Valérie Costes

Neck restaging with sentinel node biopsy in T1-T2N0 oral and oropharyngeal cancer: Why and how?

Objective: To evaluate the lack of accuracy in neck staging with the classical technique (i.e., neck dissection and routine histopathology) with the sentinel node (SN) biopsy in oral and oropharyngeal T1-T2N0 cancer. Study Design: Cross-sectional study with planned data collection. Setting: Tertiary center care. Subjects and Methods: In 50 consecutive patients, the pathological stage of sentinel node (pSN) was established after analyzing SN biopsies (n = 148) using serial sectioning and immunohistochemistry. Systematic selective neck dissection was performed. The pN stage was established with routine histopathologic analysis of both the non-SN (n = 1075) and the 148 SN biopsies. Results: The sensitivity and negative predictive value of pSN staging were 100 percent. Conversely, if one considers pSN staging procedure as the reference test for micro- and macro-metastasis diagnosis, the sensitivity of the classical pN staging procedure was 50 percent (9/1; 95% CI 26.9-73.1) and its negative predictive value was 78 percent (95% CI 61.9-88.8). Fifteen patients (30%) were upstaged, including nine cases from pN0 to pSN ≥ 1 and six cases from pN1 to pSN2. Two of the pN0-pSN1 upstaged patients died with relapsed neck disease. Conclusion: The SN biopsy technique appeared to be the best staging method in cN0 patients and provided evidence that routinely undiagnosed lymph node invasion may have clinical significance.

Human papillomavirus (HPV) prevalence and type distribution, and HPV-associated cytological abnormalities in anal specimens from men infected with HIV who have sex with men

Human papillomavirus (HPV) detection and typing using the PapilloCheck® test and cytological examination were carried out in anal samples collected from 67 men seropositive for human immunodeficiency virus (HIV) who have sex with men. Fifty (74.6%) patients had anal HPV infection, 46 (68.7%) had high‐risk (HR) HPV infection, and 38 (56.7%) had multiple infection involving 2–9 (median, 3) HPV types. The HPV types identified most frequently were HPV 44/55 (19.4%), HPV 53 (19.4%), HPV 16 (16.4%), HPV 39 (16.4%), and HPV 42 (14.9%). Thirty‐two of the 66 interpretable smears (48.5%) revealed cytological abnormalities: 9 (13.4%) atypical cells of undetermined significance, 20 (30.3%) low‐grade intraepithelial lesions, and 3 (4.5%) high‐grade intraepithelial lesions. Cytological abnormalities were associated significantly with HPV detection (Pu2009<u20090.001), multiple HPV infection (Pu2009<u20090.001), and increased number of HPV types (Pu2009<u20090.001). The HPV types associated most frequently with cytological abnormalities were HPV 39 (28.1%), HPV 42 (28.1%), HPV 53 (28.1%), HPV 16 (25.0%), HPV 44/55 (25.0%), and HPV 59 (21.9%). HPV DNA detection as well as cytological abnormalities were associated neither with HIV RNA detection in plasma nor with CD4+ T‐cell count. Differences in age or in time since HIV acquisition were not observed in patients with or without cytological abnormalities. The present study confirms the high prevalence of anal HR‐HPV infection and cytological abnormalities in men infected with HIV who have sex with men. HPV testing and/or cytological analysis may be helpful in selecting the patients to be referred to proctological examination. J. Med. Virol. 82:592–596, 2010.

A new autoinflammatory and autoimmune syndrome associated with NLRP1 mutations: NAIAD (NLRP1-associated autoinflammation with arthritis and dyskeratosis)

Objectives Inflammasomes are multiprotein complexes that sense pathogens and trigger biological mechanisms to control infection. Nucleotide-binding oligomerisation domain-like receptor (NLR) containing a PYRIN domain 1 (NLRP1), NLRP3 and NLRC4 plays a key role in this innate immune system by directly assembling in inflammasomes and regulating inflammation. Mutations in NLRP3 and NLRC4 are linked to hereditary autoinflammatory diseases, whereas polymorphisms in NLRP1 are associated with autoimmune disorders such as vitiligo and rheumatoid arthritis. Whether human NLRP1 mutation is associated with autoinflammation remains to be determined. Methods To search for novel genes involved in systemic juvenile idiopathic arthritis, we performed homozygosity mapping and exome sequencing to identify causative genes. Immunoassays were performed with blood samples from patients. Results We identified a novel disease in three patients from two unrelated families presenting diffuse skin dyskeratosis, autoinflammation, autoimmunity, arthritis and high transitional B-cell level. Molecular screening revealed a non-synonymous homozygous mutation in NLRP1 (c.2176C>T; p.Arg726Trp) in two cousins born of related parents originating from Algeria and a de novo heterozygous mutation (c.3641C>G, p.Pro1214Arg) in a girl of Dutch origin. The three patients showed elevated systemic levels of caspase-1 and interleukin 18, which suggested involvement of NLRP1 inflammasome. Conclusions We demonstrate the responsibility of human NLRP1 in a novel autoinflammatory disorder that we propose to call NAIAD for NLRP1-associated autoinflammation with arthritis and dyskeratosis. This disease could be a novel autoimmuno-inflammatory disease combining autoinflammatory and autoimmune features. Our data, combined with that in the literature, highlight the pleomorphic role of NLRP1 in inflammation and immunity. Trial registration number NCT02067962; Results.

Prognostic value of a three-grade classification in primary epithelial parotid carcinoma: Result of a histological review from a 20-year experience of total parotidectomy with neck dissection in a single institution

BACKGROUNDnThe tumour grading of primary parotid cancers (PPCs) remains controversial.nnnMETHODSnA 20-year standardised single centre treatment has been assessed retrospectively. The histological review of 155 consecutively treated parotid malignancies identified 96 suitable cases for univariate and multivariate survival analyses.nnnRESULTSnTreatment involved total parotidectomy, neck dissection and post-operative radiotherapy in, respectively, 91.7%, 83.3% and 70.4% of cases. The 5-year overall survival, disease-specific and recurrence-free survival rates were 79.4%, 83.5% and 70.8%, respectively. Univariate analysis confirmed the classical prognostic factors, i.e. age>60 years, male gender, facial palsy, hardness of the tumour, clinical stage, tumour grade, facial nerve invasion and lymph node metastases. Multivariate analysis identified a three-grade classification just after the clinical stage as the most important prognostic factor.nnnCONCLUSIONnThis study identifies the prognostic significance of intermediate grade tumours.

Pemphigus vulgaris antigen mRNA quantification for the staging of sentinel lymph nodes in head and neck cancer

Background:Molecular diagnosis has been proposed to enhance the intra-operative diagnosis of sentinel lymph node (SLN) invasion in head and neck squamous cell carcinoma (HNSCC). Although cytokeratin (CK) mRNA quantification with real-time reverse transcriptase-PCR (QRT–PCR) has produced encouraging results, the more discriminating markers remain to be identified.Methods:Pemphigus vulgaris antigen (PVA), squamous cell carcinoma antigen (SCCA), and CK17 mRNA were quantified using QRT–PCR, and the results were compared with an extensive histopathological examination of the entire SLNs on 78 SLNs harvested from 22 patients with HNSCC.Results:SCCA and CK17 quantification showed significantly higher mRNA values for macrometastases (MAs) than for either negative or isolated tumour cell (ITC) SLNs (P<0.01). Pemphigus vulgaris antigen allowed the discrimination of all MAs and micrometastases from both negative and ITC SLNs (P<0.001). For the neck staging of patients, considering metastatic vs non-metastatic status, receiver-operating characteristic curve analysis found areas under the curve of 93.8, 97.9, and 100% for CK17, SCCA, and PVA, respectively. With PVA, a cutoff value of 562 copies per 100u2009ng of cDNA permitted the correct distinction between patients with positive as opposed to negative neck nodes in all cases.Conclusion:PVA seems to be a highly promising marker for accurate intra-operative SLN staging in HNSCC by QRT–PCR.

Comparison of the PapilloCheck® assay with the digene HC2 HPV DNA assay for the detection of 13 high-risk human papillomaviruses in cervical and anal scrapes

The PapilloCheck® assay was compared with the Digene HC2 HPV DNA assay for the detection of 13 high‐risk human papillomaviruses (HPV) in 240 samples, including 181 cervical scrapes and 59 anal scrapes. Overall, 75 (30.5%) samples were positive by the Digene HC2 HPV DNA assay: 34 (18.8%) cervical scrapes and 41 (69.5%) anal scrapes. By considering only the 13 high‐risk HPV types detected by the Digene HC2 HPV DNA assay, 66 (27.5%) samples were positive by the PapilloCheck® assay: 27 (14.9%) cervical scrapes and 39 (66.1%) anal scrapes. Concordant results between the two assays were obtained for 225 (93.8%) samples with a Kappa coefficient value of 0.85, indicating an excellent agreement. By considering all the HPV types detectable by the PapilloCheck® assay, the overall prevalence of HPV was 34.2% (82/240): 21.0% (38/181) in cervical scrapes and 74.6% (44/59) in anal scrapes. Among the samples positive by the PapilloCheck® assay, a multiple HPV infection (2–9 HPV types) was identified in 43 of 82 (52.4%) samples, including 7 of 38 (18.4%) cervical samples, and 36 of 44 (81.8%) anal samples. The prevalence of high‐risk HPV, as determined by the PapilloCheck® assay, was 17.6% (36/205) in samples with normal cytology, 83.9% (26/31) in samples with low‐grade squamous intraepithelial lesions or atypical squamous cells of undetermined significance, and 100% (4/4) in samples with high‐grade squamous intraepithelial lesions. The results obtained indicate that the PapilloCheck® assay may be considered as a reliable screening test for HPV detection and typing. J. Med. Virol. 83:1377–1382, 2011.

γδ T Lymphocytes Count Is Normal and Expandable in Peripheral Blood of Patients with Follicular Lymphoma, Whereas It Is Decreased in Tumor Lymph Nodes Compared with Inflammatory Lymph Nodes

γδ T lymphocytes are attractive effector cells for immunotherapy. In vitro, they can be expanded and kill efficiently a variety of tumor cells. The frequency and distribution of γδ T lymphocytes were compared in tumor lymph nodes of 51 patients with follicular lymphoma lymph nodes (FL-LNs) and 28 patients with inflammatory lymph nodes (I-LNs). γδ and CD8 T lymphocytes were less abundant in FL-LNs than in I-LNs (p ≤ 10−7). These lymphocytes were localized in the perifollicular zone outside of the tumor follicles. Perifollicular γδ T lymphocytes expressed CCR7, in contrast to peripheral blood γδ T lymphocytes and both perifollicular and peripheral blood γδ T lymphocytes expressed CXCR4. The very low number of perifollicular γδ T lymphocytes in FL-LNs could be explained in part by migratory problems because of absence of CCL19 expression in FL-LNs compared with I-LNs. Conversely, CCL21 and CXCL12 were similarly expressed in both FL-LNs and I-LNs. CCL19 and CCL21 were expressed in high endothelial venules and lymphatic vessels, whereas CXCL12 was expressed by stromal cells surrounding high endothelial venules and lymphatic vessels. Peripheral γδ T lymphocytes from 34 patients with FL, expanded with Phosphostim and IL-2 in vitro, had the same expansion capacity as those from healthy individuals. Thus, γδ T lymphocytes can be an attractive source for adoptive immunotherapy in patients with FL, providing they may home in tumor LNs.

Viral load and physical status of human papillomavirus (HPV) 18 in cervical samples from female sex workers infected with HPV 18 in Burkina Faso.

Viral DNA load and physical status might be predictive of either high‐grade cervical lesions or disease progression among women infected by human papillomavirus (HPV) 16, but these virological markers have rarely been studied in HPV 18 infections. The relationships between HPV 18 DNA load, viral genome physical status and cervical squamous intraepithelial lesions were analyzed among female sex workers infected with HPV18 in Burkina Faso. HPV 18 E2 and E6 genes were quantitated by real‐time PCR. Among 21 women infected with HPV 18, 67% of whom were HIV‐1‐seropositive, 11 (52.4%) had a normal cytology, 8 (38.1%) had low‐grade squamous intraepithelial lesions, and 2 (9.5%) had high‐grade squamous intraepithelial lesions. Total viral load and integrated viral load were higher in women with squamous intraepithelial lesions than in women with normal cytology (Pu2009=u20090.01 for both parameters). Total viral load and integrated viral load were higher in HIV‐1‐seropositive women than in those who were not infected with HIV (Pu2009=u20090.01, and P, 0.01, respectively). Total viral load or integrated viral load >1,000u2009copies/ng of DNA were more frequent in women with squamous intraepithelial lesions than in women with normal cytology (7/10 vs. 1/11; Pu2009=u20090.007) and in HIV‐1‐seropositive women (8/14 vs. 0/7 in HIV‐uninfected women; Pu2009=u20090.02). Both HPV 18 DNA and integrated DNA loads might represent markers of cervical lesions. Prospective evaluations are needed to establish the value of these parameters to predict high‐grade lesion or lesion progression. J. Med. Virol. 81:1786–1791, 2009.

Performance of careHPV for detecting high-grade cervical intraepithelial neoplasia among women living with HIV-1 in Burkina Faso and South Africa: HARP study.

Background:The careHPV assay is a test for high-risk (HR) human papillomaviruses (HPV) detection designed to be affordable in resource-poor settings. We evaluated the performance of careHPV screening among 1052 women living with HIV/AIDS included in the HARP (HPV in Africa Research Partnership) study in Burkina Faso (BF) and South Africa (SA).Methods:Cervical samples were tested for HR-HPV by the careHPV and the INNO-LiPA HPV genotyping Extra assays. All women had Pap smear testing, visual inspection with acetic acid/Lugol’s iodine (VIA/VILI) and colposcopy. Cervical biopsies were obtained for participants who were HR-HPV DNA positive by careHPV or who had abnormalities detected on cytology, VIA/VILI or colposcopy.Results:Overall, 45.1% of women had a positive careHPV test (46.5% in BF, 43.8% in SA). The careHPV positivity rate increased with the grade of cytological lesions. Sensitivity and specificity of careHPV for the diagnosis of CIN2+ (n=60, both countries combined) were 93.3% (95% confidence interval (CI): 83.8–98.2) and 57.9% (95% CI: 54.5–61.2), respectively. Specificity increased with CD4 count. careHPV had a similar clinical sensitivity but higher specificity than the INNO-LiPA assay for detection of CIN2+.Conclusions:Our results suggest that careHPV testing is a reliable tool for cervical cancer screening in HIV-1-infected women in sub-Saharan Africa.

Comparative evaluation of the new FDA approved THxID™-BRAF test with high resolution melting and sanger sequencing

BackgroundSince patients diagnosed with BRAF V600E and V600K mutated advanced melanoma show response to treatment with MAP kinase inhibitors, several sensitive methods have been developed to determine the V600 allele status of melanoma patients. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar) are specific BRAF V600 inhibitors recently approved by the US FDA as single agent treatments for unresectable or metastatic melanoma in patients with the BRAF V600 mutation.MethodsWe assessed the new CE THxID™-BRAF diagnostic test, which is also FDA-approved as a companion diagnostic test in the US under a specific reference and compared the results of this assay with both High Resolution Melting (HRM) and Sanger sequencing in 113 melanoma FFPE samples.ResultsInvalid results were observed in 0/113 specimen with HRM, 5/113 (4.4%) with Sanger sequencing, and 1/113 (0.9%) with the THxID™-BRAF test. Positive percentage agreement (PPA) was 93.5% (95% CI 82.5 - 97.8) for V600E and V600K mutations combined for the THxID™-BRAF test and HRM, and negative percentage agreement (NPA) was 100.0% (95% CI 94.5 - 100.0). For the THxID™-BRAF test and Sanger, PPA was 100.0% (95% CI 92.1 - 100.0) and NPA 100.0% (95% CI 94.2 - 100.0). One V600E sample identified by THxID™-BRAF test was detected as wild-type by HRM and uninterpretable by Sanger. All V600K (nu2009=u20093) were detected using the 3 different approaches. Finally, percent agreement values were not significantly different when using punches (nu2009=u200977) vs. slides (nu2009=u200936) or depending on samples characteristics such as pigmentation, necrosis, and tumor content.ConclusionsThis study demonstrated the high agreement between the FDA approved THxID™-BRAF assay, HRM, and Sanger sequencing. It has also highlighted the potential of THxID™-BRAF to be applied to a broader range of sample types than claimed in the current “instructions for use”, an extension that would require the ad hoc validation and approval.