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EA2406, Histology and Molecular Pathology of Tumours, University of Bordeaux, Bordeaux, FranceTumor Bank and Tumor Biology Laboratory, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France
EA2406, Histology and Molecular Pathology of Tumours, University of Bordeaux, Bordeaux, FranceTumor Bank and Tumor Biology Laboratory, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France
EA2406, Histology and Molecular Pathology of Tumours, University of Bordeaux, Bordeaux, FranceDepartment of Dermatology, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France
EA2406, Histology and Molecular Pathology of Tumours, University of Bordeaux, Bordeaux, FranceDepartment of Pathology, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France
EA2406, Histology and Molecular Pathology of Tumours, University of Bordeaux, Bordeaux, FranceDepartment of Dermatology, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France
EA2406, Histology and Molecular Pathology of Tumours, University of Bordeaux, Bordeaux, FranceTumor Bank and Tumor Biology Laboratory, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France
EA2406, Histology and Molecular Pathology of Tumours, University of Bordeaux, Bordeaux, FranceTumor Bank and Tumor Biology Laboratory, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France
The molecular events underlying the oncogenesis of cutaneous T-cell lymphomas (CTCLs) remain largely unknown, especially when considering primary or driver mutations. Global genomic approaches have revealed the existence of recurrent chromosomal or genetic alterations, some of which having potential diagnosis or prognosis value (
Loss of heterozygosity on 10q and microsatellite instability in advanced stages of primary cutaneous T-cell lymphoma and possible association with homozygous deletion of PTEN.
recently identified somatic mutations of the Phospholipase C Gamma 1 (PLCG1) gene in about 20% of epidermotropic CTCLs, being notably more frequent in transformed/tumoral mycosis fungoides (T-MF; 8/30 cases) and the Sézary syndrome (SS; 1/2 cases) than in erythrodermic and folliculotropic mycosis fungoides (1/20 cases). They reported a c.1034C>T/p.S345F mutation in exon 11 in nine patients (eight T-MF and one SS) and a c.1559C>T/p.S520F mutation in exon 15 (in one erythrodermic mycosis fungoides case). Such mutations, especially the p.S345F mutation, conferred enhanced signaling capacity and transforming property. Finally, PLCG1 mutations were suggested to be possibly associated with a higher rate of disease-related death (
These findings prompted us to investigate the PLCG1 gene status in our series of CTCLs, including T-MF (n=37) and SS (n=39). We also studied other CTCL subtypes including lymphomatoid papulosis (LyP; n=4), cutaneous anaplastic large cell lymphomas (c-ALCL, n=14), and the following T cell or CTCL cell lines: MyLa, SeAX, HH, Hut78, FEPD, and 1301 (for origin see
All cases were retrieved from the Aquitaine database of cutaneous lymphomas, with approval from the regional bioethics committee and informed written consent, in accordance with the Declaration of Helsinki Principles. Skin or blood* samples (*for patients with SS) were analyzed. The selected cases contained at least 40% of tumor cells, as found by histo/cytopathological or flow cytometric evaluation. DNA was extracted from frozen CTCL samples or cell lines with the DNA easy kit (Qiagen, Courtaboeuf, France). All exhibited a dominant monoclonal rearrangement of the T-cell receptor gamma (TCRG) gene (
b). In cases with PLCG1 mutation, constitutional DNA was extracted from histologically normal tissue and checked for the absence of the monoclonal TCRG gene rearrangement. The mutational status of PLCG1 exon 11 and 15 was determined by high-resolution melting (HRM) analysis on a LC480 device (Roche Diagnostics, Meylan, France), followed by Sanger sequencing for samples exhibiting variant profiles, as reported for BRAF status determination (
). The primers used were the following: 5′-GCCCATCTGACCATACCTAC-3′ and 5′-TGGACCCCACGCACACTCA-3′ (exon 11) and 5′-CTCACAAGTCCCTCTTTGGTC-3′ and 5′-GACCTGAGCTGGTTCCTCAC-3′ (exon 15).
Only one of the 37 tested T-MF cases (2.7%) from our series harbored a mutation in exon 11 (c.1025A>G/p.D342G; case 48, Figure 1, Table 1). Two out of the 39 tested SS cases (5%) exhibited exon 11 mutations, the c.1024 G>A/p.D342N mutation (case 75) and the c.1034C>T/p.S345F mutation (case 58; Figure 1,Table 1), the latter being previously reported to be a recurrent event in CTCLs (
). All three mutations were somatic, as not detected in the control constitutional DNA. Interestingly, the p.D342G and p.D342N mutations had not been reported either in CTCLs (
) or in other types of cancers (http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=PLCG1#dist). According to the PROVEAN (http://provean.jcvi.org/seq_submit.php) or PolyPhen (http://genetics.bwh.harvard.edu/pph2/) software analysis, they were predicted to alter the function of the PLCG1 protein catalytic domain. In the two mutated SS cases, the same mutation was detected in blood samples at two time points of the disease, with a 5-year (case 58) and a 1-year (case 75) interval, respectively (Figure 1, Table 1). Although all cases contained at least 40% of tumor cells, the mutated allele was more prominent in samples from the T-MF case 48 and the SS case 75 than in the SS case 58 (Figure 1), suggesting a possible tumor cell heterogeneity for the PCLG1 status in this case. In addition, none of our T-MF and SS cases exhibited PLCG1 exon 15 mutation, and none of the tested c-ALCL and LyP cases and T-cell leukemia/CTCL cell lines showed mutations of PLCG1 exons 11 or 15.
Figure 1Identification of Phospholipase C Gamma 1 (PLCG1) exon 11 mutations in cutaneous T-cell lymphoma (CTCL) samples. (a) High-resolution melting (HRM) curves and (b) Sanger sequencing profiles (partial sequence, sense strand) of PLCG1 exon 11 PCR products obtained from normal and CTCL DNA samples. The three CTCL cases (one transformed/tumoral mycosis fungoides (T-MF) and two Sézary syndrome (SS)) in which PLCG1 exon 11 mutations were identified are illustrated.
We thus report a low frequency of PLCG1 mutations in our T-MF (1 of 37, 2.7%) and SS (2 of 39, 5%) cases and the fact that the c.1034C>T/p.S345F mutation is quite uncommon in CTCLs (one out of the three mutations found in the 94 CTCL patients tested). We also document the absence of PLCG1 exon 11 and 15 mutations in the tested c-ALCL and LyP cases, as well as in the main CTCL cell lines.
For SS, differences in sample numbers can explain the discrepancy between frequencies of PLCG1 mutations found in our study (2/39 cases) and the first one (1/2 cases;
). For T-MF, we and the previous report analyzed comparable number of samples; thus, the distinct mutation frequencies (1/37 vs. 8/30) may come from technical aspects. Indeed, in the previous study, five out of the nine cases harboring the c.1034C>T/p.S345F were identified only with a sensitive allele-specific quantitative PCR assay (
). On the other hand, our HRM/Sanger sequencing strategy identified two previously unknown PLCG1 mutations affecting the catalytic domain that would have been missed by the allele-specific mutation analysis used by our colleagues (
To exclude technical bias, we checked the sensitivity of our methodology by diluting the mutated samples within normal DNA. As illustrated for sample 2 from case 75, containing 40% of Sézary cells (Figure 1 and Supplementary Figure S1 online -Pure-), dilution up to 4-fold still allowed a clear detection of variant HRM profile, as well as a mutant peak on Sanger sequencing profiles (Supplementary Figure S1A and S1B online). Higher dilutions (8-fold) yielded normal HRM profiles, and the mutation was barely detected by Sanger sequencing. Therefore, our approach is capable to detect PLCG1 mutation in samples with 10% of tumor cells (5% of mutant allele) in accordance with our previous evaluation of this technique for BRAF analysis in melanomas (
Using allele-specific mutation analysis, the same group reported the presence of the PLCG1S345F in about 13% of peripheral T-cell lymphoma, with significant association with CD30 expression and p50 nuclear expression, suggesting an increased NF-kB activity (
). Such an association was not observed in our CTCL series, which also includes CD30+ T-MF cases and CD30+ cutaneous lymphoproliferative disorders with abundant tumor cell content (
Assessment of diagnostic criteria between primary cutaneous anaplastic large cell lymphoma and CD30-rich transformed mycosis fungoides. A study of 66 cases.
If assessment of the PLCG1 mutations, notably the p.S345F mutation, is only feasible with highly sensitive methods, especially in our samples containing more than 40% of tumor cells, this would suggest that PLCG1 mutations do not represent an initiating oncogenic event but may be acquired by some aggressive subclones with possible prognosis impact on the overall survival in CTCL and PTCL (
). Further investigations by other groups and next-generation sequencing techniques are required to establish the real prevalence of PLCG1 mutations, which appeared unusual in our series of otherwise well-characterized CTCL subtypes (
Assessment of diagnostic criteria between primary cutaneous anaplastic large cell lymphoma and CD30-rich transformed mycosis fungoides. A study of 66 cases.
This work was supported by grants from the Ligue Contre le Cancer, Comité de Gironde, the Cancéropôle Grand Sud-Ouest and the Institut National du Cancer (INCA) for supporting the Aquitaine database of cutaneous lymphoma and the Tumor Bank of CHU de Bordeaux. We also thank Nathalie Carrere, and Séverine Verdon (Tumor Bank and Tumor Biology Laboratory, Centre Hospitalier Universitaire de Bordeaux, Pessac, France) for help in the preparation of control and patient DNA samples and Christine Alfaro (Department of Dermatology, Centre Hospitalier Universitaire de Bordeaux, Hôpital Haut-Lévêque, Pessac, France) for help in collecting clinicopathological data.
Assessment of diagnostic criteria between primary cutaneous anaplastic large cell lymphoma and CD30-rich transformed mycosis fungoides. A study of 66 cases.
Loss of heterozygosity on 10q and microsatellite instability in advanced stages of primary cutaneous T-cell lymphoma and possible association with homozygous deletion of PTEN.
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