Audrey Gros

Parallel FISH and immunohistochemical studies of ALK status in 3244 non-small-cell lung cancers reveal major discordances.

Introduction: Anaplastic lymphoma kinase (ALK) rearrangements occur in 1% to 7% of non–small-cell lung cancers (NSCLCs). Crizotinib, an ALK inhibitor, has been demonstrated to provide dramatic clinical benefits in ALK-positive advanced-stage NSCLC. Fluorescent in situ hybridization (FISH) has been established in clinical trials as the standard procedure method for detecting ALK rearrangements. Although the detection of ALK by immunohistochemistry (IHC) has been proposed for the screening of patients, large-scale studies are warranted to validate such a hierarchical approach. Methods: In this article, we report the largest series thus far of parallel FISH and IHC ALK testing in 3244 consecutive NSCLC cases analyzed at two independent French centers. Results: FISH-positive and/or IHC-positive results were demonstrated in 150 of 3244 cases (4.6%). An imbalanced sex ratio was detected, with women exhibiting a 2.2-fold relative risk for an alteration. Strikingly, only 80 of 150 specimens were classified as ALK positive by both techniques. The specimens with discordant FISH/IHC analyses were FISH-positive/IHC-negative (36), FISH-negative/IHC-positive (19), or FISH-noncontributive/IHC-positive (15). Thus, a single FISH or IHC analysis performed alone would have failed to detect approximately one-fourth of the ALK-positive cases with similar findings in our two centers. Conclusions: This study highlights the feasibility of systematic NSCLC testing by both FISH and IHC in routine practice. Many preanalytical factors may account for the apparent discrepancies between both methods, suggesting that hierarchical screening may underscore ALK-positive cases. This significant level of discrepancy supports the need of combined testing to optimize the detection of ALK-inhibitor–eligible patients given that some patients with discordant testing were found to respond to crizotinib.

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.

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. IHC is useful in pre-screening for ALK rearrangement, with FISH for confirmation of diagnosis http://ow.ly/Iuu5j

MYD88 somatic mutation is a diagnostic criterion in primary cutaneous large B-cell lymphoma

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. MYD88 somatic mutation is a diagnostic criterion in primary cutaneous large B-cell lymphoma Sarah Menguy, Audrey Gros, Anne Pham-Ledard, Maxime Battistella, Nicolas Ortonne, François Comoz, Brigitte Balme, Vanessa Szablewski, Laurence Lamant, Agnès Carlotti, et al.

Clinical and genomic analysis of a randomised phase II study evaluating anastrozole and fulvestrant in postmenopausal patients treated for large operable or locally advanced hormone-receptor-positive breast cancer

Background:The aim of this study was to assess the efficacy of neoadjuvant anastrozole and fulvestrant treatment of large operable or locally advanced hormone-receptor-positive breast cancer not eligible for initial breast-conserving surgery, and to identify genomic changes occurring after treatment.Methods:One hundred and twenty post-menopausal patients were randomised to receive 1 mg anastrozole (61 patients) or 500 mg fulvestrant (59 patients) for 6 months. Genomic DNA copy number profiles were generated for a subgroup of 20 patients before and after treatment.Results:A total of 108 patients were evaluable for efficacy and 118 for toxicity. The objective response rate determined by clinical palpation was 58.9% (95% CI=45.0–71.9) in the anastrozole arm and 53.8% (95% CI=39.5–67.8) in the fulvestrant arm. The breast-conserving surgery rate was 58.9% (95% CI=45.0–71.9) in the anastrozole arm and 50.0% (95% CI=35.8–64.2) in the fulvestrant arm. Pathological responses >50% occurred in 24 patients (42.9%) in the anastrozole arm and 13 (25.0%) in the fulvestrant arm. The Ki-67 score fell after treatment but there was no significant difference between the reduction in the two arms (anastrozole 16.7% (95% CI=13.3–21.0) before, 3.2% (95% CI=1.9–5.5) after, n=43; fulvestrant 17.1% (95%CI=13.1–22.5) before, 3.2% (95% CI=1.8–5.7) after, n=38) or between the reduction in Ki-67 in clinical responders and non-responders. Genomic analysis appeared to show a reduction of clonal diversity following treatment with selection of some clones with simpler copy number profiles.Conclusions:Both anastrozole and fulvestrant were effective and well-tolerated, enabling breast-conserving surgery in over 50% of patients. Clonal changes consistent with clonal selection by the treatment were seen in a subgroup of patients.

Neuroendocrine phenotype as an acquired resistance mechanism in ALK-rearranged lung adenocarcinoma.

A 63-year-old caucasian woman, presenting with metastatic primitive lung adenocarcinoma was treated with ALK inhibitor crizotinib treatment for six month. After rapid regression of all known lesions, tumor progression appeared six month later on all the already known lesions. A biopsy of subclavicular lymphadenopathy revealed a carcinoma with neuroendocrine phenotype with both immunohistochemical expression of ALK protein and ALK-rearrangement. It was associated with acquired resistance to crizotinib with ALK-rearrangement but without point mutation or amplification of the ALK gene. We herein report the first case of histological neuroendocrine transformation after ALK inhibitor crizotinib treatment, associated with acquired resistance to crizotinib.

Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

Challenging a dogma: co-mutations exist in MAPK pathway genes in colorectal cancer

Sequencing of genes encoding mitogen-activated protein kinase (MAPK) pathway proteins in colorectal cancer (CRC) has established as dogma that of the genes in a pathway only a single one is ever mutated. We searched for cases with a mutation in more than one MAPK pathway gene (co-mutations). Tumor tissue samples of all patients presenting with CRC, and referred between 01/01/2008 and 01/06/2015 to three French cancer centers for determination of mutation status of RAS/RAF+/−PIK3CA, were retrospectively screened for co-mutations using Sanger sequencing or next-generation sequencing. We found that of 1791 colorectal patients with mutations in the MAPK pathway, 20 had a co-mutation, 8 of KRAS/NRAS, and some even with a third mutation. More than half of the mutations were in codons 12 and 13. We also found 3 cases with a co-mutation of NRAS/BRAF and 9 with a co-mutation of KRAS/BRAF. In 2 patients with a co-mutation of KRAS/NRAS, the co-mutation existed in the primary as well as in a metastasis, which suggests that co-mutations occur early during carcinogenesis and are maintained when a tumor disseminates. We conclude that co-mutations exist in the MAPK genes but with low frequency and as yet with unknown outcome implications.

Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas

In nodal diffuse large B-cell lymphoma, the search for double-hit with MYC and BCL2 and/or BCL6 rearrangements or for dual expression of BCL2 and MYC defines subgroups of patients with altered prognosis that has not been evaluated in primary cutaneous large B-cell lymphoma. Our objectives were to assess the double-hit and dual expressor status in a cohort of 44 patients with primary cutaneous large B-cell lymphoma according to the histological subtype and to evaluate their prognosis relevance. The 44 cases defined by the presence of more than 80% of large B-cells in the dermis corresponded to 21 primary cutaneous follicle centre lymphoma with large cell morphology and 23 primary cutaneous diffuse large B-cell lymphoma, leg type. Thirty-one cases (70%) expressed BCL2 and 29 (66%) expressed MYC. Dual expressor profile was observed in 25 cases (57%) of either subtypes (n = 6 or n = 19, respectively). Only one primary cutaneous follicle centre lymphoma, large-cell case had a double-hit status (2%). Specific survival was significantly worse in primary cutaneous diffuse large B-cell lymphoma, leg type than in primary cutaneous follicle centre lymphoma, large cell (p = 0.021) and for the dual expressor primary cutaneous large B-cell lymphoma group (p = 0.030). Both overall survival and specific survival were worse for patients belonging to the dual expressor primary cutaneous diffuse large B-cell lymphoma, leg type subgroup (p = 0.001 and p = 0.046, respectively). Expression of either MYC and/or BCL2 negatively impacted overall survival (p = 0.017 and p = 0.018 respectively). As the differential diagnosis between primary cutaneous follicle centre lymphoma, large cell and primary cutaneous diffuse large B-cell lymphoma, leg type has a major impact on prognosis, dual-expression of BCL2 and MYC may represent a new diagnostic criterion for primary cutaneous diffuse large B-cell lymphoma, leg type subtype and further identifies patients with impaired survival. Finally, the double-hit assessment does not appear clinically relevant in primary cutaneous large B-cell lymphoma.

TP53 alterations in primary and secondary Sézary syndrome: A diagnostic tool for the assessment of malignancy in patients with erythroderma

Recent massive parallel sequencing data have evidenced the genetic diversity and complexity of Sézary syndrome mutational landscape with TP53 alterations being the most prevalent genetic abnormality. We analyzed a cohort of 35 patients with SS and a control group of 8 patients with chronic inflammatory dermatoses. TP53 status was analyzed at different clinical stages especially in 9 patients with a past-history of mycosis fungoides (MF), coined secondary SS. TP53 mutations were only detected in 10 patients with either primary or secondary SS (29%) corresponding to point mutations, small insertions and deletions which were unique in each case. Interestingly, TP53 mutations were both detected in sequential unselected blood mononuclear cells and in skin specimens. Cytogenetic analysis of blood specimens of 32 patients with SS showed a TP53 deletion in 27 cases (84%). Altogether 29 out of 35 cases exhibited TP53 mutation and/or deletion (83%). No difference in prognosis was observed according to TP53 status while patients with secondary SS had a worse prognosis than patients with primary SS. Interestingly, patients with TP53 alterations displayed a younger age and the presence of TP53 alteration at initial diagnosis stage supports a pivotal oncogenic role for TP53 mutation in SS as well as in erythrodermic MF making TP53 assessment an ancillary method for the diagnosis of patients with erythroderma as patients with inflammatory dermatoses did not display TP53 alteration.