PRAME is a useful marker for the differential diagnosis of melanocytic tumours and histological mimics.

AIMS: Although the morphological assessment of melanoma is generally straightforward, diagnosis can be especially difficult when the significant morphological and immunohistochemical results overlap with those of benign and malignant melanocytic tumours and histological mimics. This study assessed the potential diagnostic utility of measuring PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemically in naevi, melanomas and clear cell sarcomas (CCSs) in Chinese patients. METHODS: We examined the immunohistochemical expression of PRAME in 317 melanocytic naevi, 178 primary melanomas, 72 metastatic melanomas and 19 CCSs and compared the sensitivity and specificity of PRAME immunohistochemistry (IHC) in the differential diagnosis of melanocytic tumours and histological mimics. RESULTS: Of the 317 melanocytic naevi, 98.1%were completely negative for PRAME; six cases showed focal PRAME immunoreactivity in a minor population of lesional melanocytes. Diffuse nuclear immunoreactivity for PRAME was found in 89.9% of primary melanomas and 93.1% of metastatic melanomas. Regarding melanoma subtypes, PRAME was expressed in 100% of superficial spreading melanomas, 100% of melanomas arise in congenital naevus, 91.4% of nodular melanomas, 87.8% of acral lentigo melanomas, 80.0% of lentigo malignant melanomas, 60.0% of Spitz melanomas, 96.2% of mucosal melanomas and 80.0% of uveal melanomas. None of the two desmoplastic melanomas expressed PRAME. Of the 19 CCS cases, 89.5% were negative for PRAME and 10.5% showed focal weak PRAME immunoreactivity in a minor population of tumour cells. CONCLUSIONS: Our findings indicate that PRAME may be a useful marker to support a suspected diagnosis of melanoma. In addition, lack of PRAME expression is a valuable hint to CCS in a suspected case, and then molecular confirmation of the presence of EWSR1 rearrangement is necessary.

Clinicopathological and prognostic features of hepatitis B virus-associated diffuse large B-cell lymphoma: a single-center retrospective study in China.

BACKGROUND: While the epidemiologic association between hepatitis B virus (HBV) infection and diffuse large B-cell lymphoma (DLBCL) is established, little is known about the pathological characteristics and outcome of DLBCL arising in patients with HBV infection. METHODS: We retrospectively studied a cohort of 420 patients with DLBCL for the incidence of HBV infection, and the clinicopathologic features and prognostic factors in HBsAg-positive DLBCL patients in China, a hepatitis B endemic area. RESULTS: In our study, 127 (30.2%) patients were HBsAg-positive. HBsAg-positive DLBCL displayed a younger median onset age (50 vs. 54 years, P = 0.002), more frequent involvement of the spleen (19.7% vs. 6.1%, P < 0.001), less frequent involvement of the small and large intestine (2.3% vs. 11.2%, P = 0.003), more advanced disease (stage III/IV: 56.7% vs. 45.1%, P = 0.028), and lower expression rate of MYC (49.1% vs. 66.7%, P = 0.026). The median follow-up time was 61.9 months. Univariate analysis showed that there was no significant difference in overall survival (OS) between HBsAg-negative and -positive DLBCL (P = 0.577). In the HBsAg-positive DLBCL subgroup, age older than 60 years, advanced disease, elevated lactate dehydrogenase (LDH), spleen involvement, B symptoms (fever, night sweats, weight loss), and double expressers of MYC and BCL2 had a significantly worse outcome, and patients treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) had a better prognosis. Multivariate analysis further confirmed that spleen involvement and rituximab use were independent prognostic factors in HBsAg-positive DLBCL patients. CONCLUSIONS: Our study indicates that HBsAg-positive DLBCL has unique clinicopathological features and independent prognostic factors. Moreover, under antiviral prophylaxis, the survival of DLBCL patients with HBV infections was comparable to that of HBV-negative patients, and the use of rituximab significantly improved OS in HBsAg-positive DLBCL patients.

Recurrent KRAS, KIT and SF3B1 mutations in melanoma of the female genital tract.

BACKGROUND: Malignant melanoma of the female genital tract is relatively uncommon and accounts for 3-7% of all melanoma localizations. This study aimed to identify driver genes in melanoma of the female genital tract with the purpose of enhancing understanding of disease pathogenesis and identifying potential new therapeutic targets to develop effective therapies. METHODS: KIT (CD117) and BRAF expression were detected immunohistochemically. Polymerase Chain Reaction (PCR) and Sanger sequencing techniques were performed to identify the mutational status of BRAF, NRAS, KRAS, NF1, KIT, PDGFRA and SF3B1 on 19 melanomas of the female genital tract, paired with 25 cutaneous melanomas, 18 acral melanomas and 11 melanomas of nasal cavity. RESULTS: Somatic variant analysis identified KRAS (6/19; 32%) as the most commonly mutated gene, followed by KIT (4/19; 21%), SF3B1 (3/19; 16%) and NRAS (1/19; 5%). None of the cases were found to harbor BRAF, NF1 and PDGFRA mutations in melanomas of the female genital tract. However, none of the cases were found to harbor SF3B1 and KIT mutations in cutaneous melanomas, acral melanomas and melanomas of nasal cavity. Recurrent KIT mutations, as well as mutations in the less frequently mutated genes NRAS and SF3B1, were exclusively detected in vulvovaginal melanomas, but not in tumors arising in the cervix. However, recurrent KRAS mutations were detected in similar frequencies in tumors of the vulva, vagina, and cervix. Additionally, recurrent KRAS and KIT mutations occurred predominantly in polygonal and epithelioid cell types of melanoma in the female genital tract. Immunohistochemistry revealed moderate or strong cytoplasmic CD117 expression in 6 of the 19 cases (31.6%). CONCLUSIONS: We observed that gynecologic melanoma harbored distinct mutation rates in the KIT, BRAF, SF3B1, KRAS, and NRAS genes. Our findings support the notion that gynecologic melanoma is a distinct entity from non-gynecologic melanoma, and these findings offer insights into future therapeutic options for these patients.

Prevalence And Clinical Significance Of Oncogenic CD79B And MYD88 Mutations In Primary Testicular Diffuse Large B-Cell Lymphoma: A Retrospective Study In China.

PURPOSE: In this study, we investigated the prevalence of CD79B and MYD88 mutations and their relation to clinical characteristics in a cohort of Chinese patients with primary testicular diffuse large B cell lymphoma (PT-DLBCL). PATIENTS AND METHODS: We examined the mutational status of CD79B and MYD88 by Sanger sequencing, and the gene amplification and protein expression of MYD88 in tissue samples from 30 cases of PT-DLBCL by quantitative polymerase chain reaction and immunohistochemistry, respectively. Western blotting was used to analyze phosphorylated STAT3 (p-STAT3) and phosphorylated p65 (p-p65) protein expression in cell lines harboring retroviral constructs for WT MYD88 or MYD88 mutant. RESULTS: Immunophenotypically, MYD88 protein staining was positive in 26/30 (86.67%) cases, and 23/30 (76.7%) cases tested positive for p65 in the nucleus. Genetically, CD79B mutation was found in 13/30 (43.3%) cases, whereas the MYD88 L265P mutation was found in 18/30 (60.0%) cases. Interestingly, CD79B and MYD88 mutations were more prevalent in the non-germinal center B cell (GCB) subtype (83.3% and 76.9%, respectively) and were relatively rare in the GCB subtype (16.7% and 23.1%, respectively). Furthermore, although MYD88 was significantly amplified in PT-DLBCL, the amplification status showed no correlation with its mutational status and protein expression. Clinicopathological comparison between the mutant and wild-type group showed that both CD79B mutation and MYD88 L265P were not significantly correlated with age, anatomical site, Ann Arbor stage, non-GCB/GCB subtype, p65 protein expression, BCL-2 protein expression, or BCL-2/c-MYC double expression (P>0.05). Survival analyses showed that high IPI and advanced stage (stage III-IV) associated with worse outcome (P<0.05). The expression of p-STAT3 and p-p65 protein was upregulated in the mutant group, indicating that MYD88 mutant activated NF-κB and JAK-STAT3 signaling. CONCLUSION: Our results suggest that MYD88 and CD79B mutations are important drivers of immune-privileged site-associated DLBCL and highlight potential therapeutic targets for personalized treatment.

[A novel missense mutation resulting in X-linked adrenoleukodystrophy in female heterozygotes of a Chinese family].

OBJECTIVE: To identify ABCD1 gene mutation in a Chinese family with three heterozygous female patients. METHODS: Four fragments covering the entire coding sequence of the ABCD1 gene from one of the female patients were amplified by reverse transcription-PCR. The PCR products were directly sequenced. The result of sequencing was confirmed by restriction enzyme digestion of PCR products from genomic DNA. Human ABCD1 gene and ALD protein were aligned with those of rat, monkey, mouse and cattle by Clustal X 1.83. Softwares of Motif Scan, TMpred and ESYpred3D were used to predict the effect of the mutation on the structure of the ALD protein. RESULTS: A novel missense mutation, CAC to CGC, was found at codon 283 of the ABCD1 gene from the patient, resulting in the replacement of histidine by arginine. This mutation abolished an Msl I site in the gene. Her son was free from this mutation. The mutated amino acid residue (283H) was highly conservative in evolution, and the mutation caused a dramatic change in the structure of the ALD protein. CONCLUSION: Three female patients heterozygous for ABCD1 gene mutation were first reported in China, and a novel mutation, p.H283R, was identified in this X-ALD family.

[Mutation analysis of SMN gene in a patient and his family with spinal muscular atrophy].

OBJECTIVE: To perform mutation analysis and describe the genotype of the SMN gene in a patient with spinal muscular atrophy (SMA) and his family. METHODS: Deletion analysis of the SMN1 exon 7 by conventional PCR-restriction fragment length polymorphism (RFLP) and allele-specific PCR, and gene dosage of SMN1 and SMN2 by multiplex ligation-dependent probe amplification (MLPA) were performed for the patient and his parents; reverse transcriptase (RT)-PCR and sequencing were performed for the patient. To determine whether the SMN variant was exclusive to transcripts derived from SMN1, the RT-PCR product of the patient was subcloned and multiple clones were sequenced directly; PCR of SMN exon 5 from the genomic DNA of the parents and direct sequencing were performed to confirm the mutation. RESULTS: In SMN1 exon 7 deletion analysis, no homozygous deletion of the SMN1 was observed in the family; the gene dosage analysis by MLPA showed that the patient had 1 copy of SMN1 and 1 copy of SMN2 his father had 2 copies of SMN1 and 2 copies of SMN2, and his mother had 1 copy of SMN1 and no SMN2. A previously unreported missense mutation of S230L was identified from the patient and this mutation was also found in his father. CONCLUSION: A novel missense mutation of S230L was identified in the SMA family and the genotype of the family members were investigated.

[Molecular diagnosis of a Chinese pedigree with osteogenesis imperfecta type I].

OBJECTIVE: To perform molecular diagnosis for a Chinese pedigree with osteogenesis imperfecta type I. METHODS: Thirty pairs of primers were designed to amplify all the 52 exons, exon boundaries and promoter region of the COL1A1 gene from genomic DNA of peripheral blood cells of the family members. The PCR products were purified and directly sequenced. To check the mutation in normal controls, the genomic DNA from peripheral blood cells of the index patient, his mother and 60 normal controls were analyzed by amplification refractory mutation system. RESULTS: A missense mutation of GAT>CAT was identified at codon 1441 of the COL1A1 gene from the family, which resulted in the replacement of aspartic acid by histidine (D1441H). This mutation was not found in a group of 60 normal controls. CONCLUSION: The method for molecular diagnosis of osteogenesis imperfecta was established and a novel COL1A1 gene mutation, D1441H, was identified in the Chinese pedigree with osteogenesis imperfecta type I.

[Prenatal molecular diagnosis of four fetuses at high risk for X-linked adrenoleukodystrophy].

OBJECTIVE: To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy (X-ALD). METHODS: The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells. Maternal contamination was evaluated by paternity test. PCR-RFLP, sequencing and denaturing high performance liquid chromatography (DHPLC) were used to detect the ABCD1 gene of fetal genome. RESULTS: In the pedigree 1, the PCR product (799 bp) of the fetus 1 and her father (normal control) could be digested with BcnI. No P560L mutation, which was present in the index patient, was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing. In the pedigree 2, the PCR product (232 bp) of the fetus 2 and her father could not be digested with MaeI and no Q177X mutation, which was present in the propositus, was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing. In the pedigree 3, the PCR product (271 bp) was digested with AciI, the pattern of the fetus 3 and the propositus being the same, and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing. In the pedigree 4, the PCR product (269 bp) was analyzed with the DHPLC, and the pattern of elution peaks of the fetus 4 and her father was similar, but different from that of the propositus. No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing. Judging from the sex of the fetuses, fetuses 1 and 2 were normal homozygotes, fetus 3 was an ALD hemizygote, and fetus 4 was a normal hemizygote. CONCLUSION: A new protocol for X-ALD prenatal molecular diagnosis is proposed, which would ensure the accuracy of prenatal diagnosis.

[Molecular diagnosis of spinal muscular atrophy by multiplex ligation-dependent probe amplification].

OBJECTIVE: To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA). METHODS: Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR. RESULTS: In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P < 0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P < 0.01). Two of the 10 fetuses had homozygous deletion of SMN1. CONCLUSION: The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

[Prenatal diagnosis of 5 fetuses with high risk of developing spinal muscular atrophy].

OBJECTIVE: To perform prenatal diagnosis for 5 pregnant women who had given birth to children with spinal muscular atrophy (SMA). METHODS: Thirty to forty mililiters of amniotic fluid was obtained by amniocentesis under ultrasonic monitoring. DNA was extracted directly from sediment of amniotic fluid. Short tandem repeat (STR) profiling was carried out to evaluate the contamination of amniotic DNA by maternal genomic DNA. Two methods, PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR, were used to analyze exon 7 of SMN gene from amniotic DNA. RESULTS: Comparing the 16 STR sites of each fetus with those of his/her parents, there was no or little contamination of amniotic DNA by maternal genomic DNA. In conventional PCR-RFLP, part of the PCR product (189 bp) from amniotic DNA of fetus A, C, or D remained intact after digestion with Dra I, while the PCR product from amniotic DNA of fetus B or E was completely digested by Dra I. In allele-specific PCR, exon 7 of both SMN1 and SMN2 gene could be seen when amniotic DNA of fetuses A, C, or D was analyzed, while only exon 7 of SMN2 could be seen when amniotic DNA of fetuses B or E was analyzed. CONCLUSION: Homozygous deletion of SMN1 is not detected in fetuses A, C, and D, predicting that the risk of developing SMA after birth would be extremely low. Homozygous deletion of SMN1 was present in fetuses B and E suggesting high risk of developing SMA after birth.