BL-MOL-AR Project, Preliminary Results about Liquid Biopsy: Molecular Approach Experience and Research Activity in Oncological Settings.

Background Liquid biopsy is mainly used to identify tumor cells in pulmonary neoplasms. It is more often used in research than in clinical practice. The BL-MOL-AR study aims to investigate the efficacy of next-generation sequencing (NGS) and clinical interpretation of the circulating free DNA (cfDNA) levels. This study reports the preliminary results from the first samples analyzed from patients affected by various neoplasms: lung, intestinal, mammary, gastric, biliary, and cutaneous. Methods The Biopsia Liquida-Molecolare-Arezzo study aims to enroll cancer patients affected by various malignancies, including pulmonary, intestinal, advanced urothelial, biliary, breast, cutaneous, and gastric malignancies. Thirty-nine patients were included in this preliminary report. At time zero, a liquid biopsy is executed, and two types of NGS panels are performed, comprising 17 genes in panel 1, which is already used in the routine tissue setting, and 52 genes in panel 2. From the 7th month after enrollment, 10 sequential liquid biopsies are performed up to the 17th month. The variant allele frequency (%) and cfDNA levels (ng/mL) are measured in every plasmatic sample. Results The NGS results obtained by different panels are similar even though the number of mutations is more concordant for lung pathologies. There are no significant differences in the actionability levels of the identified variants. Most of the molecular profiles of liquid biopsies reflect tissue data. Conclusions Preliminary data from this study confirm the need to clarify the limitations and potential of liquid biopsy beyond the lung setting. Overall, parameters related to cfDNA levels and variant allele frequency could provide important indications for prognosis and disease monitoring.

Lab tests for MPN.

Molecular laboratory investigations for myeloproliferative neoplasm (MPN) can ideally be divided into two distincts groups, those for the detection of the BCR-ABL rearrangement (suspect of chronic myeloid leukemia) and those for the variants determination of the driver genes of the negative Philadelphia forms (MPN Ph neg). The BCR-ABL detection is based on RT-Polymerase Chain Reaction techniques and more recently on droplet digital PCR (ddPCR). For this type of analysis, combined with chromosome banding analysis (CBA) and Fluorescent in situ hybridization (FISH), it is essential to quantify BCR-ABL mutated copies by standard curve method. The investigation on driver genes for MPN Ph neg forms includes activity for erythroid forms such as Polycythemia Vera (test JAK2V617F and JAK2 exon 12), for non-erythroid forms such as essential thrombocythemia and myelofibrosis (test JAK2V617F, CALR exon 9, MPL exon 10), for "atypical" ones such as mastocytosis (cKIT D816V test) and for hypereosinophilic syndrome (FIP1L1-PDGFRalpha test). It's crucial to assign prognosis value through calculating allelic burden of JAK2 V617F variant and determining CALR esone 9 variants (type1/1like, type2/2like and atypical ones). A fundamental innovation for investigating triple negative cases for JAK2, CALR, MPL and for providing prognostic score is the use of Next Generation Sequencing panels containing high molecular risk genes as ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2. This technique allows to detect additional or subclonal mutations which are usually acquired in varying sized sub-clones of hematopoietic progenitors. These additional variants have a prognostic significance and should be indagated to exclude false negative cases.

Experience of Uncommon EGFR Mutation in Lung Scheme Quality Program: Discussing Risks and Opportunities for the Improvement of Laboratory Response.

Background The quality programs can be considered to be a valuable tool for global and individual growth. Each result, obtained by a single laboratory, contributes to define the standardization of the response. In the case of the uncommon epidermal growth factor receptor (EGFR) mutations, the molecular result is sometimes difficult to interpret in terms of biological significance and therapy choosing. The standardization effort in the diagnostic lung setting also consists of active quality program participation. Materials and Methods The quality control analysis, which is defined as a clinical case, was performed by the extraction of DNA from FFPE sections and by RT-PCR on the EGFR (exons 19, 20, 21), BRAF, and KRAS genes. The laboratory performed a validation sequencing of EGFR exon 20 with the help of the Sanger method. Results The laboratory reported positivity for EGFR exon 20 insertions and negative results for BRAF and KRAS. The quality test finished with the redaction of a report containing the recommendation to consider the efficacy of therapy with tyrosine kinase inhibitors (TKI). This specific interpretation has determined poor performance judgment by the quality provider, which explained why most of these mutations are TKI-resistant. Conclusions This experience provides an opportunity to reflect on the critical aspects of this diagnostic setting. The detection of some uncommon EGFR mutations should entail the mutation characterization, especially for the rare exon 20 insertions, of which are not classifiable as "resistant." Moreover, this experience allows reflecting on the quality program design, mandatory actions for the laboratory, and routine activity in the oncologic multidisciplinary team.

HIPK2 deficiency causes chromosomal instability by cytokinesis failure and increases tumorigenicity.

HIPK2, a cell fate decision kinase inactivated in several human cancers, is thought to exert its oncosuppressing activity through its p53-dependent and -independent apoptotic function. However, a HIPK2 role in cell proliferation has also been described. In particular, HIPK2 is required to complete cytokinesis and impaired HIPK2 expression results in cytokinesis failure and tetraploidization. Since tetraploidy may yield to aneuploidy and chromosomal instability (CIN), we asked whether unscheduled tetraploidy caused by loss of HIPK2 might contribute to tumorigenicity. Here, we show that, compared to Hipk2+/+ mouse embryo fibroblasts (MEFs), hipk2-null MEFs accumulate subtetraploid karyotypes and develop CIN. Accumulation of these defects inhibits proliferation and spontaneous immortalization of primary MEFs whereas increases tumorigenicity when MEFs are transformed by E1A and Harvey-Ras oncogenes. Upon mouse injection, E1A/Ras-transformed hipk2-null MEFs generate tumors with genetic alterations resembling those of human cancers derived by initial tetraploidization events, such as pancreatic adenocarcinoma. Thus, we evaluated HIPK2 expression in different stages of pancreatic transformation. Importantly, we found a significant correlation among reduced HIPK2 expression, high grade of malignancy, and high nuclear size, a marker of increased ploidy. Overall, these results indicate that HIPK2 acts as a caretaker gene, whose inactivation increases tumorigenicity and causes CIN by cytokinesis failure.

Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations.

TAR DNA-binding protein 43 (TDP-43) is a major component of the pathologic inclusions observed in the motor neurons of amyotrophic lateral sclerosis (ALS) patients. We examined TDP-43 expression in primary fibroblasts cultures from 22 ALS patients, including cases with SOD1 (n = 4), TARDBP (n = 4), FUS (n = 2), and C9ORF72 (n = 3) mutations and 9 patients without genetic defect. By using a phosphorylation-independent antibody, 15 patients showed notable alterations of TDP-43 level in the nuclear or cytoplasmic compartments. In particular, a marked accumulation of TDP-43 was observed in the cytoplasm of all cases with C9ORF72 and TARDBP mutations, 1 patient with FUS mutation and 3 patients without genetic defect. Patients with SOD1 mutations revealed a significant reduction of TDP-43 in the nuclei without cytoplasmic mislocalization. These changes were associated with the presence of truncated and phosphorylated TDP-43 species. Our results show that fibroblasts recapitulate some of hallmark TDP-43 abnormalities observed in neuronal cells. The reduction of full-length TDP-43 level in mutant SOD1 cells indicates that at least some SOD1 mutations alter TDP-43 metabolism.

Novel de novo heterozygous loss-of-function variants in MED13L and further delineation of the MED13L haploinsufficiency syndrome.

MED13L haploinsufficiency has recently been described as responsible for syndromic intellectual disability. We planned a search for causative gene variants in seven subjects with intellectual disability and overlapping dysmorphic facial features such as bulbous nasal tip, short mouth and straight eyebrows. We found two de novo frameshift variants in MED13L, consisting in single-nucleotide deletion (c.3765delC) and duplication (c.607dupT). A de novo nonsense variant (c.4420A>T) in MED13L was detected in a further subject in the course of routine whole-exome sequencing. By analyzing the clinical data of our patients along with those recently described in the literature, we confirm that there is a common, recognizable phenotype associated with MED13L haploinsufficiency, which includes intellectual disability and a distinctive facial appearance. Congenital heart diseases are found in some subjects with various degree of severity. Our observation of cleft palate, ataxia, epilepsy and childhood leukemia observed in single cases broadens the known clinical spectrum. Haploinsufficiency for MED13L should be considered in the differential diagnosis of the 1p36 microdeletion syndrome, due to overlapping dysmorphic facial features in some patients. The introduction of massive parallel-sequencing techniques into clinical practice is expected to allow for detection of other causative point variants in MED13L. Analysis of genomic data in connection with deep clinical evaluation of patients could elucidate genetic heterogeneity of the MED13L haploinsufficiency phenotype.

ATXN2 polyQ intermediate repeats are a modifier of ALS survival.

OBJECTIVE: To analyze the frequency and clinical characteristics of patients with amyotrophic lateral sclerosis (ALS) with intermediate-length (CAG) expansion (encoding 27-33 glutamines, polyQ) in the ATXN2 gene, in a population-based cohort of Italian patients with ALS (discovery cohort), and to replicate the findings in an independent cohort of consecutive patients from an ALS tertiary center (validation cohort). METHODS: PolyQ repeats were assessed in 672 patients with incident ALS in Piemonte and Valle d'Aosta regions, Italy, in the 2007-2012 period (discovery cohort); controls were 509 neurologically healthy age- and sex-matched subjects resident in the study area. The validation cohort included 661 patients with ALS consecutively seen between 2001 and 2013 in the ALS Clinic Center of the Catholic University in Rome, Italy. RESULTS: In the discovery cohort, the frequency of ≥31 polyQ ATNX2 repeats was significantly more common in ALS cases (19 patients vs 1 control, p = 0.0001; odds ratio 14.8, 95% confidence interval 1.9-110.8). Patients with an increased number of polyQ repeats had a shorter survival than those with <31 repeats (median survival, polyQ ≥31, 1.8 years, interquartile range [IQR] 1.3-2.2; polyQ <31, 2.7 years, IQR 1.6-5.1; p = 0.001). An increased number of polyQ repeats remained independently significant at multivariable analysis. In the validation cohort, patients with ≥31 polyQ repeats had a shorter survival than those with <31 repeats (median survival, polyQ ≥31, 2.0 years, IQR 1.5-3.4; polyQ <31, 3.2 years, IQR 2.0-6.4; p = 0.007). CONCLUSIONS: ATXN2 polyQ intermediate-length repeat is a modifier of ALS survival. Disease-modifying therapies targeted to ATXN2 represent a promising therapeutic approach for ALS.

Mutations in the 3' untranslated region of FUS causing FUS overexpression are associated with amyotrophic lateral sclerosis.

Mutations in the gene encoding fused-in-sarcoma (FUS) have been identified in a subset of patients with sporadic and familial amyotrophic lateral sclerosis (ALS). Variants in the 3' untranslated region (3'UTR) of FUS have also been reported in ALS patients, but their pathogenic role has not been assessed. We sequenced the whole 3'UTR of FUS in 420 ALS patients who were negative for mutations in the currently known ALS genes and in 480 ethnically matched controls. We detected four 3'UTR variants (c.*48 G>A, c.*59 G>A, c.*108 C>T and c.*110 G>A) in four sporadic and in one familial ALS patients compared with none in controls (P = 0.02).We investigated whether these variants impaired FUS expression in primary fibroblast cultures from three patients harbouring the c.*59 G>A, c.*108 C>T and c.*110 G>A variants, respectively. The pattern of FUS expression was also investigated in fibroblasts from one ALS patient with FUS R521C mutation, in two ALS patients without mutations in the known ALS genes and in four control individuals. By immunostaining and immunoblotting, large amounts of FUS were observed in both the cytoplasm and nuclei of mutant 3'UTR FUS fibroblasts. In FUS R521C mutant fibroblasts, we observed a slight increase of FUS in the cytoplasm associated with a remarkable loss of detection in nuclei. Our findings show that mutations in 3'UTR of FUS are overrepresented in ALS patients and result into translation de-regulation of FUS. Overexpression and mislocalization of wild-type FUS likely contribute to ALS pathogenesis in these cases.

HIPK2 controls cytokinesis and prevents tetraploidization by phosphorylating histone H2B at the midbody.

Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.

HIPK2 regulation by MDM2 determines tumor cell response to the p53-reactivating drugs nutlin-3 and RITA.

In the past few years, much effort has been devoted to show the single-target specificity of nongenotoxic, p53 reactivating compounds. However, the divergent biological responses induced by the different compounds, even in the same tumor cells, demand additional mechanistic insights, whose knowledge may lead to improved drug design or selection of the most potent drug combinations. To address the molecular mechanism underlying induction of mitotic arrest versus clinically more desirable apoptosis, we took advantage of two MDM2 antagonists, Nutlin-3 and RITA, which respectively produce these two outcomes. We show that, along with p53 reactivation, the proapoptotic p53-activator HIPK2 is degraded by MDM2 in Nutlin-3-treated cells, but activated by transiently reduced MDM2 levels in RITA-treated ones. Gain- and loss-of-function experiments revealed the functional significance of MDM2-mediated HIPK2 regulation in cell decision between mitotic arrest and apoptosis in both types of p53 reactivation. These data indicate that strategies of p53 reactivation by MDM2 inhibition should also take into consideration MDM2 targets other than p53, such as the apoptosis activator HIPK2.