated the incidence of MSI in GEP-NEC/MANECs and characterized their clinicopathologic and molecular features. MSI analysis and immunohistochemistry for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) were performed in 89 GEP-NEC/MANECs (six esophageal, 77 gastrointestinal, three pancreatic, and three of the gallbladder). Methylation of 34 genes was studied by methylation-specific multiplex ligation probe amplification. Mutation analysis of BRAF and KRAS was assessed by PCR-pyrosequencing analysis. MSI was observed in 11 NEC/MANECs (12.4%): seven intestinal and four gastric. All but two MSI-cases showed MLH1 methylation and loss of MLH1 protein. The remaining two MSI-cancers showed lack of MSH2 or PMS2 immunohistochemical expression. MSI-NEC/MANECs showed higher methylation levels than microsatellite stable NEC/MANECs (40.6% vs 20.2% methylated genes respectively, P<0.001). BRAF mutation was detected in six out of 88 cases (7%) and KRAS mutation was identified in 15 cases (17%). BRAF mutation was associated with MSI (P<0.0008), while KRAS status did not correlate with any clinicopathologic or molecular feature. Vascular invasion (P=0.0003) and MSI (P=0.0084) were identified as the only independent prognostic factors in multivariate analysis. We conclude that MSI identifies a subset of gastric and intestinal NEC/MANECs with distinct biology and better prognosis. MSI-NEC/MANECs resemble MSI-gastrointestinal adenocarcinomas for frequency, molecular profile and pathogenetic mechanisms.

Up-regulation of the hypoxia-inducible factor–1 transcriptional pathway in colorectal carcinomas

The aim of the study was to identify the impact on prognosis of hypoxia-inducible factor-1 genetic program in colorectal carcinomas and to develop an experimental procedure that would allow a reliable quantitative gene expression analysis in formalin-fixed and paraffin-embedded tissue. The expression of hypoxia-inducible factor-1alpha and 13 hypoxia-inducible factor-1 target genes (AMF, CAIX, VEGF, VEGFR1, VEGFR2, HGF, MET, TGFalpha, EGFR, IGF2, MMP2, PLAUR, NIX) was quantified by real-time polymerase chain reaction in 18 colorectal, poorly differentiated neuroendocrine carcinomas and in 60 invasive colorectal carcinomas. Moreover, hypoxia-inducible factor-1alpha protein expression was evaluated by immunohistochemistry. High levels of hypoxia-inducible factor-1alpha were positively associated with poorly differentiated neuroendocrine carcinoma histology (P < .005), poor differentiation (P < .025), presence of necrosis, and presence of microsatellite instability (P < .05). AMF, TGFalpha, IGF2, NIX, VEGF, and VEGFR2 transcripts were significantly higher in the very aggressive poorly differentiated neuroendocrine carcinomas than in exocrine colorectal carcinomas and TGFalpha expression was significantly associated with presence of lymph nodal metastases (P < .05). High levels of TGFalpha and NIX were significantly associated with decreased overall survival (P < .001; P < .01). The multivariate analysis showed that advanced stage, presence of lymph node metastases, and high TGFalpha expression had an independent effect on survival (P < .006; P < .01; P < .0006). Our study suggests an up-regulation of the hypoxia-inducible factor-1 transcriptional pathway in colorectal carcinomas. hypoxia-inducible factor-1alpha overexpression alone, has no impact on the prognosis of colorectal carcinomas likely because the consequences of hypoxia-inducible factor-1alpha expression/stabilization strongly depend on the genetic background of the transformed cells. Mechanisms leading to increased synthesis of hypoxia-inducible factor-1alpha mRNA via autocrine growth factor loops may play a crucial role in invasive growth in this site.

Prognostic relevance of aberrant DNA methylation in g1 and g2 pancreatic neuroendocrine tumors.

Background/Aims: The occurrence and clinical relevance of DNA hypermethylation and global hypomethylation in pancreatic neuroendocrine tumours (PanNETs) are still unknown. We evaluated the frequency of both epigenetic alterations in PanNETs to assess the relationship between methylation profiles and chromosomal instability, tumour phenotypes and prognosis. Methods: In a well-characterized series of 56 sporadic G1 and G2 PanNETs, methylation-sensitive multiple ligation-dependent probe amplification was performed to assess hypermethylayion of 33 genes and copy number alterations (CNAs) of 53 chromosomal regions. Long interspersed nucleotide element-1 (LINE-1) hypomethylation was quantified by pyrosequencing. Results: Unsupervised hierarchical clustering allowed to identify a subset of 22 PanNETs (39%) exhibiting high frequency of gene-specific methylation and low CNA percentages. This tumour cluster was significantly associated with stage IV (p = 0.04) and with poor prognosis in univariable analysis (p = 0.004). LINE-1 methylation levels in PanNETs were significantly lower than in normal samples (p < 0.01) and were approximately normally distributed. 12 tumours (21%) were highly hypomethylated, showing variable levels of CNA. Interestingly, only 5 PanNETs (9%) were observed to show simultaneously LINE-1 hypomethylation and high frequency of gene-specific methylation. LINE-1 hypomethylation was strongly correlated with advanced stage (p = 0.002) and with poor prognosis (p < 0.0001). In the multivariable analysis, low LINE-1 methylation status and methylation clusters were the only independent significant predictors of outcome (p = 0.034 and p = 0.029, respectively). Conclusion: The combination of global DNA hypomethylation and gene hypermethylation analyses may be useful to define distinct subsets of PanNETs. Both alterations are common in PanNETs and could be directly correlated with tumour progression.

Hierarchical clustering analysis of pathologic and molecular data identifies prognostically and biologically distinct groups of colorectal carcinomas

This work has evaluated the potential superiority of a morphomolecular classification based on the combination of clinicopathologic and molecular features of colorectal cancers. A cohort of 126 colorectal carcinomas was investigated by unsupervised hierarchical clustering analysis to combine 13 routinely assessed clinicopathologic features and all five molecular markers recently suggested by Jass’ classification to distinguish four molecular subtypes of sporadic colorectal carcinomas. Survival analysis was assessed by a Cox proportional hazards model. A clear separation into three prognostically significant groups was identified: cluster A and cluster C were associated with good prognosis and cluster B with poor prognosis (P=0.006). Clinicopathologic and molecular features of cluster A and cluster B tumors were strongly concordant with colorectal cancer profiles characterized by microsatellite instability or by chromosomal instability, respectively. The clinicopathologic features of cluster C tumors were suggestive of a less aggressive disease than cluster B tumors. Genetically, they appeared intermediate between cluster A and cluster B tumors, as they were mainly microsatellite stable tumors showing high levels of both MGMT methylation and loss of heterozygosity. Chromosomal instability was significantly lower in cluster C than in cluster B tumors. A more accurate tumor classification should combine the prognostic power of clinicopathologic parameters with molecular biomarkers that provide information regarding the natural history of the cancer. Hierarchical clustering seems to be a useful, promising and powerful tool for further translational studies and should lead us to define a diagnostic and prognostic signature for different carcinomas.

ALK Testing in Lung Adenocarcinoma Technical Aspects to Improve FISH Evaluation in Daily Practice

Introduction: Anaplastic lymphoma kinase (ALK) gene rearrangement characterizes a subgroup of patients with lung adenocarcinoma who may benefit from ALK inhibitors. Fluorescence in situ hybridization (FISH) with a break-apart/split-signal strategy is the gold standard to investigate ALK. The cutoff to define ALK positivity has been settled at 15% or greater. A subset of patients has ALK borderline status, showing 15% ± 5% positive cells. Several aspects, both biological and technical, might influence signals evaluation, making FISH interpretation a challenging task. To improve ALK evaluation, we classified the different FISH patterns on the basis of the type of the split signals, namely short, long, far away, and deleted. Methods: We investigated ALK gene status by FISH in 244 lung adenocarcinomas and in a series of ALK negative cell lines samples, collected in three Institutions. Results: ALK positive profile was found in 12% of patients; long, deleted, and far-away splits were the primary patterns observed. ALK borderline profile characterized 10% of samples; long and deleted splits were significantly more frequent in those borderline finally classified as ALK positive, whereas short split were mostly detected in those borderline patients finally classified as ALK negative (p = 3.4 × 10−3). In the ALK negative control series, short split was the predominant pattern. Concordance was observed among different operators and probes for both samples and controls. Conclusions: Difficulties in ALK FISH signal interpretation might be bypassed using this detailed scoring system, which is highly reproducible, helps clarify borderline samples (according to split type), and provides experimental evidence that 15% is a reasonable cutoff to overcome the assay-dependent background noise.

EGFR and KRAS Mutations in ALK-Positive Lung Adenocarcinomas: Biological and Clinical Effect.

INTRODUCTION In lung adenocarcinoma (ADC), anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements are mutually exclusive with epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. However, the existence of double-positive (DP) patients have been sporadically described. We identified DP cases in therapy-naive ALK-rearranged ADC and characterized the biology of these tumors to better understand the clinical response to tyrosine kinase inhibitors (TKIs). MATERIALS AND METHODS We selected 42 ALK-positive ADCs from a multicentric series of 301 cases of ADCs. A mutational analysis was performed using Sanger and/or pyrosequencing to address exons 18-21 of EGFR and codons 12-13 of the KRAS gene. In addition, the KRAS and EGFR copy number was investigated using fluorescent in situ hybridization. DP patients were treated with TKIs, and their response was evaluated according to the Response Evaluation Criteria in Solid Tumors criteria. RESULTS Eight of 42 ALK-positive ADCs (19%) demonstrated a concomitant mutation in the EGFR (3 cases) or KRAS (5 cases) genes and were classified as DP. All DP cases displayed copy number gains in the EGFR or KRAS gene because of polysomy or gene amplification. In the latter cases, a mutant allele-specific imbalance was observed. Four patients were treated with TKIs. The 2 EGFR-mutant DP patients demonstrated a better response to crizotinib compared with erlotinib. The 2 KRAS-mutant DP patients experienced opposite responses to crizotinib. CONCLUSION The incidence of DP ADC is not negligible. Patients with ALK/EGFR might benefit more from crizotinib compared with erlotinib administration, although the efficacy of TKIs in patients with ALK/KRAS remains unclear. An integrated targeted therapy should be considered for patients with DP ADC.

Molecular Features and Methylation Status in Early Onset (≤40 Years) Colorectal Cancer: A Population Based, Case-Control Study

Colorectal cancer is usually considered a disease of the elderly. However, a small fraction of patients develops colorectal cancer earlier. The aim of our study was to define the frequency of known hereditary colorectal syndromes and to characterise genetic and epigenetic features of early nonhereditary tumors. Thirty-three patients ≤40 years with diagnosis of colorectal cancer and 41 patients with disease at >60 years of age were investigated for MSI, Mismatch Repair proteins expression, KRAS and BRAF mutations, hypermethylation, and LINE-1 hypomethylation. Detection of germline mutations was performed in Mismatch Repair, APC and MUTYH genes. Early onset colorectal cancer showed a high incidence of hereditary forms (18%). KRAS mutations were detected in 36% of early nonhereditary tumors. Early onset colorectal cancer disclosed an average number of methylated genes significantly lower when compared to the controls (p = 0.02). Finally both of the two groups were highly methylated in ESR1, GATA5, and WT1 genes and were similar for LINE-1 hypomethylation. The genetic make-up of carcinomas differs from young to elderly patients. Early onset tumors showed more frequently a constitutional defective of Mismatch Repair System and a minor number of methylated genes. Hypermethylation of ESR1, GATA5, and WT1 genes suggests possible markers in the earlier diagnosis of colorectal tumorigenesis.

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells.

Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated ‘ex-vivo’ system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.