Gene expression profiles of gliomas in formalin-fixed paraffin-embedded material

Background:

We have recently demonstrated that expression profiling is a more accurate and objective method to classify gliomas than histology. Similar to most expression profiling studies, our experiments were performed using fresh frozen (FF) glioma samples whereas most archival samples are fixed in formalin and embedded in paraffin (FFPE). Identification of the same, expression-based intrinsic subtypes in FFPE-stored samples would enable validation of the prognostic value of these subtypes on these archival samples. In this study, we have therefore determined whether the intrinsic subtypes identified using FF material can be reproduced in FFPE-stored samples.

Methods:

We have performed expression profiling on 55 paired FF-FFPE glioma samples using HU133 plus 2.0 arrays (FF) and Exon 1.0 ST arrays (FFPE). The median time in paraffin of the FFPE samples was 14.1 years (range 6.6–26.4 years).

Results:

In general, the correlation between FF and FFPE expression in a single sample was poor. We then selected the most variable probe sets per gene (n=17 583), and of these, the 5000 most variable probe sets on FFPE expression profiles. This unsupervised selection resulted in a better concordance (R²=0.54) between expression of FF and FFPE samples. Importantly, this probe set selection resulted in a correct assignment of 87% of FFPE samples into one of seven intrinsic subtypes identified using FF samples. Assignment to the same molecular cluster as the paired FF tissue was not correlated to time in paraffin.

Conclusion:

We are the first to examine a large cohort of paired FF and FFPE samples. We show that expression data from FFPE material can be used to assign samples to intrinsic molecular subtypes identified using FF material. This assignment allows the use of archival material, including material derived from large-randomised clinical trials, to determine the predictive and/or prognostic value of ‘intrinsic glioma subtypes'' on Exon arrays. This would enable clinicians to provide patients with an objective and accurate diagnosis and prognosis, and a personalised treatment strategy.     

Droplet digital PCR measurement of HER2 in patients with gastric cancer

Background:

Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator.

Methods:

We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer.

Results:

The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients.

Conclusions:

Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.     

Independent and functional validation of a multi-tumour-type proliferation signature

Background:

Previously we demonstrated that an mRNA signature reflecting cellular proliferation had strong prognostic value. As clinical applicability of signatures can be controversial, we sought to improve our marker''s clinical utility by validating its biological relevance, reproducibility in independent data sets and applicability using an independent technique.

Methods:

To facilitate signature evaluation with quantitative PCR (qPCR) a novel computational procedure was used to reduce the number of signature genes without significant information loss. These genes were validated in different human cancer cell lines upon serum starvation and in a 168 xenografts panel. Analyses were then extended to breast cancer and non-small-cell lung cancer (NSCLC) patient cohorts.

Results:

Expression of the qPCR-based signature was dramatically decreased under starvation conditions and inversely correlated with tumour volume doubling time in xenografts. The signature validated in breast cancer (hazard ratio (HR)=1.63, P<0.001, n=1820) and NSCLC adenocarcinoma (HR=1.64, P<0.001, n=639) microarray data sets. Lastly, qPCR in a node-negative, non-adjuvantly treated breast cancer cohort (n=129) showed that patients assigned to the high-proliferation group had worse disease-free survival (HR=2.25, P<0.05).

Conclusion:

We have developed and validated a qPCR-based proliferation signature. This test might be used in the clinic to select (early-stage) patients for specific treatments that target proliferation.     

Gene expression profiling reveals GC and CEACAM1 as new tools in the diagnosis of lung carcinoids

Background:

Classification of lung carcinoids into typical and atypical is a diagnostic challenge since no immunohistochemical tools are available to support pathologists in distinguishing between the two subtypes. A differential diagnosis is essential for clinicians to correctly discuss therapy, prognosis and follow-up with patients. Indeed, the distinction between the two typical and atypical subtypes on biopsies/cytological specimens is still unfeasible and sometimes limited also after radical surgeries. By comparing the gene expression profile of typical (TC) and atypical carcinoids (AC), we intended to find genes specifically expressed in one of the two subtypes that could be used as diagnostic markers.

Methods:

Expression profiling, with Affymetrix arrays, was performed on six typical and seven atypical samples. Data were validated on an independent cohort of 29 tumours, by means of quantitative PCR and immunohistochemistry (IHC).

Results:

High-throughput gene expression profiling was successfully used to identify a gene signature specific for atypical lung carcinoids. Among the 273 upregulated genes in the atypical vs typical subtype, GC (vitamin D-binding protein) and CEACAM1 (carcinoembryonic antigen family member) emerged as potent diagnostic markers. Quantitative PCR and IHC on a validation set of 17 ACs and 12 TCs confirmed their reproducibility and feasibility.

Conclusions:

GC and CEACAM1 can distinguish between TC and AC, defining an IHC assay potentially useful for routine cytological and histochemical diagnostic procedures. The high sensitivity and reproducibility of this new diagnostic algorithm strongly support a further validation on a wider sample size.     

A microRNA-based prediction algorithm for diagnosis of non-small lung cell carcinoma in minimal biopsy material

Background:

Diagnosis is jeopardised when limited biopsy material is available or histological quality compromised. Here we developed and validated a prediction algorithm based on microRNA (miRNA) expression that can assist clinical diagnosis of lung cancer in minimal biopsy material to improve clinical management.

Methods:

Discovery utilised Taqman Low Density Arrays (754 miRNAs) in 20 non-small cell lung cancer (NSCLC) tumour/normal pairs. In an independent set of 40 NSCLC patients, 28 miRNA targets were validated using qRT–PCR. A prediction algorithm based on eight miRNA targets was validated blindly in a third independent set of 47 NSCLC patients. The panel was also tested in formalin-fixed paraffin-embedded (FFPE) specimens from 20 NSCLC patients. The genomic methylation status of highly deregulated miRNAs was investigated by pyrosequencing.

Results:

In the final, frozen validation set the panel had very high sensitivity (97.5%), specificity (96.3%) and ROC-AUC (0.99, P=10⁻¹⁵). The panel provided 100% sensitivity and 95% specificity in FFPE tissue (ROC-AUC=0.97 (P=10⁻⁶)). DNA methylation abnormalities contribute little to the deregulation of the miRNAs tested.

Conclusion:

The developed prediction algorithm is a valuable potential biomarker for assisting lung cancer diagnosis in minimal biopsy material. A prospective validation is required to measure the enhancement of diagnostic accuracy of our current clinical practice.     

Highly sensitive molecular diagnosis of prostate cancer using surplus material washed off from biopsy needles

Introduction:

Currently, final diagnosis of prostate cancer (PCa) is based on histopathological analysis of needle biopsies, but this process often bears uncertainties due to small sample size, tumour focality and pathologist''s subjective assessment.

Methods:

Prostate cancer diagnostic signatures were generated by applying linear discriminant analysis to microarray and real-time RT–PCR (qRT–PCR) data from normal and tumoural prostate tissue samples. Additionally, after removal of biopsy tissues, material washed off from transrectal biopsy needles was used for molecular profiling and discriminant analysis.

Results:

Linear discriminant analysis applied to microarray data for a set of 318 genes differentially expressed between non-tumoural and tumoural prostate samples produced 26 gene signatures, which classified the 84 samples used with 100% accuracy. To identify signatures potentially useful for the diagnosis of prostate biopsies, surplus material washed off from routine biopsy needles from 53 patients was used to generate qRT–PCR data for a subset of 11 genes. This analysis identified a six-gene signature that correctly assigned the biopsies as benign or tumoural in 92.6% of the cases, with 88.8% sensitivity and 96.1% specificity.

Conclusion:

Surplus material from prostate needle biopsies can be used for minimal-size gene signature analysis for sensitive and accurate discrimination between non-tumoural and tumoural prostates, without interference with current diagnostic procedures. This approach could be a useful adjunct to current procedures in PCa diagnosis.     

ASC amino-acid transporter 2 (ASCT2) as a novel prognostic marker in non-small cell lung cancer

Background:

ASC amino-acid transporter 2 (ASCT2) is a major glutamine transporter that has an essential role in tumour growth and progression. Although ASCT2 is highly expressed in various cancer cells, the clinicopathological significance of its expression in non-small cell lung cancer (NSCLC) remains unclear.

Methods:

One hundred and four patients with surgically resected NSCLC were evaluated as one institutional cohort. Tumour sections were stained by immunohistochemistry (IHC) for ASCT2, Ki-67, phospho-mTOR (mammalian target of rapamycin), and CD34 to assess the microvessel density. Two hundred and four patients with NSCLC were also validated by IHC from an independent cohort.

Results:

ASC amino-acid transporter 2 was expressed in 66% of patients, and was closely correlated with disease stage, lymphatic permeation, vascular invasion, CD98, cell proliferation, angiogenesis, and mTOR phosphorylation, particularly in patients with adenocarcinoma (AC). Moreover, two independent cohorts confirmed that ASCT2 was an independent marker for poor outcome in AC patients.

Conclusions:

ASC amino-acid transporter 2 expression has a crucial role in the metastasis of pulmonary AC, and is a potential molecular marker for predicting poor prognosis after surgery.     

Downregulation of MTSS1 expression is an independent prognosticator in squamous cell carcinoma of the lung

Background:

The metastasis suppressor 1 (MTSS1) is a newly discovered protein putatively involved in tumour progression and metastasis.

Material and Methods:

Immunohistochemical expression of MTSS1 was analysed in 264 non-small-cell lung carcinomas (NSCLCs).

Results:

The metastasis suppressor 1 was significantly overexpressed in NSCLC compared with normal lung (P=0.01). Within NSCLC, MTSS1 expression was inversely correlated with pT-stage (P=0.019) and histological grading (P<0.001). NSCLC with MTSS1 downregulation (<20%) showed a significantly worse outcome (P=0.007). This proved to be an independent prognostic factor in squamous cell carcinomas (SCCs; P=0.041), especially in early cancer stages (P=0.006).

Conclusion:

The metastasis suppressor 1 downregulation could thus serve as a stratifying marker for adjuvant therapy in early-stage SCC of the lung.     

Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene

Background:

There is a need to develop robust and clinically applicable gene expression signatures. Hypoxia is a key factor promoting solid tumour progression and resistance to therapy; a hypoxia signature has the potential to be not only prognostic but also to predict benefit from particular interventions.

Methods:

An approach for deriving signatures that combine knowledge of gene function and analysis of in vivo co-expression patterns was used to define a common hypoxia signature from three head and neck and five breast cancer studies. Previously validated hypoxia-regulated genes (seeds) were used to generate hypoxia co-expression cancer networks.

Results:

A common hypoxia signature, or metagene, was derived by selecting genes that were consistently co-expressed with the hypoxia seeds in multiple cancers. This was highly enriched for hypoxia-regulated pathways, and prognostic in multivariate analyses. Genes with the highest connectivity were also the most prognostic, and a reduced metagene consisting of a small number of top-ranked genes, including VEGFA, SLC2A1 and PGAM1, outperformed both a larger signature and reported signatures in independent data sets of head and neck, breast and lung cancers.

Conclusion:

Combined knowledge of multiple genes'' function from in vitro experiments together with meta-analysis of multiple cancers can deliver compact and robust signatures suitable for clinical application.     

High expression of kinesin light chain-2, a novel target of miR-125b,is associated with poor clinical outcome of elderly non-small-cell lung cancer patients

Background:

MiR-125b has critical role in non-small-cell lung cancer (NSCLC) cell migration, and its target genes have not been elucidated. Kinesin-1 light chain (KLC)-2 was predicted as one of miR-125b''s targets by bioinformatics analysis. This study is to identify the function of KLC2 and its interaction with miR-125b in NSCLC.

Methods:

Kinesin-1 light chain-2 protein expression and its clinical relevance were analysed in 140 matched NSCLC and adjacent non-neoplastic lung tissues. Both KLC2 gain- and loss-of-function analyses were performed in NSCLC cell lines by transient transfection. The direct interaction between KLC2 and miR-125b was confirmed by a luciferase reporter assay and a transient co-transfection assay as well as an analysis of eight matched clinical samples.

Results:

KLC2 protein was upregulated in NSCLC cell lines and tissues, and was an independent predictor of poor prognosis for elderly NSCLC patients. Kinesin-1 light chain-2 remarkably enhanced the invasive and migratory ability of NSCLC cells. MiR-125b inhibited KLC2 3′-untranslated region luciferase activity and protein expression, and inversely correlated with KLC2 expression in clinical samples. Kinesin-1 light chain-2 almost completely reversed miR-125b-induced inhibition on migration and invasion.

Conclusions:

Kinesin-1 light chain-2 protein overexpression predicts poor survival in elderly NSCLC patients. Kinesin-1 light chain-2 acts as a proto-oncogene and a functional target of miR-125b in NSCLC cells.     

Prognostic role of FGFR1 amplification in early-stage non-small cell lung cancer

Background:

Recently, fibroblast growth factor receptor 1 (FGFR1) was discovered in squamous cell carcinomas (SCC) of the lung with FGFR1 amplification described as a promising predictive marker for anti-FGFR inhibitor treatment. Only few data are available regarding prevalence, prognostic significance and clinico-pathological characteristics of FGFR1-amplified and early-stage non-small cell lung carcinomas (NSCLC). We therefore investigated the FGFR1 gene status in a large number of well-characterised early-stage NSCLC.

Methods:

FGFR1 gene status was evaluated using a commercially available fluorescent in situ hybridisation (FISH) probe on a tissue microarray (TMA). This TMA harbours 329 resected, formalin-fixed and paraffin-embedded, nodal-negative NSCLC with a UICC stage I–II. The FISH results were correlated with clinico-pathological features and overall survival (OS).

Results:

The prevalence of an FGFR1 amplification was 12.5% (41/329) and was significantly (P<0.0001) higher in squamous cell carcinoma (SCC) (20.7%) than in adenocarcinoma (2.2%) and large cell carcinoma (13%). Multivariate analysis revealed significantly (P=0.0367) worse 5-year OS in patients with an FGFR1-amplified NSCLC.

Conclusions:

FGFR1 amplification is common in early-stage SCC of the lung and is an independent and adverse prognostic marker. Its potential role as a predictive marker for targeted therapies or adjuvant treatment needs further investigation.     

TGFBI expression is an independent predictor of survival in adjuvant-treated lung squamous cell carcinoma patients

Background:

Transforming growth factor β-induced protein (TGFBI) is a secreted protein that mediates cell anchoring to the extracellular matrix. This protein is downregulated in lung cancer, and when overexpressed, contributes to apoptotic cell death. Using a small series of stage IV non-small cell lung cancer (NSCLC) patients, we previously suggested the usefulness of TGFBI as a prognostic and predictive factor in chemotherapy-treated late-stage NSCLC. In order to validate and extend these results, we broaden the analysis and studied TGFBI expression in a large series of samples obtained from stage I–IV NSCLC patients.

Methods:

TGFBI expression was assessed by immunohistochemistry in 364 completely resected primary NSCLC samples: 242 adenocarcinomas (ADCs) and 122 squamous cell carcinomas (SCCs). Kaplan–Meier curves, log-rank tests and the Cox proportional hazards model were used to analyse the association between TGFBI expression and survival.

Results:

High TGFBI levels were associated with longer overall survival (OS, P<0.001) and progression-free survival (PFS, P<0.001) in SCC patients who received adjuvant platinium-based chemotherapy. Moreover, multivariate analysis demonstrated that high TGFBI expression is an independent predictor of better survival in patients (OS: P=0.030 and PFS: P=0.026).

Conclusions:

TGFBI may be useful for the identification of a subset of NSCLC who may benefit from adjuvant therapy.     

Statin use and risk of nonmelanoma skin cancer: a nationwide study in Denmark

Background:

Evidence is conflicting regarding statin use and risk of basal cell (BCC) and squamous cell skin cancer (SCC).

Methods:

Using Danish nationwide registries, we identified all patients with incident BCC/SCC during 2005–2009 and matched them to population controls. We computed odds ratios (ORs) for BCC and SCC associated with statin use.

Results:

We identified 38 484 cases of BCC and 3724 cases of SCC. Statin ever use was associated with ORs of 1.09 (CI: 1.06–1.13) for BCC and 1.01 (CI: 0.91–1.11) for SCC.

Conclusions:

Statin use was not associated with risk of SCC. Residual confounding plausibly explains the marginally increased risk of BCC.     

Patients with usual vulvar intraepithelial neoplasia-related vulvar cancer have an increased risk of cervical abnormalities

Background:

Vulvar squamous cell carcinoma (SCC) originates the following two pathways, related to differentiated (d) vulvar intraepithelial neoplasia (VIN) or to human papillomavirus (HPV)-related usual (u) VIN. Multicentric HPV infections (cervix, vagina and vulva) are common. We hypothesise that patients with a uVIN-related vulvar SCC more often have cervical high-grade squamous intraepithelial lesions (HSILs) compared with women with dVIN-related vulvar SCC.

Methods:

All vulvar SCCs (201) were classified to be dVIN- (n=164) or uVIN related (n=37). Data with regard to the smear history and cervical histology were retrieved from PALGA, the nationwide Netherlands database of histo- and cytopathology. For HSIL cervical smears of which histology was taken, HPV DNA analysis on both the vulvar and cervical specimens was performed.

Results:

At least one smear was available in 145 (72%) of the 201 patients. Patients with a uVIN-related vulvar SCC more often had an HSIL compared with patients with a dVIN-related SCC (35 vs 2%, P<0.001). A total of 10 of the 13 HSILs were histologically assessed and identical HPV types were found in the vulva and cervix.

Conclusion:

These data emphasise the necessity to differentiate between dVIN- and uVIN-related vulvar tumours and to examine the entire lower female ano-genital tract once an uVIN-related lesion is found.