p38MAPK builds a hyaluronan cancer niche to drive lung tumorigenesis.

Expansion of neoplastic lesions generates the initial signal that instigates the creation of a tumor niche. Nontransformed cell types within the microenvironment continuously coevolve with tumor cells to promote tumorigenesis. Here, we identify p38MAPK as a key component of human lung cancer, and specifically stromal interactomes, which provides an early, protumorigenic signal in the tissue microenvironment. We found that lung cancer growth depends on short-distance cues produced by the cancer niche in a p38-dependent manner. We identified fibroblast-specific hyaluronan synthesis at the center of p38-driven tumorigenesis, which regulates early stromal fibroblast activation, the conversion to carcinoma-associated fibroblasts (CAFs), and cancer cell proliferation. Systemic down-regulation of p38MAPK signaling in a knock-in model with substitution of activating Tyr182 to phenylalanine or conditional ablation of p38 in fibroblasts has a significant tumor-suppressive effect on K-ras lung tumorigenesis. Furthermore, both Kras-driven mouse lung tumors and orthotopically grown primary human lung cancers show a significant sensitivity to both a chemical p38 inhibitor and an over-the-counter inhibitor of hyaluronan synthesis. We propose that p38MAPK-hyaluronan-dependent reprogramming of the tumor microenvironment plays a critical role in driving lung tumorigenesis, while blocking this process could have far-reaching therapeutic implications.

Quantitative multiplexed imaging technologies for single-cell analysis to assess predictive markers for immunotherapy in thoracic immuno-oncology: promises and challenges.

The past decade has witnessed a revolution in cancer treatment by the shift from conventional drugs (chemotherapies) towards targeted molecular therapies and immune-based therapies, in particular the immune-checkpoint inhibitors (ICIs). These immunotherapies selectively release the host immune system against the tumour and have shown unprecedented durable remission for patients with cancers that were thought incurable such as advanced non-small cell lung cancer (aNSCLC). The prediction of therapy response is based since the first anti-PD-1/PD-L1 molecules FDA and EMA approvals on the level of PD-L1 tumour cells expression evaluated by immunohistochemistry, and recently more or less on tumour mutation burden in the USA. However, not all aNSCLC patients benefit from immunotherapy equally, since only around 30% of them received ICIs and among them 30% have an initial response to these treatments. Conversely, a few aNSCLC patients could have an efficacy ICIs response despite low PD-L1 tumour cells expression. In this context, there is an urgent need to look for additional robust predictive markers for ICIs efficacy in thoracic oncology. Understanding of the mechanisms that enable cancer cells to adapt to and eventually overcome therapy and identifying such mechanisms can help circumvent resistance and improve treatment. However, more than a unique universal marker, the evaluation of several molecules in the tumour at the same time, particularly by using multiplex immunostaining is a promising open room to optimise the selection of patients who benefit from ICIs. Therefore, urgent further efforts are needed to optimise to individualise immunotherapy based on both patient-specific and tumour-specific characteristics. This review aims to rethink the role of multiplex immunostaining in immuno-thoracic oncology, with the current advantages and limitations in the near-daily practice use.

European Real-World Assessment of the Clinical Validity of a CE-IVD Panel for Ultra-Fast Next-Generation Sequencing in Solid Tumors.

Molecular profiling of solid tumors facilitates personalized, targeted therapeutic interventions. The ability to perform next-generation sequencing (NGS), especially from small tissue samples, in a short turnaround time (TAT) is essential to providing results that enable rapid clinical decisions. This multicenter study evaluated the performance of a CE in vitro diagnostic (IVD) assay, the Oncomine Dx Express Test, on the Ion Torrent Genexus System for detecting DNA and RNA variants in solid tumors. Eighty-two archived formalin-fixed paraffin embedded (FFPE) tissue samples from lung, colorectal, central nervous system, melanoma, breast, gastric, thyroid, and soft tissue cancers were used to assess the presence of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variations (CNVs), gene fusions, and splice variants. These clinical samples were previously characterized at the various academic centers using orthogonal methods. The Oncomine Dx Express Test showed high performance with 100% concordance with previous characterization for SNVs, indels, CNVs, gene fusions, and splice variants. SNVs and indels with allele frequencies as low as 5% were correctly identified. The test detected all the expected ALK, RET, NTRK1, and ROS1 fusion isoforms and MET exon 14-skipping splice variants. The average TAT from extracted nucleic acids to the final variant report was 18.3 h. The Oncomine Dx Express Test in combination with the Ion Torrent Genexus System is a CE-IVD-compliant, performant, and multicenter reproducible method for NGS detection of actionable biomarkers from a range of tumor samples, providing results in a short TAT that could support timely decision- making for targeted therapeutic interventions.

Accurate Detection of SARS-CoV-2 by Next-Generation Sequencing in Low Viral Load Specimens.

As new SARS-CoV-2 variants emerge, there is an urgent need to increase the efficiency and availability of viral genome sequencing, notably to detect the lineage in samples with a low viral load. SARS-CoV-2 genome next-generation sequencing (NGS) was performed retrospectively in a single center on 175 positive samples from individuals. An automated workflow used the Ion AmpliSeq SARS-CoV-2 Insight Research Assay on the Genexus Sequencer. All samples were collected in the metropolitan area of the city of Nice (France) over a period of 32 weeks (from 19 July 2021 to 11 February 2022). In total, 76% of cases were identified with a low viral load (Ct ≥ 32, and ≤200 copies/µL). The NGS analysis was successful in 91% of cases, among which 57% of cases harbored the Delta variant, and 34% the Omicron BA.1.1 variant. Only 9% of cases had unreadable sequences. There was no significant difference in the viral load in patients infected with the Omicron variant compared to the Delta variant (Ct values, p = 0.0507; copy number, p = 0.252). We show that the NGS analysis of the SARS-CoV-2 genome provides reliable detection of the Delta and Omicron SARS-CoV-2 variants in low viral load samples.

Fusion-positive non-small cell lung carcinoma: Biological principles, clinical practice, and diagnostic implications.

Based on superior efficacy and tolerability, targeted therapy is currently preferred over chemotherapy and/or immunotherapy for actionable gene fusions that occur in late-stage non-small cell lung carcinoma (NSCLC). Consequently, current clinical practice guidelines mandate testing for ALK, ROS1, NTRK, and RET gene fusions in all patients with newly diagnosed advanced non-squamous NSCLC (NS-NSCLC). Gene fusions can be detected using different approaches, but today RNA next-generation sequencing (NGS) or combined DNA/RNA NGS is the method of choice. The discovery of other gene fusions (involving, eg, NRG1, NUT, FGFR1, FGFR2, MET, BRAF, EGFR, SMARC fusions) and their partners has increased progressively in recent years, leading to the development of new and promising therapies and mandating the development and implementation of comprehensive detection methods. The purpose of this review is to focus on recent data concerning the main gene fusions identified in NSCLC, followed by the discussion of major challenges in this domain.

A Single Dose of BNT162b2 Messenger RNA Vaccine Induces Airway Immunity in Severe Acute Respiratory Syndrome Coronavirus 2 Naive and Recovered Coronavirus Disease 2019 Subjects.

BACKGROUND: Mucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and therefore virus shedding. Parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior mucosal infection compared with injections of a parenteral vaccine in a mucosally naive subject. We investigated whether this was also the case for the BNT162b2 coronavirus disease 2019 (COVID-19) messenger RNA vaccine. METHODS: Twenty recovered COVID-19 subjects (RCSs) and 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-naive subjects were vaccinated with, respectively, 1 and 2 doses of the BNT162b2 COVID-19 vaccine. Nasal epithelial lining fluid (NELF) and plasma were collected before and after vaccination and assessed for immunoglobulin G (IgG) and IgA antibody levels to Spike and for their ability to neutralize binding of Spike to angiotensin-converting enzyme-2 receptor. Blood was analyzed 1 week after vaccination for the number of Spike-specific antibody-secreting cells (ASCs) with a mucosal tropism. RESULTS: All RCSs had both nasal and blood SARS-CoV-2-specific antibodies at least 90 days after initial diagnosis. In RCSs, a single dose of vaccine amplified preexisting Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including Delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2-naive subjects after 2 vaccine doses compared with RCSs after 1 dose. CONCLUSIONS: Mucosal immune response to the SARS-CoV-2 Spike protein is higher in RCSs after a single vaccine dose compared with SARS-CoV-2-naive subjects after 2 doses.

"Interchangeability" of PD-L1 immunohistochemistry assays: a meta-analysis of diagnostic accuracy.

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.

Prospective evaluation of NGS-based liquid biopsy in untreated late stage non-squamous lung carcinoma in a single institution.

BACKGROUND: NGS from plasma samples in non-squamous cell lung carcinoma (NSCC) can aid in the detection of actionable genomic alterations. However, the absolute clinical value of NGS in liquid biopsy (LB) made at baseline is currently uncertain. We assessed the impact of plasma-based NGS using an in-house test and an outsourced test in comparison to a routine molecular pathology workflow. METHODS: Twenty-four advanced/metastatic treatment-naïve NSCC patients were prospectively included. NGS analyses were conducted both in-house using the Oncomine cfTNA Panel and in an external testing center using the Foundation Liquid assay. NGS analysis and/or specific molecular based assays were conducted in parallel on tissue or cytological samples. RESULTS: Both LB tests were well correlated. Tissue NGS results were obtained in 67% of patients and demonstrated good correlation with LB assays. Activating EGFR mutations were detected using LB tests in three patients. PD-L1 expression assessed in tissue sections enabled the initiation of pembrolizumab treatment in five patients. CONCLUSION: NGS from LB is feasible in routine clinical practice using an in-house or an outsourced test at baseline. However, the impact on therapy selection was limited in this small series of patients and LB was not able to replace tissue-based testing in our hands.

Correction: Prognostic and predictive role of CD8 and PD-L1 determination in lung tumor tissue of patients under anti-PD-1 therapy.

Since the publication of this paper, the authors noticed that Corentin Richard was not credited as contributing equally to the paper. He should be considered as a joint first author with Jean-David Fumet.

Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition.

Extracellular vesicles (EVs) have been shown to play an important role in intercellular communication as carriers of DNA, RNA and proteins. While the intercellular transfer of miRNA through EVs has been extensively studied, the stability of extracellular miRNA (ex-miRNA) once engulfed by a recipient cell remains to be determined. Here, we identify the ex-miRNA-directed phenotype to be transient due to the rapid decay of ex-miRNA. We demonstrate that the ex-miR-223-3p transferred from polymorphonuclear leukocytes to cancer cells were functional, as demonstrated by the decreased expression of its target FOXO1 and the occurrence of epithelial-mesenchymal transition reprogramming. We showed that the engulfed ex-miRNA, unlike endogenous miRNA, was unstable, enabling dynamic regulation and a return to a non-invasive phenotype within 8 h. This transient phenotype could be modulated by targeting XRN1/PACMAN exonuclease. Indeed, its silencing was associated with slower decay of ex-miR-223-3p and subsequently prolonged the invasive properties. In conclusion, we showed that the 'steady step' level of engulfed miRNA and its subsequent activity was dependent on the presence of a donor cell in the surroundings to constantly fuel the recipient cell with ex-miRNAs and of XRN1 exonuclease, which is involved in the decay of these imported miRNA.

[The age of artificial intelligence in lung cancer pathology: Between hope, gloom and perspectives]. / La pathologie cancéreuse pulmonaire à l'heure de l'intelligence artificielle : entre espoir, désespoir et perspectives.

Histopathology is the fundamental tool of pathology used for more than a century to establish the final diagnosis of lung cancer. In addition, the phenotypic data contained in the histological images reflects the overall effect of molecular alterations on the behavior of cancer cells and provides a practical visual reading of the aggressiveness of the disease. However, the human evaluation of the histological images is sometimes subjective and may lack reproducibility. Therefore, computational analysis of histological imaging using so-called "artificial intelligence" (AI) approaches has recently received considerable attention to improve this diagnostic accuracy. Thus, computational analysis of lung cancer images has recently been evaluated for the optimization of histological or cytological classification, prognostic prediction or genomic profile of patients with lung cancer. This rapidly growing field constantly demonstrates great power in the field of computing medical imaging by producing highly accurate detection, segmentation or recognition tasks. However, there are still several challenges or issues to be addressed in order to successfully succeed the actual transfer into clinical routine. The objective of this review is to emphasize recent applications of AI in pulmonary cancer pathology, but also to clarify the advantages and limitations of this approach, as well as the perspectives to be implemented for a potential transfer into clinical routine.

PREVALENCE AND INTENSITY OF DIGESTIVE AND PULMONARY PARASITES IN WILD BOARS IN ROMANIA.

The prevalence and intensity of infection with digestive, liver, and pulmonary parasites in wild boars in Romania was determined by examination of 280 cadavers from 26 hunting grounds during the period 2012-2016. Eleven genera of parasites were recovered: nine within the digestive system (Eimeria, Ascaris, Globocephalus, Gongylonema, Hyostrongylus, Oesophagostomum, Physocephalus, Trichuris, and Macracanthorinchus); and two (Dicrocoelium, Metastrongylus ) located in the hepatic and pulmonary systems. The overall prevalence of infection was 80.7% (n = 280). Polyparasitism was found in 82.8% of positive cases. The mean intensity of parasitism was highest for pulmonary parasites (Metastrongylus salmi, 25.95). Regarding gastrointestinal parasites, the highest mean intensity occurred in the case of Oesophagastomum dentatum infections (22.14), whilst the lowest was that of Macracanthorhynchus hirudinaceus (1.66). Wild boars are an important source of infection for domestic pigs in Romania and neighboring countries where extensive breeding systems occur.

Prognostic and predictive role of CD8 and PD-L1 determination in lung tumor tissue of patients under anti-PD-1 therapy.

BACKGROUND: No study has evaluated the predictive and prognostic role of CD8 and PD-L1 coexpression in non-small-cell lung cancer (NSCLC). METHODS: We analyzed RNA sequencing and/or immunohistochemistry staining in NSCLC patients from The Cancer Genome Atlas (n = 1016), and 34 metastatic NSCLC samples not treated by immunotherapy as prognostic cohorts. As predictive aspect of CD8 and PD-L1, we used 85 NSCLC patients treated with anti-PD-1. Two validation cohorts were used including 44 NSCLC patients treated with anti-PD-1 and an external cohort with different tumor types. RESULTS: In prognostic cohorts, high CD8A expression was associated with longer OS (p = 0.02), while high CD274 mRNA was associated with poor prognosis (p = 0.05). In predictive cohort, high CD8 expression and CD8A mRNA were associated with longer progression-free survival (PFS) (p = 0.0002). There was no significant association between PD-L1 expression and PFS while high CD274 mRNA was associated with longer PFS (p = 0.009). A combination of CD8A and CD274 was highly predictive of outcome. These results were confirmed in the validation cohorts. This two-genes signature demonstrated similar results compared to gold standard signatures. CONCLUSION: CD8 represents both a prognostic and predictive factor of outcomes, while PD-L1 share different prognostic and predictive roles.

Use of circulating tumor cells in prospective clinical trials for NSCLC patients - standardization of the pre-analytical conditions.

BACKGROUND: Circulating tumor cells (CTCs) hold potential for noninvasive diagnosis, prognosis and prediction testing in non-small cell lung cancer (NSCLC) patients. Minimizing degradation or loss of CTCs is pivotal for detection and profiling of the low abundance and fragile CTCs, particularly in clinical trials. We prospectively investigated (NCT02372448) whether a new blood collection device performed better compared to commonly used K3EDTA tubes, when subjected to long-term sample storage. METHODS: Blood samples were drawn into K3EDTA and blood collection tubes (BCT) (Streck), and filtered by the Isolation by SizE of Tumor/Trophoblastic Cells (ISET® system), for CTC detection in two study populations of NSCLC patients; the training set of 14 patients with stage II/IV NSCLC, and the validation set of 36 patients with stage IV NSCLC). MET expression was evaluated by immunocytochemistry (ICC) and anaplastic lymphoma kinase (ALK) gene rearrangement by break-apart fluorescence in situ hybridization (FISH) on ISET-enriched CTCs. RESULTS: Blood processed after 24 h and 48 h in BCT tubes showed stable CTCs counts and integrity, whereas CTCs in K3EDTA tubes showed an altered morphology in all patients. CTCs recovered in BCT or K3EDTA tubes at 24 and 48 h were evaluable by ICC for MET expression and by FISH for ALK rearrangement. CONCLUSIONS: The BCT tubes gave a high yield and preserved the integrity of CTCs after 24 and 48 h of storage at room temperature, which facilitate their molecular characterization in NSCLC patients entering clinical trials.

[PD1/PD-L1 immunohistochemistry in thoracic oncology: Where are we?] / Immunohistochimie PD-1/PD-L1 en oncologie thoracique : où en sommes-nous ?

The assays for the assessment of the PD-L1 status by immunohistochemistry are available in clinical studies in thoracic oncology to predict response to immunotherapies targeting the PD-1/PD-L1 pathway. With the arrival of this new class of molecules in second line and very soon in first line of treatment for patients with advanced or metastatic non-small cell lung cancer, these tests will certainly be required in routine once these new drugs will be granted marketing authorization. The rapid introduction of these "companion" or "complementary" tests seems essential to select patients to benefit from these effective but also expensive and sometimes toxic therapies. Although challenged by some oncologists (as some patients not expressing PD-L1 may sometimes respond to PD-1/PD-L1 blockade), the anti-PD-L1 immunohistochemically approach seems inevitable in 2017. This new activity developed in the pathology laboratories raises several questions: which anti-PD-L1 clone should be used? On which device? What threshold of positivity should be considered? Should PD-L1 expression be assessed on tumor cells as well as on the immune cells? What controls should be used? Comparative studies are underway or have been already implemented in order to answer some of these questions. This review addresses the different evaluation criteria for immunohistochemistry using the main anti-PD-L1 antibodies used to date as well the recently published studies using these antibodies in thoracic oncology.

PD-L1 expression in basaloid squamous cell lung carcinoma: Relationship to PD-1+ and CD8+ tumor-infiltrating T cells and outcome.

PD-1/PD-L1 inhibitors demonstrated durable clinical responses in patients with lung squamous cell carcinoma. However, the expression pattern of PD-L1 and the presence of CD8+ and PD-1+ tumor-infiltrating T cells in the basaloid variant of squamous cell carcinoma remain unknown. immunohistochemistry analysis of PD-L1 expression, with three recently validated monoclonal antibodies used in clinical trials (clones SP142, SP263, and 28-8), and detection of CD8+ and PD-1+ tumor-infiltrating T cells was performed on whole-tissue sections from 56 patients following surgery for basaloid squamous cell carcinoma. Data were correlated to clinicopathological parameters and outcome. Fair to poor concordance was observed between the SP142 vs SP263 clones, and SP142 vs 28-8 (κ range, 0.018-0.412), while the 28-8 and SP263 demonstrated a strong correlation in both the tumor cell and immune cell compartments (κ=0.883, and κ=0.721). Expression of PD-L1 correlated with a high content of CD8+ and PD-1+ tumor-infiltrating T cells when using SP142 (P=0.012; P=0.022), but not with SP263 or 28-8 (P=0.314; P=0.611). In the multivariate analysis, we found significantly better disease-free and overall survival rates for high PD-L1 expression with SP142, CD8+ and PD-1+ tumor-infiltrating T cells (P=0.003; P=0.007). No significant prognosis value was observed for SP263 and 28-8 clones, except a correlation between improved overall survival and SP263 in the univariate analysis (P=0.039), not confirmed in the multivariate model. In conclusion, we report that the expression of PD-L1 and the content of CD8+ and PD-1+ tumor-infiltrating T cells is an independent indicator of better outcome in basaloid squamous cell carcinoma patients, although the observed effect is dependent on the PD-L1 immunohistochemistry assay.

French multicentric validation of ALK rearrangement diagnostic in 547 lung adenocarcinomas.

Anaplastic lymphoma kinase (ALK) gene rearrangements in lung adenocarcinoma result in kinase activity targetable by crizotinib. Although fluorescence in situ hybridisation (FISH) is the reference diagnostic technique, immunohistochemistry (IHC) could be useful for pre-screening. Diagnostic yields of ALK IHC, FISH and quantitative reverse transcriptase PCR performed in 14 French pathology/molecular genetics platforms were compared. 547 lung adenocarcinoma specimens were analysed using 5A4 and D5F3 antibodies, two break-apart FISH probes and TaqMan kits. Clinicopathological data were recorded. 140 tumours were ALK rearranged (FISH with ≥15% of rearranged cells) and 400 were ALK FISH negative (<15%). FISH was not interpretable for seven cases. ALK patients were young (p=0.003), mostly females (p=0.007) and light/nonsmokers (p<0.0001). 13 cases were IHC negative but FISH ≥15%, including six cases with FISH between 15% and 20%; eight were IHC positive with FISH between 10% and 14%. Sensitivity and specificity for 5A4 and D5F3 were 87% and 92%, and 89% and 76%, respectively. False-negative IHC, observed in 2.4% of cases, dropped to 1.3% for FISH >20%. Variants were undetected in 36% of ALK tumours. Discordances predominated with FISH ranging from 10% to 20% of rearranged cells and were centre dependent. IHC remains a reliable pre-screening method for ALK rearrangement detection.

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.