Tindaro Giardina

Thapsigargin Modulates Osteoclastogenesis Through the Regulation of RANKL‐Induced Signaling Pathways and Reactive Oxygen Species Production

The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase‐3 activity, cytosolic Ca2+, and RANKL‐induced activation of NF‐κB and AP‐1 activities.

Detection of EGFR mutational profile by direct dideoxy sequencing in cytology and non-cytology biopsy samples

Summary Epidermal growth factor receptor (EGFR) mutational analysis is recommended in the diagnostic work-up of non-small cell lung carcinoma. The first diagnostic biopsy is usually obtained by a minimally invasive procedure, especially in patients with unresectable disease. This paper aims to compare the types of somatic EGFR mutations detected by cytology and non-cytology samples by direct dideoxy sequencing and propose practical guidelines for handling such material. Only samples with sufficient polymerase chain reaction (PCR) product were considered, a total 310 samples (302 patients), of which 168 samples were cytology material and 142 samples were non-cytology biopsy material. All samples were assessed for tumour content and bidirectional direct sequencing was performed on exons 18, 19, 20 and 21. There were 49 cases with EGFR mutation detected (16.2%), without a significant difference in the detection of mutations between either cytology or non-cytology material. EGFR mutation was detected in most sample types including endoscopic ultrasound guided FNA, bronchial washings/brushings and pleural/peritoneal fluid samples. Cytology material can provide an adequate source of material for EGFR mutational analysis, with coordinated effort between clinicians and pathologists critical for best outcome.

Molecular Profiling Reveals a Clonal Relationship Between Ovarian Mucinous Tumors and Corresponding Mural Carcinomatous Nodules

Benign or malignant mural nodules rarely occur in mucinous tumors (MTs) of the ovary and malignant nodules can show mesenchymal or epithelial differentiation. The histogenesis of mural nodules is unclear and it has been suggested that these may evolve through divergent differentiation of the mucinous neoplasm or alternatively represent a collision phenomenon. To test these possibilities we compared the molecular profile of 7 ovarian MTs with their matched mural carcinomatous nodules (MCNs) by next-generation sequencing. We found identical KRAS mutations in paired MTs and MCNs in 6 cases, one of which also showed identical CDH1 mutations in both components. In 1 tumor a KRAS mutation was detected in the mucinous neoplasm but not in the MCN; however, identical p53 mutations were present in both tumor elements. Unpaired p53 and PTEN mutations were detected only in the MCN in 2 cases, while mutations in p53 and PIK3CA genes were observed only in the MT in 2 cases. The overall comparative genomic profile was consistent with the neoplastic nature of the MCNs and strongly supported their clonal relationship with the more differentiated mucinous neoplasms. MCNs possibly develop through the acquisition of additional genomic alterations, such as p53 and PTEN mutations, resulting in an anaplastic morphologic phenotype. Our findings also suggest that ovarian MTs with MCNs often arise in KRAS mutant neoplasms. However, mutations in other genes such as PIK3CA and CDH1 may play a role in the neoplastic evolution of a subset of these tumors.

Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma

Background Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wild-type cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis. Methods A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C>T and chr5:1,295,250 C>T TERT promoter mutations. The assay was validated using 39 melanoma cell lines and 22 matched plasma and tumor samples. In addition, plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations. Results The established ddPCR assay detected TERT promoter mutations with a lower limit of detection (LOD) of 0.17%. Total concordance was demonstrated between ddPCR and Sanger sequencing in all cell lines except one, which carried a second mutation within the probe binding-site. Concordance between matched plasma and tumor tissue was 68% (15/22), with a sensitivity of 53% (95% CI, 27%-79%) and a specificity of 100% (95% CI, 59%-100%). A significantly longer PFS (p=0.028) was evident in ctDNA negative patients. Importantly, our TERT promoter mutations ddPCR assay allowed detection of ctDNA in 11 BRAF wild-type cases. Conclusions The TERT promoter mutation ddPCR assay offers a sensitive test for molecular analysis of melanoma tumors and ctDNA, with the potential to be applied to other cancers.

Locus‐specific concordance of genomic alterations between tissue and plasma circulating tumor DNA (ctDNA) in metastatic melanoma

Circulating tumor DNA (ctDNA) may serve as a surrogate to tissue biopsy for noninvasive identification of mutations across multiple genetic loci and for disease monitoring in melanoma. In this study, we compared the mutation profiles of tumor biopsies and plasma ctDNA from metastatic melanoma patients using custom sequencing panels targeting 30 melanoma‐associated genes. Somatic mutations were identified in 20 of 24 melanoma biopsies, and 16 of 20 (70%) matched‐patient plasmas had detectable ctDNA. In a subgroup of seven patients for whom matching tumor tissue and plasma were sequenced, 80% of the mutations found in tumor tissue were also detected in ctDNA. However, TERT promoter mutations were only detected by ddPCR, and promoter mutations were consistently found at lower concentrations than other driver mutations in longitudinal samples. In vitro experiments revealed that mutations in promoter regions of TERT and DPH3 are underrepresented in ctDNA. While the results underscore the utility of using ctDNA as an alternative to tissue biopsy for genetic profiling and surveillance of the disease, our study highlights the underrepresentation of promoter mutations in ctDNA and its potential impact on quantitative liquid biopsy applications.

19. Folliculotropic mycosis fungoides with cysts and comedones

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. Folliculotropic MF (FMF) is an uncommon variant, characterised by folliculocentric T-cell infiltrates. FMF shows a tendency towards more rapid progression than conventional MF and a relative resistance to skin targeted therapy, warranting its separation from conventional MF, which is reflected in the current WHO classification. The majority of cases of FMF present clinically with follicular papules, alopecia, mucinorrhoea and pruritis. In uncommon cases there can be an acneiform appearance. These cases show histological evidence of prominent milia, infundibular cysts and comedones. This uncommon form of an uncommon disease poses a significant diagnostic challenge, as there is close clinical and pathological mimicry of a number of benign folliculocentric inflammatory processes. We present a case of FMF with prominent follicular cysts, comedones and secondary granulomatous inflammation which was clinically and histologically mistaken for a benign process, including epidermal cyst, on two separate biopsies reported two years apart on two continents.

Diffuse large B cell lymphoma evolving from extranodal marginal zone lymphoma as the first presentation of complicated coeliac disease: case report and review of the literature

Complicated coeliac disease (CD) is known to predispose to enteropathy-associated T cell lymphoma (EATL) [1–3], but its association with an increased risk of B cell non-Hodgkin lymphoma (B-NHL) is not as well recognised [4]. Data on the association of B-NHL with CD are sparse, and the pathogenesis of B cell lymphoma in the context of CD remains unknown. It has been hypothesised that chronic B cell stimulation and antigenic drive play a role in lymphomagenesis. This occurs with chronic inflammation in the setting of Helicobacter pylori-associated extranodal marginal zone lymphoma and in a variety of autoimmune-related diseases. Hence, CD may favour the development of B-NHL in a similar way. The incidence of B-NHL of the small intestine arising in previously undiagnosed CD appears to be very low with only a few previous similar cases reported in the literature [5, 6]. This case highlights the importance of considering underlying undiagnosed CD in young patients developing small intestinal B-NHL.