Validity of whole genomes sequencing results in neoplasms in precision medicine.

OBJECTIVE: To compare the whole genomes sequencing (WGS) results in the 100K Genomes project with the results of routine molecular diagnostics in precision medicine. MATERIALS AND METHODS: We analysed 374 cancers including a high tumour mutational burden (TMB-high) subgroup, defined as >10 non-synonymous single nucleotide variations per megabase. Colon cancers were evaluated for microsatellite instability (MSI), mismatch repair (MMR) genes and NRAS, KRAS and BRAF mutations using routine molecular diagnostics. Fluorescence in-situ hybridisation/immunohistochemistry was used to evaluate the Her2Neu status in breast cancers. RESULTS: There was high correlation between WGS and routine diagnostic testing results irrespective of TMB status in colon cancers. Her2Neu status was discordant in 3 out of the 5 TMB-high breast cancers (p=0.049). The presence of ductal carcinoma in-situ correlated significantly with discordance (p=0.04). There were 3 (5%) discordant colorectal cases, all in the KRAS gene, 2 of which were from the non-invasive adenomatous component (p=0.0058). Of the 374 cases we identified 24 tumours with a TMB >10; comprising (colorectal carcinomas (CRCs) n=16, breast carcinomas n=5, bladder urothelial cell cancers n=3). Of the 16 TMB-high colorectal adenocarcinomas, 13 had MSI-high status. The same 13 had defective MMR protein expression. TMB-high colorectal cancers had 100% concordant results between WGS and NGS testing for KRAS, BRAF and NRAS (16/16). CONCLUSION: The microsatellite and mutational status of colorectal cancers evaluated by WGS seem to correlate well with the routine diagnostic testing if it is ensured that the invasive component is sequenced. Evaluation of WGS results need to be carefully correlated with histomorphology, as tumour heterogeneity/contamination with pre-malignant components needs to be taken into account.

KRAS Mutant Status May Be Associated with Distant Recurrence in Early-stage Rectal Cancer.

BACKGROUND/AIM: Total mesorectal excision combined with neo-adjuvant chemoradiotherary (CRT) and adjuvant chemotherapy, has been the standard treatment of locally advanced rectal cancer (LARC). Although TNM (Tumor, Node, Metastasis) classification for malignant Tumors is still the cornerstone in rectal cancer staging, there has been an effort to identify molecular biomarkers with additional prognostic or predictive value. MATERIALS AND METHODS: We retrospectively analyzed molecular biomarkers on prospectively collected histological specimens and clinical data from a cohort of 135 consecutive rectal cancer cases who underwent radical excision in a tertiary center between 2011-2014 (males=87, females=48, age range=22-89 years, mean=64,67 years, SD=13.40). Radiological, histopathological, molecular staging, treatment stratification by the multidisciplinary team (MDT), as well as prognostic outcome data were compared with various biomarkers including KRAS, BRAF, p16, b-catenin, MSI, MMR and MGMT. RESULTS: The mean follow-up was 39.21 months (range=5-83 months, SD=21.34). Twenty-eight cases were Stage I (20.9%), n=30 Stage II (22.4%), n=45 Stage III (33.6%) and n=31 Stage IV (23.1%). Forty specimens were KRAS-mutant (mt) (37.4%) while n=67 (62.6%) wild type (wt). KRAS mt status was associated with female sex (n=20, p=0.021) and older age (69.62 vs. 62.27, p=0.005). Stage I Early Cancer Subgroup analysis showed that KRAS mt status is associated with distant recurrence of disease (n=4, p=0.045). CONCLUSION: KRAS mt status may affect the prognosis of early rectal cancer, as this is linked with distant recurrence.

KRAS Mutant Status, p16 and ß-catenin Expression May Predict Local Recurrence in Patients Who Underwent Transanal Endoscopic Microsurgery (TEMS) for Stage I Rectal Cancer.

BACKGROUND/AIM: Transanal endoscopic microsurgery (TEMS) is emerging as an alternative treatment for rectal cancer Stage I. There remains a risk of local recurrence. The Aim of the study was to study the effect of biomarkers in local recurrence for Stage I rectal cancer following TEMS plus or minus radiotherapy. MATERIALS AND METHODS: This is a case control study where we compared 10 early rectal cancers that had recurred, against 19 cases with no recurrence, total 29 patients (age=28.25-86.87, mean age=67.92 years, SD=14.91, Male, N=18, Female, N=11). All patients underwent TEMS for radiological Stage I rectal cancer (yT1N0M0 or yT2N0M0) established with combination of magnetic resonance imaging (MRI) and endorectal ultrasound. We prospectively collected all data on tumour histology, morphological features, as well as follow-up parameters. Molecular analysis was performed to identify their status on BRAF, KRAS, p16 O6-methylguanine-DNA methyltransferase (MGMT) and ß-catenin. RESULTS: Out of 29 specimens analyzed, 19 were KRAS wild type (65.9%) and 10 mutant (34.5%). Recurrence of the tumour was noted in 10 cases (34.5%) from which 60% were pT1 (N=6) and 40% pT2 (N=4). There was a statistically significant association between KRAS mutant status and local recurrence (N=6, p=0.037). P16 expression greater than 5% (mean=10.8%, min=0, max=95) is linked with earlier recurrence within 11.70 months (N=7, p=0.004). Membranous ß-catenin expression (N=12, 48%) was also related with KRAS mutant status (p=0.006) but not with survival (p>0.05). BRAF gene was found to be wild type in all cases tested (N=23). CONCLUSION: KRAS/p16/ß-catenin could be used as a combined biomarker for prediction of local recurrence and stratification of the risk for further surgery.

BRAF V600E mutation in colorectal cancer is associated with right-sided tumours and iron deficiency anaemia.

BACKGROUND: BRAF gene encodes a serinethreonine kinase that inhibits the RAS/MAPK intracellular pathway. BRAF mutations occur at an early stage of colorectal cancer and their presence, 10-20% of colorectal cancer (CRC), is usually associated with inferior prognosis. MATERIALS AND METHODS: From 41 consecutive CRC confirmed referrals from 1,446 suspected cancer cases (mean age=67.99+13.451, male=21, female=20), we retrospectively analyzed collected data from haemoglobin (Hb) and symptoms at presentation, location of tumor and stage of the disease, including lymphovascular invasion (LVI). Gene profile analysis data (KRAS, BRAF) were retrospectively collected and associated with the presentation profile above. RESULTS: There was no significant correlation in presentation Hb levels and eventual disease staging (p>0.05 for all associations). Patients with right-sided tumours were found to have a lower Hb level than patients with either left-sided colonic or rectal tumours. Hb levels were also significantly lower in patients with the BRAF V600E mutation. KRAS status or LVI status did not have a specific correlation with Hb levels. CONCLUSION: BRAF V600E mutation might be associated with right-sided tumors and subsequently related unexplained iron-deficiency anaemia (IDA) at presentation. This finding may affect the choice of clinical strategy for investigation of unexplained IDA. Further research should be conducted in order to identify and support the potential biological explanation of the findings above.

Interaction with vascular endothelium enhances survival in primary chronic lymphocytic leukemia cells via NF-kappaB activation and de novo gene transcription.

Chronic lymphocytic leukemia (CLL) cells rapidly undergo apoptosis in vitro, suggesting that the in vivo microenvironment provides crucial antiapoptotic signals. Overexpression of the antiapoptotic proteins Bcl-2 and Mcl-1 is a hallmark of CLL, and their expression is further enhanced in the lymphoid tissues. However, the high levels of Mcl-1 found in peripheral blood samples, coupled with its short half-life, led us to hypothesize that it must be actively maintained in the peripheral circulation. Coculture of CLL cells with human vascular endothelial cells significantly enhanced tumor cell survival, an effect that was not observed with normal B cells. This was associated with elevated levels of the antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-X(L) and marked increased expression of CD38 and CD49d, both of which are associated with clinically aggressive disease. Because CD38, CD49d, and some Bcl-2 family genes are transcriptional targets for NF-κB, we assessed NF-κB activation following coculture with endothelial cells. DNA binding of the NF-κB subunit Rel A was significantly increased and strongly correlated with changes in transcription of CD38, CD49d, BCL2, MCL1, and BCLXL, effects that were reversed by a peptide inhibitor of Rel A. These effects were not observed following coculture with nonendothelial cell lines. Therefore, CLL cells receive specific survival signals following interaction with endothelial cells mediated through the activation of NF-κB and the induction of downstream target genes. This type of interaction in the peripheral vasculature may explain the constitutive NF-κB activation and the overexpression of Bcl-2 family proteins commonly seen in this disease.

Treatment of autoimmune hyperthyroidism in a murine model of Graves' disease with tumor necrosis factor-family ligand inhibitors suggests a key role for B cell activating factor in disease pathology.

Hyperthyroid Graves' disease is a common autoimmune disorder mediated by agonistic antibodies to the TSH receptor, termed thyroid stimulating antibodies (TSAbs). Recently members of the TNF superfamily, B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), have been identified along with their receptors, B cell maturation antigen and transmembrane activator and calcium-modulator and cyclophilin ligand interactor, and the BAFF-specific receptor. BAFF is a fundamental B cell survival/maturation factor, and both BAFF and APRIL have been implicated in antibody production. We investigated the effect of interfering with BAFF- and APRIL-mediated signals in an induced model of Graves' disease by blockade of these factors using soluble decoy receptors. In a therapeutic setting in mice with established hyperthyroidism, we show that blockade of BAFF or BAFF+APRIL with BAFF-specific receptor-Fc and B cell maturation antigen-Fc, respectively, leads to significant reductions in the induced hyperthyroidism. This was supported by a parallel pattern of declining TSAbs in the responding animals. Histopathological analysis of splenic sections from treated animals revealed marked reductions in the B cell follicle regions, but staining with anti-CD138 revealed the persistence of plasma cells. Thus, the reductions in TSAbs in the treated animals were not related to overall plasma cell numbers in the secondary lymphoid organs. Our results are the first to demonstrate attenuation of established hyperthyroidism by therapeutic intervention aimed at autoreactive B cells and indicate that both BAFF and APRIL appear to play important roles in the development and survival of the autoantibody producing cells in this model.

Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways.

The thyroid target Ag for disease-inducing autoantibodies in Graves' disease is the receptor for thyroid-stimulating hormone (TSH), but little is known about the molecular basis of this pathogenic Ab response. We describe the characteristics of two high- affinity mAbs developed from an experimental murine model of hyperthyroid Graves' disease that exhibit potent thyroid-stimulating activity. Nanogram concentrations of the IgG mAbs KSAb1 and KSAb2 and their Fab induce full stimulation of the TSH receptor that is matched by the ligand TSH and, thus, act as full agonists for the receptor. However, KSAb1 and KSAb2 display differential activities in their ability to block TSH-mediated stimulation of the receptor, indicating subtle differences in their biological properties. In displacement studies, IgG and Fabs of KSAb1 and KSAb2 compete with Graves' disease autoantibodies as well as thyroid-blocking Abs present in some hypothyroid patients, indicating a close relationship between these autoimmune determinants on the receptor. In passive transfer studies, single injections of microgram quantities of KSAb1 or KSAb2 IgG led to rapid elevation of serum thyroxine and a hyperthyroid state that was maintained for a number of days. The thyroid glands showed evidence of cell necrosis, but there was no accompanying mononuclear cell infiltrate. In studying their receptor activation pathways, both KSAb1 and KSAb2 provoked phosphorylation of the intracellular ERK1/2 pathway in primary thyrocytes, indicating that multiple signaling pathways may participate in the pathogenesis of Graves' disease. In summary, our findings emphasize the similarities of the experimental mouse model in reproducing the human disorder and provide improved means for characterizing the molecular basis of this pathogenic response.

Expression of Ras GTPase isoforms in normal and diseased pancreas.

BACKGROUND: Ki-Ras is well studied in its oncogenic form in relation to pancreatic pathologies. However, the individual contribution of each of the wild-type Ras isoforms (Ha-, Ki-, and N-) in pancreatic cells in health and disease is unknown. METHODS: Archival formalin-fixed, paraffin-embedded specimens of normal (n = 6) and malignant pancreas (n = 35) were used for immuno-histochemical detection of Ras isoforms using a modified polymer system. In addition, immunogold labelling for Ras isoforms was done for subcellular localisation under electron microscopy. RESULTS: Pancreatic ductal cells expressed Ha-Ras in the cytoplasm, with Ki-Ras in the apical region and N-Ras (50% of cases) in a supranuclear distribution. Pancreatic acinar cells express all three isoforms with some nuclear expression of Ki-Ras and supranuclear expression of N-Ras. Islets show Ki- and Ha-Ras mainly with differential expression of Ha-Ras (beta cells showing less Ha-Ras and more Ki-Ras than alpha cells). Electron microscopy shows that Ha-Ras is mainly localised in the endoplasmic reticulum and Golgi apparatus of the acinar cells with some plasma membrane localisation of Ki-Ras in the ductal cells. There was no change in any of the Ras isoform expression in the ductal or acinar cells in various malignancies studied (Mann-Whitney U test, p > 0.1). CONCLUSIONS: Ras isoforms have distinct and separate cellular and subcellular distribution that may persist even in the malignantly transformed state. Understanding this distinct functional distribution patterns in detail is an essential step if mutant Ki-Ras is to be targeted in the pancreas by genetic or molecular therapeutic tools.

Placental expression of interferon-gamma (IFN-gamma) and its receptor IFN-gamma R2 fail to switch from early hypoxic to late normotensive development in preeclampsia.

The inability of the mother to switch from T helper cell type 1 (Th1) to Th2 cytokine profiles at the fetal-maternal interface has been proposed as one of the primary causes of miscarriage, intrauterine growth restriction (IUGR), and preeclampsia (PE). The Th1 [interferon-gamma (IFN-gamma), TNF-alpha, and IL-12] and Th2 (IL-4 and IL-10) cytokines have opposite effects on human pregnancy. Leukemia inhibitory factor (LIF) promotes embryo implantation and sustains pregnancy, whereas IFN-gamma and TNF-alpha are detrimental to pregnancy. Both IFN-gamma and LIF are produced by maternal cells and tissues at the fetal-maternal interface, whereas the IFN-gamma receptors (IFN-gamma R1 and IFN-gamma R2) and LIF receptor are abundantly expressed on the surface of placental trophoblasts. The effect of IFN-gamma on T lymphocyte activation is influenced by the relative membrane density of its two receptors, particularly IFN-gamma R2. In this study we report that in PE (25-40 wk gestation) and PE complicated by IUGR, IFN-gamma R2 protein expression is severely down-regulated and is similar to that observed in early placenta (7-10 wk gestation) developing under low O(2) tension. IFN-gamma production was found to be inversely related to the IFN-gamma R2 protein expression, and LIF receptor protein expression in PE mimicked that in early placental development. These results show that in PE, placental trophoblasts fail to establish an early to late switch with respect to IFN-gamma and IFN-gamma R2 expression. This supports the hypothesis that trophoblasts control the polarization of maternal immune effectors and cytokine profiles at the fetal-maternal interface that could be subject to oxidative stress in PE.

Expression of Ras GTPases in normal kidney and in glomerulonephritis.

BACKGROUND: Small monomeric Ras GTPases play critical and specific roles in the control of cellular proliferation and apoptosis but the expression of the three Ras isoforms (Ha-Ras, Ki-Ras and N-Ras) in human renal tissue is unknown. This work is an immunohistochemical study of Ras expression in normal renal tissue and in membranous glomerulonephritis (MGN), IgA nephropathy (IgAN) and IgA-negative mesangioproliferative glomerulonephritis (MPGN). METHODS: Formalin-fixed, paraffin-embedded tissue was stained using pan-Ras monoclonal antibody (mAb) and Ras isoform-specific mAb. Detection employed a (DAKO Envision) modified polymer system. RESULTS: The expression of Ras isoforms in normal human kidney was cell-specific. For example, N-Ras was detected in tubule epithelial cells but not in glomerular or interstitial cells. Ki-Ras was expressed in mesangial cells, interstitial cells and in proximal convoluted tubule cells (PCT) (particularly localized at brush borders) and in collecting duct cells (CD) (localized to cell membranes) but not in podocytes. Cytoplasmic Ha-Ras was detected in all the above cell types except podocytes. MGN was associated with podocyte expression of all three Ras isoforms and with reduced mesangial cell expression of Ha-Ras and Ki-Ras. IgAN was characterized by podocyte expression of Ha-Ras (but not Ki-Ras) and reduced mesangial cell expression of Ki-Ras without alterations in mesangial Ha-Ras expression. MPGN was associated with reduced mesangial cell Ha-Ras and Ki-Ras expression without significant podocyte Ras expression. CONCLUSION: These disease-specific and isoform-specific alterations in Ras expression may be of significance in pathogenesis and warrant further functional investigation.