Next-generation sequencing: advances and applications in cancer diagnosis

Simona Serratì, Simona De Summa, Brunella Pilato, Daniela Petriella, Rosanna Lacalamita, Stefania Tommasi, Rosamaria Pinto Molecular Genetics Laboratory, IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy Abstract: Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment. Keywords: hereditary breast cancer, melanoma, prostate cancer, thyroid cancer, lung cancer, colorectal cancer, hematologic cance

Unsuspected Diagnosis of Uterine Leiomyosarcoma after Laparoscopic Myomectomy

Study Objective: To evaluate the risk of peritoneal dissemination after laparoscopic myomectomy and morcellment for unsuspected sarcoma. Design: Retrospective analysis of three consecutive cases of uterine leiomyosarcoma. Setting: University-affiliated hospital. Patients: Between January 2000 and December 2008, 588 patients had laparoscopic myomectomy at the Department of Obstetrics and Gynecology, University of Bari, Italy and among these 3 cases (0.5%) of uterine leiomyosarcoma were diagnosed at pathologic examination. Intervention: Following diagnosis of malignant condition all patients were extensively staged with pelvic examination, transvaginal ultrasound, hysteroscopy, Pap smear and total-body CT scan that did not revealed any sign of persistent disease. Within 28 days from primary surgery all patients, after informed consent, were submitted to a surgical staging including total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, resection of all trocar ports, pelvic lymphadenectomy and peritoneal washings. Measurements and Main Results: Pathologic examination did not revealed persistent tumor in the uterus, and no sign of peritoneal disease (negative cytology, adnexae and nodes). Conclusion: Uterine sarcomas are rare tumors, ultrasound and clinical findings are similar to their benign counterpart (uterine fibroids) and diagnosis is almost always made after pathologic examination. With the wide development of endoscopic procedures for surgical treatment of uterine fibroids the management of an unsuspected sarcoma after laparoscopy will be more common in the future. However, based on the reported cases no increased risk of dissemination within the abdominal cavity can be anticipated. This condition raises the question wheather laparoscopic procedure (gas insufflation and morcellment) may increase the risk of tumor dissemination within the abdominal cavity

Unsuspected Diagnosis of Uterine Leiomyosarcoma after Laparoscopic Myomectomy

Study Objective: To evaluate the risk of peritoneal dissemination after laparoscopic myomectomy and morcellment for unsuspected sarcoma. Design: Retrospective analysis of three consecutive cases of uterine leiomyosarcoma. Setting: University-affiliated hospital. Patients: Between January 2000 and December 2008, 588 patients had laparoscopic myomectomy at the Department of Obstetrics and Gynecology, University of Bari, Italy and among these 3 cases (0.5%) of uterine leiomyosarcoma were diagnosed at pathologic examination. Intervention: Following diagnosis of malignant condition all patients were extensively staged with pelvic examination, transvaginal ultrasound, hysteroscopy, Pap smear and total-body CT scan that did not revealed any sign of persistent disease. Within 28 days from primary surgery all patients, after informed consent, were submitted to a surgical staging including total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, resection of all trocar ports, pelvic lymphadenectomy and peritoneal washings. Measurements and Main Results: Pathologic examination did not revealed persistent tumor in the uterus, and no sign of peritoneal disease (negative cytology, adnexae and nodes). Conclusion: Uterine sarcomas are rare tumors, ultrasound and clinical findings are similar to their benign counterpart (uterine fibroids) and diagnosis is almost always made after pathologic examination. With the wide development of endoscopic procedures for surgical treatment of uterine fibroids the management of an unsuspected sarcoma after laparoscopy will be more common in the future. However, based on the reported cases no increased risk of dissemination within the abdominal cavity can be anticipated. This condition raises the question wheather laparoscopic procedure (gas insufflation and morcellment) may increase the risk of tumor dissemination within the abdominal cavity

Six low-penetrance SNPs for the estimation of breast cancer heritability: A family-based study in Caucasian Italian patients

Breast cancer is a malignancy with a strong heritable component. Genetic counseling has been principally focused on families carrying high-penetrance breast cancer 1/2, early onset genes. Current modeling suggests that the majority of the unexplained fraction of familial risk is likely to be explained by a polygenic model. The aim of the present study was to estimate the heritability (h2) of breast cancer susceptibility through the analysis of 6 single nucleotide polymorphisms (SNPs), nuclear mitotic apparatus protein 1, cyclin D1, cytochrome C oxidase copper chaperone, fibroblast growth factor receptor 2, TOX high mobility group box family member 3 and solute carrier family 4 member 7. These 6 SNPs, previously identified by genome-wide association studies, were considered to evaluate the additive and common environmental components that contribute to the development of breast cancer in nuclear (pedigrees including only first degree relationships) and in extended families (with at most third degree relationships). A total of 22 extended pedigrees, subsequently split into 52 nuclear pedigrees were analyzed. An example of splitting process from extended to nuclear pedigree is shown in Fig. 1. Firstly, an underline latent continuous trait (Y*) using breast cancer status and information of 6 breast cancer-associated SNPs was calculated. This novel trait summarized the susceptibility of breast cancer in each individual. Secondly, the h2 of Y* was estimated using an additive polygenic-common environment-unique error model. h2 was evaluated in extended and immediate pedigrees, obtaining comparable results. h2 accounts for ~40% of the total phenotypic variance, indicating a fairly strong additive genetic effect of breast cancer susceptibility. The present study indicated the importance of the evaluation and consideration of these six SNPs, which can be used as instrumental variables in order to obtain improved genetic models that are useful for h2 analysis

Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain-loss of function of pentraxin 3 and matrix metalloproteinase 12

OBJECTIVE: Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo. METHODS: Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice. RESULTS: Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti-fibroblast growth factor 2/anti-vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene-silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice. CONCLUSION: Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge

Systemic sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a connective tissue growth factor (CCN2)/transforming growth factor β-dependent mesenchymal-to-mesenchymal transition

OBJECTIVE: Clinical evidence suggests that the vascular abnormalities of systemic sclerosis (SSc) precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive, although they have been searched for intensively. Since we had previously shown that connective tissue growth factor (CCN2), endowed with fibroblast-oriented activities, was overexpressed by endothelial cells (ECs) from SSc patients, we undertook this study to investigate its role and mechanisms in fibroblast activation. METHODS: Normal fibroblasts were challenged with conditioned medium of normal microvascular ECs (MVECs) and MVECs obtained from SSc patients with the diffuse form of the disease. Fibroblast invasion was studied using the Boyden chamber Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-smooth muscle actin (α-SMA), vimentin, and type I collagen. Matrix metalloproteinase (MMP) production was evaluated by zymography and reverse transcription-polymerase chain reaction. Signal transduction and activation of the small GTPases RhoA and Rac1 were studied by Western blotting. Inhibition of SSc MVEC conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and the transforming growth factor β (TGFβ) antagonist peptide p17. RESULTS: SSc MVEC CCN2 stimulated fibroblast activation and invasion. Such activities depended on CCN2-induced overexpression of TGFβ and its type I, II, and III receptors combined with overproduction of MMP-2 and MMP-9 gelatinases. All of these effects were reversed by the TGFβ antagonist peptide p17. Motility increase required Rac1 activation and RhoA inhibition and was inhibited by an MMP inhibitor. These features connoted a mesenchymal style of cell invasion. Since fibroblast activation also fostered overexpression of α-SMA, vimentin, and type I collagen, the CCN2-dependent increase in fibroblast activities recapitulated the characteristics of a mesenchymal-to-mesenchymal transition. CONCLUSION: SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition

Comparison of Two Different Dosing Regimens with Lymecycline, in Association with Adapalene, in Inflammatory Acne

Combined treatment with oral lymecycline and topical adapalene has been shown to induce greater and faster improvement of acne than monotherapy with lymecycline. We wanted to evaluate the effects of combined therapy with topical adapalene (cream or gel) plus oral lymecycline used at different dosages (group A: 300mg/day for 2 weeks and then 150mg/day for 14 weeks; group B: 300mg/day for 16 weeks) in 242 patients with inflammatory acne. Both dosage regimens were well tolerated in the majority of patients and significantly improved both the acne lesion count and seborrhoea. There was a trend towards a greater reduction of seborrhoea and nodules in patients of group B. After the initial 16-week phase, patients entered an 8-week follow-up phase, consisting of the use of adapalene monotherapy. The results obtained after this phase confirm the therapeutic value and the favourable tolerability of adapalene as maintenance treatment after successful treatment of inflammatory acne. At the same time, a significantly reduced count of comedones was observed in patients of group B as compared with group A, suggesting that prolonged use of high-dose lymecycline may have a more pronounced influence on comedogenesis