Elodie Long-Mira

Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients reveal major discordances. A potential issue for anti-PD-L1 therapeutic strategies

BACKGROUND High expression of programmed death ligand-1 (PD-L1) on tumor cells (TC) and/or on tumor-infiltrating immune cells (IC) is associated with a high response rate in patients with advanced nonsmall-cell lung cancer (NSCLC) treated with PD-L1 inhibitors. The use of a PD-L1 immunohistochemical (IHC) test in determining the responsiveness to immunotherapy has raised the question of the reliability and reproducibility of its evaluation in lung biopsies compared with corresponding resected surgical specimens. PATIENTS AND METHODS PD-L1 expression in TC and IC was assessed in 160 patients with operable NSCLC on both whole surgical tissue sections and matched lung biopsies, by using a highly sensitive SP142 IHC assay. The specimens were scored as TC 0-3 and IC 0-3 based on increasing PD-L1 expression. RESULTS PD-L1 expression was frequently discordant between surgical resected and matched biopsy specimens (the overall discordance rate = 48%; 95% confidence interval 4.64-13.24) and κ value was equal to 0.218 (poor agreement). In all cases, the biopsy specimens underestimated the PD-L1 status observed on the whole tissue sample. PD-L1-positive IC tumors were more common than PD-L1-positive TC tumors on resected specimens. The discrepancies were mainly related to the lack of a PD-L1-positive IC component in matched biopsies. CONCLUSIONS Our results indicate relatively poor association of the PD-L1 expression in TC and IC between lung biopsies and corresponding resected tumors. Although these results need to be further validated in larger cohorts, they indicate that the daily routine evaluation of the PD-L1 expression in diagnostic biopsies can be misleading in defining the sensitivity to treatment with PD-L1 targeted therapy.

Sentinel circulating tumor cells allow early diagnosis of lung cancer in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer. Migration of circulating tumor cells (CTCs) into the blood stream is an early event that occurs during carcinogenesis. We aimed to examine the presence of CTCs in complement to CT-scan in COPD patients without clinically detectable lung cancer as a first step to identify a new marker for early lung cancer diagnosis. The presence of CTCs was examined by an ISET filtration-enrichment technique, for 245 subjects without cancer, including 168 (68.6%) COPD patients, and 77 subjects without COPD (31.4%), including 42 control smokers and 35 non-smoking healthy individuals. CTCs were identified by cytomorphological analysis and characterized by studying their expression of epithelial and mesenchymal markers. COPD patients were monitored annually by low-dose spiral CT. CTCs were detected in 3% of COPD patients (5 out of 168 patients). The annual surveillance of the CTC-positive COPD patients by CT-scan screening detected lung nodules 1 to 4 years after CTC detection, leading to prompt surgical resection and histopathological diagnosis of early-stage lung cancer. Follow-up of the 5 patients by CT-scan and ISET 12 month after surgery showed no tumor recurrence. CTCs detected in COPD patients had a heterogeneous expression of epithelial and mesenchymal markers, which was similar to the corresponding lung tumor phenotype. No CTCs were detected in control smoking and non-smoking healthy individuals. CTCs can be detected in patients with COPD without clinically detectable lung cancer. Monitoring “sentinel” CTC-positive COPD patients may allow early diagnosis of lung cancer.

Diagnostic value of immunohistochemistry for the detection of the BRAF V600E mutation in papillary thyroid carcinoma: Comparative analysis with three DNA-based assays

BACKGROUND The aim of this study was to compare the detection of BRAF(V600E) by immunohistochemistry (IHC) using a mutation-specific antibody with molecular biology methods for evaluation of papillary thyroid carcinoma (PTC) patients. PATIENTS AND METHODS This study concerned 198 consecutive conventional PTC patients, of which the majority were women (133/198; 67%), with a mean age of 56 years (range 19-79 years). BRAF mutation analysis was performed using DNA-based (direct sequencing, pyrosequencing, and SNaPshot) and IHC (VE1 antibody) methods. The sensitivity and specificity of IHC for BRAF(V600E) was compared with the molecular biology data. RESULTS A BRAF mutational result was obtained in 194 cases. A BRAF(V600E) mutation was detected in 153/194 (79%) cases of PTC when using at least one molecular method, and in 151/194 (78%) cases with IHC. No false positive results were noted using IHC to detect the BRAF(V600E) mutation. Besides this mutation, other rare BRAF mutations (BRAF(V600K) and BRAF(K601E)), used as negative controls, were consistently negative with IHC. The sensitivity and specificity of IHC for the detection of this mutation were 98.7% and 100% respectively. The IHC test demonstrated excellent performance at a level equivalent to two DNA-based counterparts (pyrosequencing and SNaPshot). Failure to achieve a result was more frequent with the direct sequencing method than with the three other methods. CONCLUSION IHC using the VE1 antibody is a specific and sensitive method for the detection of the BRAF(V600E) mutation in PTC. IHC may be an alternative to molecular biology approaches for the routine detection of this mutation in PTC patients.

Usefulness of Immunocytochemistry for the Detection of the BRAFV600E Mutation in Circulating Tumor Cells from Metastatic Melanoma Patients

Abbreviations: CMCs, circulating melanoma tumor cells; CTCs, circulating tumor cells; ICC, immunocytochemistry; IHC, immunohistochemistry; ISET, isolation by size of epithelial tumor cells

Setting up a wide panel of patient-derived tumor xenografts of non-small cell lung cancer by improving the preanalytical steps.

With the ongoing need to improve therapy for non–small cell lung cancer (NSCLC) there has been increasing interest in developing reliable preclinical models to test novel therapeutics. Patient‐derived tumor xenografts (PDX) are considered to be interesting candidates. However, the establishment of such model systems requires highly specialized research facilities and introduces logistic challenges. We aimed to establish an extensive well‐characterized panel of NSCLC xenograft models in the context of a long‐distance research network after careful control of the preanalytical steps. One hundred fresh surgically resected NSCLC specimens were shipped in survival medium at room temperature from a hospital‐integrated biobank to animal facilities. Within 24 h post‐surgery, tumor fragments were subcutaneously xenografted into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical, aCGH and next‐generation sequencing approaches. For this model system, the tumor take rate was 35%, with higher rates for squamous carcinoma (60%) than for adenocarcinoma (13%). Patients for whom PDX tumors were obtained had a significantly shorter disease‐free survival (DFS) compared to patients for whom no PDX tumors (P = 0.039) were obtained. We established a large panel of PDX NSCLC models with a high frequency of mutations (29%) in EGFR, KRAS, NRAS, MEK1, BRAF, PTEN, and PI3KCA genes and with gene amplification (20%) of c‐MET and FGFR1. This new patient‐derived NSCLC xenograft collection, established regardless of the considerable time required and the distance between the clinic and the animal facilities, recapitulated the histopathology and molecular diversity of NSCLC and provides stable and reliable preclinical models for human lung cancer research.

Immunohistochemistry as a potential tool for routine detection of the NRAS Q61R mutation in patients with metastatic melanoma.

BACKGROUND It can be useful to assess the NRAS mutation status in patients with metastatic melanoma because NRAS-activating mutations confer resistance to RAF inhibitors, and NRAS-mutated patients appear to be sensitive to mitogen-activated protein kinase (MEK) inhibitors. OBJECTIVE We aimed to assess the diagnostic accuracy of an immunohistochemistry (IHC) approach using a novel anti-NRAS (Q61R) monoclonal antibody on formalin-fixed paraffin-embedded tissue samples from patients with metastatic melanoma. METHODS We conducted a retrospective multicenter cohort study on 170 patients with metastatic melanoma. The automated IHC assay was performed using the SP174 clone, and compared with results of the molecular testing. RESULTS Evaluation of a test cohort with knowledge of the mutation status established a specific IHC pattern for the mutation. In the independent blinded analysis of the remaining cases, the anti-NRAS (Q61R) antibody accurately identified all NRAS Q61R-mutated tumors, and demonstrated 100% sensitivity and specificity. LIMITATIONS Limitations include retrospective design and lack of multicenter interobserver reproducibility. CONCLUSION The NRAS (Q61R) IHC assay is reliable and specific for the evaluation of the Q61R mutation status in metastatic melanoma and may be an alternative to molecular biology in evaluation of metastatic melanoma in routine practice.

Sense and nonsense in the process of accreditation of a pathology laboratory

The aim of accreditation of a pathology laboratory is to control and optimize, in a permanent manner, good professional practice in clinical and molecular pathology, as defined by internationally established standards. Accreditation of a pathology laboratory is a key element in fine in increasing recognition of the quality of the analyses performed by a laboratory and in improving the care it provides to patients. One of the accreditation standards applied to clinical chemistry and pathology laboratories in the European Union is the ISO 15189 norm. Continued functioning of a pathology laboratory might in time be determined by whether or not it has succeeded the accreditation process. Necessary requirements for accreditation, according to the ISO 15189 norm, include an operational quality management system and continuous control of the methods used for diagnostic purposes. Given these goals, one would expect that all pathologists would agree on the positive effects of accreditation. Yet, some of the requirements stipulated in the accreditation standards, coming from the bodies that accredit pathology laboratories, and certain normative issues are perceived as arduous and sometimes not adapted to or even useless in daily pathology practice. The aim of this review is to elaborate why it is necessary to obtain accreditation but also why certain requirements for accreditation might be experienced as inappropriate.

Expression of MET in circulating tumor cells correlates with expression in tumor tissue from advanced-stage lung cancer patients

Given the difficulty in obtaining adequate tissue in NSCLC, we investigated the utility of circulating tumor cells (CTCs) for MET status assessment in NSCLC patients. We used two platforms for CTC capture, and assessed MET expression in CTCs and matched-bronchial biopsies in patients with advanced-stage III/IV lung adenocarcinoma. Baseline peripheral blood was collected from 256 advanced-stage III/IV NSCLC patients from Genentech clinical trials, and from 106 patients with advanced-stage III/IV lung adenocarcinoma treated at the Department of Pneumology, Pasteur Hospital, Nice. CTCs were enriched using CellSearch (Genentech), or ISET technologies (Pasteur Hospital). MET expression was evaluated by immunofluorescence on CellSearch, and by immunocytochemistry on ISET-enriched CTCs and on matched FFPE tissue sections (Pasteur Hospital). CTCs were detected in 83 of 256 (32%) patients evaluated on CellSearch, with 30 samples (12%) exhibiting ≥ 5 CTCs/7.5 ml blood. On ISET, CTC were observed in 80 of 106 patients (75%), and 79 patients (75%) exhibited ≥ 5 CTCs/4 ml blood. MET expression on ISET CTCs was positive in 72% of cases, and the MET expression on matched-patient tissue was positive in 65% patients using the Onartuzumab IHC scoring algorithm (93% concordance). Quantification of MET expression using H-scores showed strong correlation between MET expression in tissue and CTCs (Spearman correlation, 0.93). MET status in CTCs isolated on ISET filters from blood samples of advanced-stage NSCLC patients correlated strongly with MET status in tumor tissue, illustrating the potential for using CTCs as a non-invasive, real-time biopsy to determine MET status of patients entering clinical trials.

BRAFV600E mutation analysis by immunohistochemistry in patients with thoracic metastases from colorectal cancer

Summary The BRAFV600E mutation confers worse prognosis to metastatic colorectal cancer (mCRC) patients. In addition, this mutation has a negative predictive value for response to treatment with monoclonal antibodies against EGFR in patients with KRAS wild-type (wt) mCRC. The utility of immunohistochemistry (IHC) as an alternative approach for detection of BRAFV600E in the thoracic metastases of sporadic mCRC patients has not been evaluated until now. The purpose of this study was to compare BRAFV600E IHC staining with molecular biology methods and to define the diagnostic value of the VE1 antibody for the detection of BRAFV600E in this population. BRAF mutations were analysed by two DNA sequencing methods (pyrosequencing and Sanger sequencing) in a Caucasian population of 310 sporadic mCRC with thoracic metastases patients expressing KRAS wt. Detection of the BRAFV600E mutation was performed in the corresponding tumours by IHC using the VE1 antibody and compared to results of the DNA-based assays. Thirty-nine out of 310 (13%) of tumours harboured a BRAF mutation, which corresponded to either a BRAFV600E in 34 of 310 (11%) cases or a non-BRAFV600E mutation in 5 of 310 (2%) cases. IHC with VE1 was strongly positive in 32 of 34 (88%) BRAFV600E mutated tumours and negative in non-BRAFV600E mutated tumours. IHC using the VE1 clone is a specific and sensitive method for the detection of BRAFV600E and may be either a complementary or an alternative method to molecular testing in mCRC patients.

Any Place for Immunohistochemistry within the Predictive Biomarkers of Treatment in Lung Cancer Patients

The identification of certain genomic alterations (EGFR, ALK, ROS1, BRAF) or immunological markers (PD-L1) in tissues or cells has led to targeted treatment for patients presenting with late stage or metastatic lung cancer. These biomarkers can be detected by immunohistochemistry (IHC) and/or by molecular biology (MB) techniques. These approaches are often complementary but depending on, the quantity and quality of the biological material, the urgency to get the results, the access to technological platforms, the financial resources and the expertise of the team, the choice of the approach can be questioned. The possibility of detecting simultaneously several molecular targets, and of analyzing the degree of tumor mutation burden and of the micro-satellite instability, as well as the recent requirement to quantify the expression of PD-L1 in tumor cells, has led to case by case development of algorithms and international recommendations, which depend on the quality and quantity of biological samples. This review will highlight the different predictive biomarkers detected by IHC for treatment of lung cancer as well as the present advantages and limitations of this approach. A number of perspectives will be considered.