Changes in the oral microbiome, particularly Fusobacterium nucleatum, are associated with oral squamous cell carcinoma (OSCC). F. nucleatum has been reported to modulate local immunity in cancers. We ...aimed to assess the association between intratumoral F. nucleatum and clinico-pathological features, relapse, and overall survival (OS) in two independent cohorts of patients with OSCC, and to explore the interplay with immune-related genes. We retrospectively analyzed tissue samples from a first cohort of 122 patients with head and neck squamous cell carcinoma, including 61 OSCC (cohort #1), and a second cohort of 90 additional OSCC (cohort #2). We then performed a sensitivity analysis on the merged cohort of OSCC patients (N = 151). F. nucleatum 16S rRNA gene sequences were quantified using real-time quantitative PCR. The presence of gram-negative bacteria and macrophages was confirmed by LPS and CD163 immunostainings, respectively. F. nucleatum positivity was associated with older age, less alcohol and combined alcohol plus tobacco consumption, and less frequent lymph node invasion. There was a trend for a lower recurrence rate in F. nucleatum-positive cases, with less metastatic relapses compared to F. nucleatum-negative tumors, and significantly longer OS, relapse-free and metastasis-free survival. F. nucleatum status was independently associated with OS in multivariate analysis. Immune-related gene and immunohistochemistry analyses showed that gram-negative bacteria load inversely correlated with M2 macrophages. F. nucleatum-associated OSCC has a specific immune microenvironment, is more frequent in older, non-drinking patients, and associated with a favorable prognosis.
Human papillomavirus (HPV) is found in 90% of squamous cell carcinomas of the anal canal (SCCA). We investigated the clinical validity of HPV circulating tumor DNA (ctDNA) detection in patients ...enrolled in the Epitopes-HPV02 trial that demonstrated the efficacy of docetaxel, cisplatin, and 5-FU as first-line chemotherapy in advanced SCCA.
According to the protocol, serum samples were collected before chemotherapy and on completion of chemotherapy. HPV16 ctDNA was quantified by droplet digital PCR (ddPCR) and correlated with prospectively registered patient characteristics and outcomes. A landmark was set at the time of chemotherapy completion for postchemotherapy progression-free survival (PFS) analyses.
Among 57 patients with HPV16-related advanced SCCA, HPV ctDNA was detected in 91.1% (95% confidence interval, 81.1-96.2) of baseline samples. Baseline HPV ctDNA levels were not associated with any patient characteristics; baseline ctDNA level below the cutoff obtained by AUC (area under the curve) was associated with a longer PFS (HR = 2.1;
= 0.04). Among the 36 patients who completed 5 months of chemotherapy, residual HPV ctDNA was detected after chemotherapy in 38.9% of patients. Residual HPV ctDNA detected at chemotherapy completion was associated with shorter postchemotherapy PFS (median PFS 3.4 months vs. not reached; HR = 5.5;
< 0.001) and a reduction of 1-year overall survival rate (OR = 7.0;
= 0.02).
This prospective study in advanced SCCA demonstrated a significant prognostic impact of HPV ctDNA level before first-line chemotherapy and HPV ctDNA negativity after chemotherapy completion. With a limited cost and short turnaround, this assay is a promising tool to optimize the therapeutic management of SCCA.
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Activating mutations in the estrogen receptor 1 (ESR1) gene confer resistance to aromatase inhibitors (AI), and may be targeted by selective estrogen receptor downregulators. We designed a multiplex ...droplet digital PCR (ddPCR), which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with an unconventional assay interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S, or D538G mutations. Further validation was performed on plasma samples from a prospective study and compared with next generation sequencing (NGS) data. The multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them, all mutations being confirmed by NGS. In addition, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of circulating tumor DNA using this technique during treatment follow-up predicts the clinical benefit of palbociclib-fulvestrant. The multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations with good sensitivity. This method allows real-time liquid biopsy for ESR1 mutation monitoring in large cohorts of patients.
Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. We further investigated transcriptome‐genotype‐phenotype correlations in HCC. ...Global transcriptome analyses were performed on 57 HCCs and 3 hepatocellular adenomas and validated by quantitative RT‐PCR using 63 additional HCCs. We determined loss of heterozygosity, gene mutations, promoter methylation of CDH1 and CDKN2A, and HBV DNA copy number for each tumor. Unsupervised transcriptome analysis identified 6 robust subgroups of HCC (G1‐G6) associated with clinical and genetic characteristics. G1 tumors were associated with low copy number of HBV and overexpression of genes expressed in fetal liver and controlled by parental imprinting. G2 included HCCs infected with a high copy number of HBV and mutations in PIK3CA and TP53. In these first groups, we detected specific activation of the AKT pathway. G3 tumors were typified by mutation of TP53 and overexpression of genes controlling the cell cycle. G4 was a heterogeneous subgroup of tumors including TCF1‐mutated hepatocellular adenomas and carcinomas. G5 and G6 were strongly related to β‐catenin mutations that lead to Wnt pathway activation; in particular, G6 tumors were characterized by satellite nodules, higher activation of the Wnt pathway, and E‐cadherin underexpression. Conclusion: These results have furthered our understanding of the genetic diversity of human HCC and have provided specific identifiers for classifying tumors. In addition, our classification has potential therapeutic implications because 50% of the tumors were related to WNT or AKT pathway activation, which potentially could be targeted by specific inhibiting therapies. (HEPATOLOGY 2007;45:42–52.rpar;
Anal carcinomas (AC) are associated with human papillomavirus (HPV) DNA sequences, but little is known about the physical state of the viral genome in carcinoma cells. To define the integration ...status and gene(s) targeted by viral insertions in AC, tumor DNAs extracted from 35 tumor specimen samples in patients with HPV16‐associated invasive carcinoma were analyzed using the detection of integrated papillomavirus sequences‐PCR approach. The genomic status at integration sites was assessed using comparative genomic hybridization‐array assay and gene expression using reverse transcription quantitative PCR (RT‐qPCR). HPV16 DNA was found integrated in 25/35 (71%) cases and the integration locus could be determined at the molecular level in 19 cases (29 total integration loci). HPV DNA was inserted on different chromosomes, but 5 cases harbored viral sequences at 19p13.2, within the nuclear factor I X (NFIX) locus. Viral DNA mapped between the most distal and the two proximal alternatively expressed exons of this gene in three cases (CA21, CA04, and CA35) and upstream of this gene (663 kb and 2.3 Mb) in the others. CGH arrays showed genomic gains/amplifications at the NFIX region, associated with HPV within the gene and RT‐qPCR, revealed NFIX mRNA overexpression. Other genes targeted by integration were IL20RB, RPS6KA2, MSRA1, PIP5K1B, SLX4IP, CECR1, BCAR3, ATF6, CSNK1G1, APBA2, AGK, ILF3, PVT1, TRMT1, RAD51B, FASN, CCDC57, DSG3, and ZNF563. We identified recurrent targeting of NFIX by HPV16 insertion in anal carcinomas, supporting a role for this gene in oncogenesis, as reported for non‐HPV tumors.
Human papillomavirus (HPV) is recognised as the cause of precancerous and cancerous cervical lesions. Furthermore, in high-grade lesions, HPV is frequently integrated in the host cell genome and ...associated with the partial or complete loss of the E1 and E2 genes, which regulate the activity of viral oncoproteins E6 and E7. In this study, using a double-capture system followed by high-throughput sequencing, we determined the HPV integration status present in liquid-based cervical smears in an urban Gabonese population. The main inclusion criteria were based on cytological grade and the detection of the HPV16 genotype using molecular assays. The rate of HPV integration in the host genome varied with cytological grade: 85.7% (6/7), 71.4% (5/7), 66.7% (2/3) 60% (3/5) and 30.8% (4/13) for carcinomas, HSIL, ASCH, LSIL and ASCUS, respectively. For high cytological grades (carcinomas and HSIL), genotypes HPV16 and 18 represented 92.9% of the samples (13/14). The integrated form of HPV16 genotype was mainly found in high-grade lesions in 71.4% of samples regardless of cytological grade. Minority genotypes (HPV33, 51, 58 and 59) were found in LSIL samples, except HPV59, which was identified in one HSIL sample. Among all the HPV genotypes identified after double capture, 10 genotypes (HPV30, 35, 39, 44, 45, 53, 56, 59, 74 and 82) were detected only in episomal form. Our study revealed that the degree of HPV integration varies with cervical cytological grade. The integration event might be a potential clinical prognostic biomarker for the prediction of the progression of neoplastic lesions.
In most cases of cervical cancers, HPV DNA is integrated into the genome of carcinoma cells. This mutational insertion constitutes a highly specific molecular marker of tumor DNA for every patient. ...Circulating tumor DNA (ctDNA) is an emerging marker of tumor dynamics which detection requires specific molecular motif. To determine whether the sequence of the cell-viral junction could be used in clinical practice as a specific marker of ctDNA, we analyzed a series of cervical cancer patient serums.
Serum specimens of 16 patients diagnosed with HPV16/18-associated cervical cancer, and for which the viral integration locus had been previously localized, were analyzed. Sequential serum specimens, taken at different times during the course of the disease, were also available for two of these cases. ctDNA was found in 11 out of 13 patients with tumor size greater than 20 mm at diagnosis, and analysis of sequential serum specimens showed that ctDNA concentration in patients serum was related to tumor dynamics.
We report that HPV mutational insertion constitutes a highly specific molecular marker of ctDNA in HPV-associated tumor patients. Using this original approach, ctDNA was detected in most cervical cancer patients over stage I and ctDNA concentration was found to reflect tumor burden. In addition to its potential prognostic and predictive value, HPV mutation insertion is likely to constitute a new molecular surrogate of minimal residual disease and of subclinical relapse in HPV-associated tumor. This is of major importance in the perspective of specific anti-HPV therapy.
Most endocervical adenocarcinomas are human papillomavirus (HPV)-related cancers associated with p16 immunostaining. Ovarian metastasis from cervical cancer is a rare phenomenon, the mechanism of ...dissemination remains unclear. The diagnosis of metastasis may be difficult to establish when the ovarian neoplasm presents features consistent with primary tumor. Immunohistochemical expression of p16 in ovarian tumors can guide the diagnosis of metastasis from HPV-related cervical cancer, but p16 positivity is nonspecific. Identical HPV genotype in the paired endocervical and ovarian tumors is a better marker for cervical origin, which may also be confirmed by identical HPV integration site.
Two women presented with HPV18 cervical adenocarcinoma. No signs of disease were visible on MRI after treatment. After several years of follow-up, mucinous ovarian tumors were discovered in both patients. Molecular analyses showed that the ovarian lesions were HPV18-positive; indicating a primary cervical origin. A third woman was diagnosed with grade 1 ovarian endometrioid carcinoma with no peritoneal carcinomatosis. Final histological examination and HPV genotyping revealed HPV18-related in situ endometrioid adenocarcinoma in the endocervix and HPV18-related invasive endometrioid adenocarcinoma in the endometrium and both ovaries. Additional molecular analyses performed in two patients identified the same HPV integration sites in both the ovarian and cervical tumors, confirming that the ovarian mass was a metastasis from the cervical adenocarcinoma.
We report three new cases of ovarian neoplasia in which the diagnosis of metastasis from cervical cancer was supported by the same HPV genotype and the same integration site in the paired cervical and ovarian tumors. To our knowledge, this is the first report of molecular evidence of the cervical origin of an ovarian metastasis. HPV screening should be performed in ovarian tumors for all patients with history of cervical neoplasia.
Background & Aims Mosaic G-protein alpha-subunit ( GNAS )-activating mutations are responsible for the McCune–Albright (MCA) syndrome. This oncogene that activates the adenylate cyclase is also ...mutated in various tumor types leading to the accumulation of cyclic-AMP. Identification of a hepatocellular adenoma (HCA) in two MCA patients led us to search for GNAS activation in benign and malignant hepatocellular carcinogenesis. Methods GNAS mutations were screened by sequencing 164 HCA, 245 hepatocellular carcinoma (HCC), and 17 fibrolamellar carcinomas. Tumors were characterized by quantitative RT-PCR, gene mutation screening and pathological reviewing. The consequences of wild type and mutant GNAS expression were analyzed in hepatocellular cell lines. Results A somatic GNAS -activating mutation was identified in 5 benign tumors and in 2 HCC. In benign tumors, GNAS mutations were exclusive from HNF1A , CTNNB1 , and IL6ST mutations whereas one HCC demonstrated both CTNNB1 and GNAS mutations. Quantitative RT-PCR showed an activation of the IL-6 and interferon pathways in GNAS -mutated tumor tissues. Accordingly, pathological reviewing identified in GNAS -mutated tumors an inflammatory phenotype characterized by fibrosis and STAT3 activation. We further demonstrated in HCC cell lines that GNAS mutant expression induced inflammatory response and STAT3 activation. Conclusions We identified for the first time the association between two rare diseases, MCA syndrome and HCA occurrence, but also that somatic GNAS -activating mutations in sporadic benign and malignant liver tumors are characterized by an inflammatory phenotype. These results showed a cross-talk between cyclic-AMP and JAK/STAT pathways in liver tumors and they reinforce the role of STAT3 activation in liver tumorigenesis.
A prevalence of around 26% of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has been previously reported. HPV induced oncogenesis mainly involving E6 and E7 viral ...oncoproteins. In some cases, HPV viral DNA has been detected to integrate with the host genome and possibly contributes to carcinogenesis by affecting the gene expression. We retrospectively assessed HPV integration sites and signatures in 80 HPV positive patients with HNSCC, by using a double capture‐HPV method followed by next‐generation Sequencing. We detected HPV16 in 90% of the analyzed cohort and confirmed five previously described mechanistic signatures of HPV integration episomal (EPI), integrated in a truncated form revealing two HPV‐chromosomal junctions colinear (2J‐COL) or nonlinear (2J‐NL), multiple hybrid junctions clustering in a single chromosomal region (MJ‐CL) or scattered over different chromosomal regions (MJ‐SC) of the human genome. Our results suggested that HPV remained episomal in 38.8% of the cases or was integrated/mixed in the remaining 61.2% of patients with HNSCC. We showed a lack of association of HPV genomic signatures to tumour and patient characteristics, as well as patient survival. Similar to other HPV associated cancers, low HPV copy number was associated with worse prognosis. We identified 267 HPV‐human junctions scattered on most chromosomes. Remarkably, we observed four recurrent integration regions: PDL1/PDL2/PLGRKT (8.2%), MYC/PVT1 (6.1%), MACROD2 (4.1%) and KLF5/KLF12 regions (4.1%). We detected the overexpression of PDL1 and MYC upon integration by gene expression analysis. In conclusion, we identified recurrent targeting of several cancer genes such as PDL1 and MYC upon HPV integration, suggesting a role of altered gene expression by HPV integration during HNSCC carcinogenesis.
Five previously described mechanistic signatures of human papillomavirus (HPV) integration episomal, integrated in a truncated form revealing two HPV‐chromosomal junctions colinear (2J‐COL) or nonlinear (2J‐NL), multiple hybrid junctions clustering in a single chromosomal region (MJ‐CL) or scattered over different chromosomal regions (MJ‐SC) of the human genome are confirmed in our head and neck squamous cell carcinoma cohort. Two hundred sixty‐seven HPV‐human junctions scattered on most chromosomes are reported.
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