Maria Anna Smolle

Long Non-Coding RNAs in Endometrial Carcinoma

Endometrial carcinoma (EC), the second most common form of gynaecological malignancy, can be divided into two distinct sub-types: Type I tumours arise from hyperplastic endometrium and typically effect women around the time of menopause, whereas type II tumours arise in postmenopausal women from atrophic endometrium. Long non-coding RNAs (lncRNAs) are a novel class of non-protein coding molecules that have recently been implicated in the pathogenesis of many types of cancer including gynaecological tumours. Although they play critical physiological roles in cellular metabolism, their expression and function are deregulated in EC compared with paired normal tissue, indicating that they may also participate in tumour initiation and progression. For instance, the lncRNA MALAT-1 is down-regulated in EC samples compared to normal or hyperplastic endometrium, whereas the lncRNA OVAL is down-regulated in type II disease but up-regulated in type I disease. Other notatble lncRNAs such as HOTAIR, H19 and SRA become up-regulated with increasing EC tumour grade and other features associated with poor prognosis. In the current review, we will examine the growing body of evidence linking deregulated lncRNAs with specific biological functions of tumour cells in EC, we will highlight associations between lncRNAs and the molecular pathways implicated in EC tumourigenesis and we will identify critical knowledge gaps that remain to be addressed.

Noncoding RNAs and immune checkpoints—clinical implications as cancer therapeutics

A major mechanism of tumor development and progression is silencing of the patients immune response to cancer‐specific antigens. Defects in the so‐called cancer immunity cycle may occur at any stage of tumor development. Within the tumor microenvironment, aberrant expression of immune checkpoint molecules with activating or inhibitory effects on T lymphocytes induces immune tolerance and cellular immune escape. Targeting immune checkpoint molecules such as programmed cell death protein 1 (PD‐1) and its ligand PD‐L1 with specific antibodies has proven to be a major advance in the treatment of several types of cancer. Another way to therapeutically influence the tumor microenvironment is by modulating the levels of microRNAs (miRNAs), small noncoding RNAs that shuttle bidirectionally between malignant and tumor microenvironmental cells. These small RNA transcripts have two features: (a) their expression is quite specific to distinct tumors, and (b) they are involved in early regulation of immune responses. Consequently, miRNAs may be ideal molecules for use in cancer therapy. Many miRNAs are aberrantly expressed in human cancer cells, opening new opportunities for cancer therapy, but the exact functions of these miRNAs and their interactions with immune checkpoint molecules have yet to be investigated. This review summarizes recently reported findings about miRNAs as modulators of immune checkpoint molecules and their potential application as cancer therapeutics in clinical practice.

Current status of long non-coding RNAs in human cancer with specific focus on colorectal cancer

The latest investigations of long non-coding RNAs (lncRNAs) have revealed their important role in human cancers. LncRNAs are larger than 200 nucleotides in length and fulfill their cellular purpose without being translated into proteins. Though the molecular functions of some lncRNAs have been elucidated, there is still a high number of lncRNAs with unknown or controversial functions. In this review, we provide an overview of different lncRNAs and their role in human cancers. In particular, we emphasize their importance in tumorigenesis of colorectal cancer, the third most common cancer worldwide.

Current Insights into Long Non-Coding RNAs (LncRNAs) in Prostate Cancer

The importance of long non-coding RNAs (lncRNAs) in the pathogenesis of various malignancies has been uncovered over the last few years. Their dysregulation often contributes to or is a result of tumour progression. In prostate cancer, the most common malignancy in men, lncRNAs can promote castration resistance, cell proliferation, invasion, and metastatic spread. Expression patterns of lncRNAs often change during tumour progression; their expression levels may constantly rise (e.g., HOX transcript antisense RNA, HOTAIR), or steadily decrease (e.g., downregulated RNA in cancer, DRAIC). In prostate cancer, lncRNAs likewise have diagnostic (e.g., prostate cancer antigen 3, PCA3), prognostic (e.g., second chromosome locus associated with prostate-1, SChLAP1), and predictive (e.g., metastasis-associated lung adenocarcinoma transcript-1, MALAT-1) functions. Considering their dynamic role in prostate cancer, lncRNAs may also serve as therapeutic targets, helping to prevent development of castration resistance, maintain stable disease, and prohibit metastatic spread.

Genetic markers of recurrence in colorectal cancer

Colorectal cancer (CRC) worldwide belongs to one of the most frequent cancers affecting both genders. Surgery and 5-fluorouracil-based adjuvant chemotherapy are recommended for patients with high-risk stage II and stage III colon carcinoma. Mutations of genes encoding for specific proteins may have an impact on the time to recurrence. These proteins act over specific signaling pathways, are implicated in metabolic processes and regulate the cell cycle. Though many retrospective studies show strong associations between genetic mutations and the clinical outcome of patients with CRC, currently no validated biomarkers are used in clinical routine settings. Therefore, large prospective validation studies should be carried out in order to strengthen the position of genetic mutations in personalized treatment of patients with CRC.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells

BACKGROUND Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

An Easy-to-Use Prognostic Model for Survival Estimation for Patients with Symptomatic Long Bone Metastases

Background: A survival estimation for patients with symptomatic long bone metastases (LBM) is crucial to prevent overtreatment and undertreatment. This study analyzed prognostic factors for overall survival and developed a simple, easy-to-use prognostic model. Methods: A multicenter retrospective study of 1,520 patients treated for symptomatic LBM between 2000 and 2013 at the radiation therapy and/or orthopaedic departments was performed. Primary tumors were categorized into 3 clinical profiles (favorable, moderate, or unfavorable) according to an existing classification system. Associations between prognostic variables and overall survival were investigated using the Kaplan-Meier method and multivariate Cox regression models. The discriminatory ability of the developed model was assessed with the Harrell C-statistic. The observed and expected survival for each survival category were compared on the basis of an external cohort. Results: Median overall survival was 7.4 months (95% confidence interval [CI], 6.7 to 8.1 months). On the basis of the independent prognostic factors, namely the clinical profile, Karnofsky Performance Score, and presence of visceral and/or brain metastases, 12 prognostic categories were created. The Harrell C-statistic was 0.70. A flowchart was developed to easily stratify patients. Using cutoff points for clinical decision-making, the 12 categories were narrowed down to 4 categories with clinical consequences. Median survival was 21.9 months (95% CI, 18.7 to 25.1 months), 10.5 months (95% CI, 7.9 to 13.1 months), 4.6 months (95% CI, 3.9 to 5.3 months), and 2.2 months (95% CI, 1.8 to 2.6 months) for the 4 categories. Conclusions: This study presents a model to easily stratify patients with symptomatic LBM according to their expected survival. The simplicity and clarity of the model facilitate and encourage its use in the routine care of patients with LBM, to provide the most appropriate treatment for each individual patient. Level of Evidence: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

MicroRNAs in Different Histologies of Soft Tissue Sarcoma: A Comprehensive Review

Soft tissue sarcomas (STS) constitute a rare tumour entity comprising over 50 histological subtypes. MicroRNAs (miRNAs) are short non-protein coding RNA molecules that regulate gene expression by targeting the 3’-untranslated region of messenger RNAs. They are involved in a variety of human diseases, including malignancies, such as endometrial cancer, osteosarcoma, bronchial carcinoma and breast cancer. In STS, various miRNAs are differentially expressed, thus contributing to development, progression and invasion. Therefore, the aim of the present review is to summarise current knowledge on the role of miRNAs in STS. Furthermore, the potential role of miRNAs as diagnostic, prognostic and predictive biomarkers is discussed.

Association of BRAF Mutations With Survival and Recurrence in Surgically Treated Patients With Metastatic Colorectal Liver Cancer

Importance BRAF mutations are reportedly associated with aggressive tumor biology. However, in contrast with primary colorectal cancer, the association of V600E and non-V600E BRAF mutations with survival and recurrence after resection of colorectal liver metastases (CRLM) has not been well studied. Objective To investigate the prognostic association of BRAF mutations with survival and recurrence independently and compared with other prognostic determinants, such as KRAS mutations. Design, Setting, and Participants In this cohort study, all patients who underwent resection for CRLM with curative intent from January 1, 2000, through December 31, 2016, at the institutions participating in the International Genetic Consortium for Colorectal Liver Metastasis and had data on BRAF and KRAS mutational status were retrospectively identified. Multivariate Cox proportional hazards regression models were used to assess long-term outcomes. Interventions Hepatectomy in patients with CRLM. Main Outcomes and Measures The association of V600E and non-V600E BRAF mutations with disease-free survival (DFS) and overall survival (OS). Results Of 853 patients who met inclusion criteria (510 men [59.8%] and 343 women [40.2%]; mean [SD] age, 60.2 [12.4] years), 849 were included in the study analyses. Forty-three (5.1%) had a mutated (mut) BRAF/wild-type (wt) KRAS (V600E and non-V600E) genotype; 480 (56.5%), a wtBRAF/wtKRAS genotype; and 326 (38.4%), a wtBRAF/mutKRAS genotype. Compared with the wtBRAF/wtKRAS genotype group, patients with a mutBRAF/wtKRAS genotype more frequently were female (27 [62.8%] vs 169 [35.2%]) and 65 years or older (22 [51.2%] vs 176 [36.9%]), had right-sided primary tumors (27 [62.8%] vs 83 [17.4%]), and presented with a metachronous liver metastasis (28 [64.3%] vs 229 [46.8%]). On multivariable analysis, V600E but not non-V600E BRAF mutation was associated with worse OS (hazard ratio [HR], 2.76; 95% CI, 1.74-4.37; P < .001) and DFS (HR, 2.04; 95% CI, 1.30-3.20; P = .002). The V600E BRAF mutation had a stronger association with OS and DFS than the KRAS mutations (&bgr; for OS, 10.15 vs 2.94; &bgr; for DFS, 7.14 vs 2.27). Conclusions and Relevance The presence of the V600E BRAF mutation was associated with worse prognosis and increased risk of recurrence. The V600E mutation was not only a stronger prognostic factor than KRAS but also was the strongest prognostic determinant in the overall cohort.

A novel mutation in ATRX associated with intellectual disability, syndromic features, and osteosarcoma

Osteosarcomas are the most frequently diagnosed primary malignancies of bone.1 They occur sporadically, although some distinct syndromes increase the risk of development, such as Li–Fraumeni, retinoblastoma, RAPADILINO (RECQL4), Bloom, and Werner syndromes.2,3 X-linked intellectual disability syndromes have a higher incidence in males.4 Examples include Lesch–Nyhan syndrome, Coffin–Lowry syndrome, and alpha-thalassemia X-linked intellectual disability (ATRX) syndrome caused by amutation in the ATRX (XNP) gene.5 A 22-year-old Caucasian man was referred to our outpatient clinic with severe knee pain, eventually diagnosed as localized osteosarcoma. He was treated according to the European and American Osteosarcoma Study Group 1 protocol with preand postoperative chemotherapy, as well as above-knee amputation. Three years after surgery, there was no evidence of local or systemic recurrence. Born with microcephaly, the patient was intellectually disabled and in need of full-time care. His younger half-brother (8a) presented with microcephaly, growth delay, and intellectual disability. While in themother’s family a relatively high incidence ofmalignancies was evident (brother [35a; pharyngeal cancer], mother [35a], and grandmother [50a, endometrial cancer]), the family histories of both fathers were inconspicuous. After collecting blood from the brothers, all genes on the X-chromosome were enriched using the Agilient SureSelect Target Enrichment System. Illumina MiSeq sequencing and variant prioristion revealed a hemizygous sequence variant within the ATRX gene (NM_000489: c.6130C>T, p.Leu2044Phe) in both brothers. This variant, which themotherwas heterozygous for, has not been reported yet in any population frequency (ExAc, ESP, 1,000 g) ormutation database. In silico predictions (SIFT, PolyPhen, LRT, Mutation taster) consistently indicated a deleterious effect of the mutation on protein function. Therefore, according to the ACMG guidelines, the variant is classified as likely pathogenic.6 Unambiguously heterogenetic mutations are causative for ATRX syndrome, characterized by distinctive craniofacial features, genital anomalies, severe developmental delays, intellectual disability, and mild-to-moderate anemia secondary to alpha-thalassemia.7 In line with the literature,8 intellectual disability and typical skeletal deformities (Fig. 1) were present in both brothers, while the mother had unspecific symptoms compatible with this trait (i.e., major depression). Overall, the genotype–phenotype correlation is rather nonspecific in patients with ATRX syndrome8 and overlaps with syndromes caused bymutations at other gene loci.9