A Canadian guideline on the use of next-generation sequencing in oncology.

Rapid advancements in next-generation sequencing (ngs) technology have created an unprecedented opportunity to decipher the molecular profile of tumours to more effectively prevent, diagnose, and treat cancer. Oncologists now have the option to order molecular tests that can guide treatment decisions. However, to date, most oncologists have received limited training in genomics, and they are now faced with the challenge of understanding how such tests and their interpretation align with patient management. Guidance on how to effectively use ngs technology is therefore needed to aid oncologists in applying the results of genomic tests. The Canadian guideline presented here describes best practices and unmet needs related to ngs-based testing for somatic variants in oncology, including clinical application, assay and sample selection, bioinformatics and interpretation of reports performed by laboratories, patient communication, and clinical trials.

The metastatic site does not influence PD-L1 expression in advanced non-small cell lung carcinoma.

INTRODUCTION: PD-L1 expression by immunohistochemistry (IHC) testing with Tumor Proportion Score (TPS) ≥50% and ≥1% is required to be eligible for first- and second-line Pembrolizumab treatment for metastatic non-small cell lung cancer (NSCLC) respectively. Stage IV NSCLC often presents with metastasis to multiple distant sites which are easily accessible for biopsy. Knowing whether PD-L1 IHC TPS can be indifferently measured from different metastatic site is therefore an important clinical question. In this study, we evaluated PD-L1 expression in NSCLC from varied distant metastatic sites. METHODS: A total of 580 NSCLC specimens of distant metastases were retrieved for study, including 35 paired samples from two different metastatic sites. The metastatic sites included brain, bone, remote lymph nodes, serous membranes (pleura, pericardium and peritoneum), extra-thoracic solid organs and skin/soft tissues. The samples were cytology cell blocks, small biopsies or surgical resections. IHC was performed using Dako PD-L1 IHC 22C3 pharmDx. A total of 100 viable tumor cells was required for adequacy. TPS ≥ 50% and 1-49% were defined as high and low PD-L1 expression respectively. RESULTS: PD-L1 TPS scores were not significantly different across a range of distant metastatic sites nor between metastases in paired samples. CONCLUSION: Our results suggest that the PD-L1 TPS scoring is similar across different metastatic sites and any site biopsied will yield necessary information for guiding clinical management.

Evidence-based best practices for EGFR T790M testing in lung cancer in Canada.

Epidermal growth factor receptor (egfr) tyrosine kinase inhibitors (tkis) are recommended as first-line systemic therapy for patients with non-small-cell lung cancer (nsclc) having mutations in the EGFR gene. Resistance to tkis eventually occurs in all nsclc patients treated with such drugs. In patients with resistance to tkis caused by the EGFR T790M mutation, the third-generation tki osimertinib is now the standard of care. For optimal patient management, accurate EGFR T790M testing is required. A multidisciplinary working group of pathologists, laboratory medicine specialists, medical oncologists, a respirologist, and a thoracic radiologist from across Canada was convened to discuss best practices for EGFR T790M mutation testing in Canada. The group made recommendations in the areas of the testing algorithm and the pre-analytic, analytic, and post-analytic aspects of clinical testing for both tissue testing and liquid biopsy circulating tumour dna testing. The recommendations aim to improve EGFR T790M testing in Canada and to thereby improve patient care.

How iMALDI can improve clinical diagnostics.

Protein mass spectrometry (MS) is an indispensable tool to detect molecular signatures that can be associated with cellular dysregulation and disease. Despite its huge success in the life sciences, where it has led to novel insights into disease mechanisms and the identification of potential protein biomarkers, protein MS is rarely used for clinical protein assays. While conventional matrix-assisted laser desorption/ionization (MALDI) MS is not compatible with complex samples, liquid chromatography-MS (LC-MS)-based assays may be too complex and may lack the robustness and ease of automation required for routine use in the clinic. Therefore, clinical protein assays are dominated by immunohistochemistry and immunoassays which, however, often lack standardization and fully depend on antibody specificity. Immuno-MALDI (iMALDI) MS may overcome these hurdles by utilizing anti-peptide antibodies for the specific enrichment of targeted analytes and on-target detection of the captured analytes, thus combining the unique properties of MS for the unambiguous detection and quantitation of analytes with a workflow that can be fully automated. Here we discuss the requirements for clinical protein assays, the pitfalls of existing methods, how iMALDI has been successfully used to quantify endogenous peptides and proteins from clinical samples, as well as its potential as a powerful tool for companion diagnostics in the light of precision medicine.

Cytology cell blocks are suitable for immunohistochemical testing for PD-L1 in lung cancer.

Background: PD-L1 immunohistochemistry (IHC) testing is usually carried out on tissue blocks from core needle biopsy or surgical resections. In this study, we assessed the feasibility of using cytology cell blocks for PD-L1 IHC assay. Methods: A total of 1419 consecutive cases of non-small-cell lung cancer (NSCLC), including 371 cytology cell blocks, 809 small biopsies, and 239 surgical specimens, were included in the study. The cytology cell blocks were prepared with formalin only, methanol/alcohol only or both. PD-L1 expression was examined by staining with Dako PD-L1 IHC 22C3 pharmDx kit. A Tumor Proportion Score (TPS) was categorized as <1%, 1%-49% and ≥50% tumor cells. A total of 100 viable tumor cells were required for adequacy. Results: Of the cytology cell blocks, 92% of the specimens had an adequate number of tumor cells, not significantly different from small biopsies. The rate of TPS ≥50% differed between sample types and was observed in 42% of cytology cell blocks versus 36% of small biopsies (P = 0.04), and 29% of surgical resections (P = 0.001). The fixative methods did not affect the immunostaining, with overall PD-L1 high expression (TPS ≥50%) rates of 42% in formalin-fixed specimens versus 40% in specimens with combined fixation by methanol/alcohol and formalin (NS). The PD-L1 high expression rate was not associated with EGFR, ALK or KRAS molecular alterations. Higher stage (IV) was associated with higher PD-L1 TPS (P= 0.001). Conclusion: Our results show that when the TPS ≥50% is used as the end point, PD-L1 IHC performs well with cytology cell blocks. Cell blocks should be considered as a valuable resource for PD-L1 testing in advanced NSCLC. The clinical significance of higher PD-L1 IHC scores in cytology specimens needs to be evaluated prospectively.

[Prostate cancer microenvironment: Its structure, functions and therapeutic applications]. / Microenvironnement du cancer de la prostate : structure, fonctions et applications thérapeutiques.

INTRODUCTION: In the field of prostate cancer there is a growing tendency for more and more studies to emphasise the predominant role of the zone situated between the tumour and the host: the tumour microenvironment. The aim of this article is to describe the structure and the functions of the prostate cancer microenvironment as well as the principal treatments that are being applied to it. MATERIAL AND METHODS: PubMed and ScienceDirect databases have been interrogated using the association of keywords "tumour microenvironment" and "neoplasm therapy" along with "microenvironnement tumoral" and "traitements". Of the 593 articles initially found, 50 were finally included. RESULTS: The tumour microenvironment principally includes host elements that are diverted from their primary functions and encourage the development of the tumour. In it we find immunity cells, support tissue as well as vascular and lymphatic neovascularization. Highlighting the major role played by this microenvironment has led to the development of specific treatments, notably antiangiogenic therapy and immunotherapy. CONCLUSION: The tumour microenvironment, the tumour and the host influence themselves mutually and create a variable situation over time. Improvement of the knowledge of the prostate cancer microenvironment gradually enables us to pass from an approach centred on the tumour to a broader approach to the whole tumoral ecosystem. This enabled the emergence of new treatments whose place in the therapeutic arsenal still need to be found.

The validity of circulating microRNAs in oncology: five years of challenges and contradictions.

MicroRNAs (miRNAs) in circulation have received an increasing amount of interest as potential minimal invasive diagnostic tools in oncology. Several diagnostic, prognostic and predictive signatures have been proposed for a variety of cancers at different stages of disease, but these have not been subjected to a critical review regarding their validity: reproducible identification in comparable studies and/or with different platforms of miRNA detection. In this review, we will critically address the results of circulating miRNA research in oncology that have been published between January 2008 and June 2013 (5.5 years), and discuss pre-analytical challenges, technological pitfalls and limitations that may contribute to the non-reproducibility of circulating miRNA research.

Tyrosinase-related protein 1 mRNA expression in lymph node metastases predicts overall survival in high-risk melanoma patients.

BACKGROUND: Clinical outcome of high-risk melanoma patients is not reliably predicted from histopathological analyses of primary tumours and is often adjusted during disease progression. Our study aimed at extending our previous findings in skin metastases to evaluate the prognostic value of tyrosinase-related protein 1 (TYRP1) in lymph node metastases of stages III and IV melanoma patients. METHODS: TYRP1 mRNA expression in 104 lymph node metastases was quantified by real-time PCR and normalised to S100 calcium-binding protein B (S100B) mRNA expression to correct for tumour load. TYRP1/S100B ratios were calculated and median was used as cutoff value. TYRP1/S100B mRNA values were correlated to clinical follow-up and histopathological characteristics of the primary lesion. RESULTS: A high TYRP1/S100B mRNA ratio significantly correlated with reduced disease-free (DFS) and overall survival (OS; Cox regression analysis, P=0.005 and 0.01, respectively), increased Breslow thickness (Spearman's rho test, P<0.001) and the presence of ulceration (Mann-Whitney test, P=0.02) of the primaries. Moreover, high TYRP1/S100B was of better prognostic value (lower P-value) for OS than Breslow thickness and ulceration. Finally, it was well conserved during disease progression with respect to high/low TYRP1 groups. CONCLUSION: High TYRP1/S100B mRNA expression in lymph node metastases from melanoma patients is associated with unfavourable clinical outcome. Its evaluation in lymph node metastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lesions and may allow different management of the two groups of patients.

EGFR and K-ras gene mutation status in squamous cell anal carcinoma: a role for concurrent radiation and EGFR inhibitors?

BACKGROUND: There is a growing appreciation for radio-sensitiser use in multi-modal cancer treatment models. Squamous cell anal carcinoma (SCAC) is a rare gastrointestinal tumour traditionally treated with concurrent chemotherapy and radiation. Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, has demonstrated significant efficacy when combined with radiation in squamous cell carcinoma of the head and neck (SccH&N). We wanted to assess EGFR and Kirsten-ras (K-ras) status in SCAC to see whether it compares with SccH&N. METHODS: Over 90 SCAC paraffin-embedded biopsies were mounted onto a tissue microarray and were assessed for EGFR expression by immunohistochemistry. These samples were also assessed for the most frequently mutated K-ras and EGFR exons by high-resolution melting analysis. RESULTS: The EGFR was present in over 90% of samples tested. The K-ras and EGFR mutations were absent in all samples tested, although a synonymous single-nucleotide polymorphism was found in 3 out of 89 samples tested for EGFR exon 19. CONCLUSION: The low rate of K-ras and EGFR mutations, coupled with the high surface expression of EGFR, suggests similarity in the EGFR signalling pathway between SCAC and SccH&N, and thus a potential role for EGFR inhibitors in SCAC. To our knowledge this is the largest cohort of invasive SCAC samples investigated for EGFR and K-ras mutations reported to date.

The role of molecular pathology in non-small-cell lung carcinoma-now and in the future.

In recent years, better understanding of the molecular biology of non-small-cell lung carcinoma (nsclc) has led to a revolution in the work-up of these neoplasms. As a pathology diagnosis, "nsclc" without further attempt at subclassification is no longer accepted as a standard of care; separating squamous cell carcinoma from adenocarcinoma and large-cell carcinoma carries implications for prognosis and treatment decisions. Currently, detection of the presence in nsclc of mutations involving the epidermal growth factor receptor (EGFR) gene and fusion of the N-terminal portion of the protein encoded by EML4 (echinoderm microtubule-associated protein-like 4 gene) with the intracellular signaling portion of the receptor tyrosine kinase encoded by ALK (anaplastic lymphoma kinase gene)-that is, EML4-ALK-and variants has become routine in many centres because patients having tumours harbouring such alterations might benefit from tyrosine kinase inhibitors as part of their treatment regimen.The purpose of the present review is to highlight important aspects of the screening for molecular derangements in nsclc and to briefly discuss the emergence of possible future biomarkers.

EGFR tyrosine kinase mutation testing in the treatment of non-small-cell lung cancer.

BACKGROUND: Non-small-cell lung cancer (nsclc) tumours with activating mutations of the epidermal growth factor receptor (efgr) tyrosine kinase are highly sensitized to the effects of oral tyrosine kinase inhibitors such as gefitinib and erlotinib, suggesting the possibility of targeted treatment of nsclc based on EFGR mutation status. However, no standardized method exists for assessing the EGFR mutation status of tumours. Also, it is not known if available methods are feasible for routine screening. To address that question, we conducted a validation study of methods used for detecting EGFR mutations in exons 19 and 21 at molecular laboratories located in five specialized Canadian cancer centres. METHODS: The screening methods were first optimized using cell lines harbouring the mutations in question. A validation phase using anonymized patient samples followed. RESULTS: The methods used at the sites were highly specific and sensitive in detecting both mutations in cell-line dna (specificity of 100% and sensitivity of at least 1% across all centres). In the validation phase, we observed excellent concordance between the laboratories for detecting mutations in the patient samples. Concordant results were obtained in 26 of 30 samples (approximately 87%). In general, the samples for which results were discordant were also less optimal, containing small amounts of tumour. CONCLUSIONS: Our results suggest that currently available methods are capable of reliably detecting exon 19 and exon 21 mutations of EFGR in tumour samples (provided that sufficient tumour material is available) and that routine screening for those mutations is feasible in clinical practice.

Biomarkers in melanoma.

Biomarkers are tumour- or host-related factors that correlate with tumour biological behaviour and patient prognosis. High-throughput analytical techniques--DNA and RNA microarrays--have identified numerous possible biomarkers, but their relevance to melanoma progression, clinical outcome and the selection of optimal treatment strategies still needs to be established. The review discusses a possible molecular basis for predictive tissue biomarkers such as melanoma thickness, ulceration and mitotic activity, and provides a list of promising new biomarkers identified from tissue microarrays that needs confirmation by independent, prospectively collected clinical data sets. In addition, common predictive serum biomarkers--lactate dehydrogenase, S100B and melanoma-inhibiting activity--as well as selected investigational serum biomarkers such as TA90IC and YKL-40 are also reviewed. A more accurate, therapeutically predictive classification of human melanomas and selection of patient populations that would profit from therapeutic interventions are among the major challenges expected to be addressed in the future.

The Janus face of dendritic cells in cancer.

On the basis of experimental models and some human data, we can assume that tumor outgrowth results from the balance between immunosurveillance (the extrinsic tumor suppressor mechanisms) and immunosubversion dictated by transformed cells and/or the corrupted surrounding microenvironment. Cancer immunosurveillance relies mainly upon conventional lymphocytes exerting either lytic or secretory functions, whereas immunosubversion results from the activity of regulatory T or suppressor myeloid cells and soluble mediators. Although specific tools to target or ablate dendritic cells (DCs) became only recently available, accumulating evidence points to the critical role of the specialized DC system in dictating most of the conventional and regulatory functions of tumor-specific T lymphocytes. Although DC can be harnessed to silence tumor development, tumors in turn can exploit DC to evade immunity. Indeed, DCs harbor defects in their differentiation and stimulatory functions in cancer-bearing hosts and can actively promote T-cell tolerance to self-tumor antigens. In this review, we will focus on the dual role of DC during tumor progression and discuss pharmacoimmunological strategies to harness DC against cancer.

[Eruptive nevi associated with sorafenib treatment]. / Naevus éruptifs sous sorafénib.

BACKGROUND: Sorafenib is a new multikinase inhibitor recently approved for renal cell carcinoma and hepatocarcinoma. Among other targets, it blocks the kinase function of the RAF gene products including V600E mutant BRAF, which is frequently found in both melanoma and naevi. Cutaneous side effects are frequent with sorafenib, but no naevus modification has been reported until now. PATIENTS AND METHODS: Five cases of eruptive naevi in patients treated with sorafenib are reported. The mean duration of sorafenib treatment was 9.2 months when naevi eruption was noticed. The patients presented with about 100 to more than 200 small, homogenous, dark-brown naevi located mainly on the trunk and upper limbs. DISCUSSION: Eruptive melanocytic naevi have been reported in association with blistering diseases, and more generally in a setting of immunosuppression. We hypothesize that naevi appearance could be linked to an anti-senescence effect of sorafenib via its action on the MAP kinase pathway. Further prospective studies are needed to explore the relationship between sorafenib and the biology of naevi.

Heterozygous mutations in the tumor suppressor gene PATCHED provoke basal cell carcinoma-like features in human organotypic skin cultures.

Basal cell carcinoma of the skin is the most common type of cancer in humans. The majority of these tumors displays aberrant activation of the SONIC HEDGEHOG (SHH)/PATCHED pathway, triggered by mutations in the PATCHED tumor suppressor gene, which encodes a transmembrane receptor of SHH. In this study, we took advantage of the natural genotype (PATCHED(+/-)) of healthy keratinocytes expanded from patients with the nevoid basal cell carcinoma or Gorlin syndrome to mimic heterozygous somatic mutations thought to occur in the PATCHED gene early upon basal cell carcinoma development in the general population. PATCHED(+/-) epidermis developed on a dermal equivalent containing wild-type (WT) PATCHED(+/+) fibroblasts exhibited striking invasiveness and hyperproliferation, as well as marked differentiation impairment. Deciphering the phenotype of PATCHED(+/-) keratinocytes revealed slight increases of the transcriptional activators GLI1 and GLI2-the latter known to provoke basal cell carcinoma-like tumors when overexpressed in transgenic mice. PATCHED(+/-) keratinocytes also showed a substantial increase of the cell cycle regulator cyclin D1. These data show for the first time the physiological impact of constitutive heterozygous PATCHED mutations in primary human keratinocytes and strongly argue for a yet elusive mechanism of haploinsufficiency leading to cancer proneness.

Overexpression of matrix metalloproteinase 1 in dermal fibroblasts from DNA repair-deficient/cancer-prone xeroderma pigmentosum group C patients.

Xeroderma pigmentosum (XP) is a rare, recessively inherited genetic disease characterized by skin cancer proneness and premature aging in photoexposed area. The disease results from defective nucleotide excision repair of ultraviolet (UV)-induced DNA lesions. Reconstruction of group C (XP-C) skin in vitro previously suggested that patients' dermal fibroblasts might be involved in promoting skin cancer development, as they elicited microinvasions of both control and XP-C keratinocytes within dermal equivalents. Here we show that in the absence of UV exposure XP-C fibroblasts exhibit aged-like features such as an elongated and dendritic shape. We analysed the repertoire of expression of matrix metalloproteinases (MMPs) involved in skin aging and cancer. All XP-C fibroblasts tested in this study overexpressed specifically and significantly MMP1. MMP1 expression was also found increased in the dermis of XP-C skin sections suggesting the active contribution of XP-C mesenchymal cells to skin aging and exacerbated carcinogenesis. Increased MMP1 expression in cultured XP-C fibroblasts resulted from MMP1 mRNA accumulation and enhanced transcriptional activity of the MMP1 gene promoter. Deletion analysis revealed the essential role of AP-1 activation in constitutive MMP1 overexpression in XP-C primary fibroblasts. In parallel, levels of reactive oxygen species and FOSB DNA-binding activity were found increased in XP-C fibroblasts. Altogether, these observations suggest that beyond its role in nucleotide excision repair the XPC protein may be important in cell metabolism and fate in the absence of UV.

The potential contribution of fluorescent in situ hybridization analysis to the cytopathological diagnosis of Merkel cell carcinoma.

We report the cases of two patients with head and neck Merkel cell carcinoma (MCC) who developed local recurrences confirmed by cytopathology. Interphase fluorescent in situ hybridization (FISH) analysis was performed for research purposes using centromeric probes of chromosomes 6 and 8, on cytological slides. Trisomy of chromosome 6 was found in 85% of tumour cells in the first case of MCC and case 2 exhibited trisomy 8 in 77% of tumour cells. In the absence of specific molecular markers, detection of trisomy 6 and/or trisomy 8 could help in identifying MCC. FISH analysis is easily and quickly performed on interphase nuclei obtained through fine needle aspiration and may be extended to the study of other relevant genetic abnormalities.