Ramon Lago‐Leston

We aimed to identify common mCRC profiles associated with a discordant mutational status of RAS between the standard of care (SoC) tumour tissue tests and ctDNA tests to understand ctDNA detection and improve treatment responses. This was a multicentre, retrospective and prospective study. A total of 366 Spanish mCRC patients were independently recruited. BEAMing ddPCR technology was employed to detect ctDNA RAS mutations, and logistic regression analyses were performed to investigate clinicopathological factors associated with discordance. The highest concordance ratios were observed in profiles with multiple metastatic sites when the liver was present (89.7%; 95% CI 84.8–93.2), profiles with synchronous disease without primary tumour resection (90.2%; 95% CI 83.6–94.3) and profiles with mCRC originating in the left colon (91.3%; 95% CI 85.0–95.0). Metachronous disease originating in the right colon (OR = 6.1; 95% CI 1.7–26.5; p-value = 0.006) or rectum (OR = 5.0; 95% CI 1.5–17.8; p-value = 0.009) showed the highest probability of discrepancies. Primary tumour resection and a higher frequency of single metastases in the peritoneum or lungs in these patients were associated with reduced plasmatic mutation allele fractions (MAFs) and an increased probability of showing false-negative genotypes. Additional testing of patients with mCRC originating in the right colon or rectum with a single non-mutated ctDNA test is advised before the choice of therapy.

Molecular profiling of circulating cell-free DNA (cfDNA) has shown utility for the management of colorectal cancer (CRC). TruSight Tumor 170 (TST170) is a next-generation sequencing (NGS) panel that covers 170 cancer-related genes, including KRAS, which is a key driver gene in CRC. We evaluated the capacity of TST170 to detect gene variants in cfDNA from a retrospective cohort of 20 metastatic CRC patients with known KRAS variants in tumor tissue and in cfDNA previously analyzed by pyrosequencing and BEAMing, respectively. The cfDNA of most of the patients (95%) was successfully sequenced. We frequently detected variants with clinical significance in KRAS (79%, 15/19) and PIK3CA (26%, 5/19) genes. Variants with potential clinical significance were also identified in another 27 cancer genes, such as APC. The type of KRAS variant detected in cfDNA by TST170 showed high concordance with those detected in tumor tissue (77%), and very high concordance with cfDNA analyzed by BEAMing (94%). The variant allele fractions for KRAS obtained in cfDNA by TST170 and BEAMing correlated strongly. This proof-of-principle study indicates that targeted NGS analysis of cfDNA with TST170 could be useful for non-invasive detection of gene variants in metastatic CRC patients, providing an assay that could be easily implemented for detecting somatic alterations in the clinic.

Background: Recent studies showed a relevant role of hematogenous spread in ovarian cancer and the interest of circulating tumor cells (CTCs) monitoring as a prognosis marker. The aim of the present study was the characterization of CTCs from ovarian cancer patients, paying special attention to cell plasticity characteristics to better understand the biology of these cells. Methods: CTCs isolation was carried out in 38 patients with advanced high-grade serous ovarian cancer using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize these cells. Results: Epithelial CTCs were found in 21% of patients, being their presence higher in patients with extraperitoneal metastasis. Importantly, this population was characterized by the expression of epithelial markers as MUC1 and CK19, but also by genes associated with mesenchymal and more malignant features as TIMP1, CXCR4 and the stem markers CD24 and CD44. In addition, we evidenced the relevance of TIMP1 expression to promote tumor proliferation, suggesting its interest as a therapeutic target. Conclusions: Overall, we evidenced the utility of the molecular characterization of EpCAM⁺ CTCs from advanced ovarian cancer patients to identify biomarkers with potential applicability for disseminated disease detection and as therapeutic targets such as TIMP1.

Circulating tumor cell (CTC) enumeration has emerged as a powerful biomarker for the assessment of prognosis and the response to treatment in metastatic breast cancer (MBC). Moreover, clinical evidences show that CTC-cluster counts add prognostic information to CTC enumeration, however, their significance is not well understood, and more clinical evidences are needed. We aim to evaluate the prognostic value of longitudinally collected single CTCs and CTC-clusters in a heterogeneous real-world cohort of 54 MBC patients. Blood samples were longitudinally collected at baseline and follow up. CTC and CTC-cluster enumeration was performed using the CellSearch^® system. Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Cox proportional hazards modelling. Elevated CTC counts and CTC-clusters at baseline were significantly associated with a shorter survival time. In joint analysis, patients with high CTC counts and CTC-cluster at baseline were at a higher risk of progression and death, and longitudinal analysis showed that patients with CTC-clusters had significantly shorter survival compared to patients without clusters. Moreover, patients with CTC-cluster of a larger size were at a higher risk of death. A longitudinal analysis of a real-world cohort of MBC patients indicates that CTC-clusters analysis provides additional prognostic value to single CTC enumeration, and that CTC-cluster size correlates with patient outcome.

Traditionally, studies to address the characterization of mechanisms promoting tumor aggressiveness and progression have been focused only on primary tumor analyses, which could provide relevant information but have limitations to really characterize the more aggressive tumor population. To overcome these limitations, circulating tumor cells (CTCs) represent a noninvasive and valuable tool for real-time profiling of disseminated tumor cells. Therefore, the aim of the present study was to explore the value of CTC enumeration and characterization to identify markers associated with the outcome and the aggressiveness of triple-negative breast cancer (TNBC). For that aim, the CTC population from 32 patients diagnosed with TNBC was isolated and characterized. This population showed important cell plasticity in terms of expression of epithelia/mesenchymal and stemness markers, suggesting the relevance of epithelial to mesenchymal transition (EMT) intermediate phenotypes for efficient tumor dissemination. Importantly, the CTC signature demonstrated prognostic value to predict the patients’ outcome and pointed to a relevant role of tissue inhibitor of metalloproteinases 1 (TIMP1) and androgen receptor (AR) for TNBC biology. Furthermore, we also analyzed the usefulness of the AR and TIMP1 blockade to target TNBC proliferation and dissemination using in vitro and in vivo zebra fish and mouse models. Overall, the molecular characterization of CTCs from advanced TNBC patients identifies highly specific biomarkers with potential applicability as noninvasive prognostic markers and reinforced the value of TIMP1 and AR as potential therapeutic targets to tackle the most aggressive breast cancer.

We detail here the clinical description and the family genetic study of a male patient with global developmental delay, disruptive and obsessive behaviors and minor dysmorphic features and a combination of two rare genetic variants: a maternally inherited 16p13.11‐p12.3 duplication and a de novo 12p12.1 deletion affecting SOX5. The 16p13.11 microduplication has been implicated in several neurodevelopmental and behavioral disorders and is characterized by variable expressivity and incomplete penetrance. The causes of this variation in phenotypic expression are not fully clear, representing a challenge in genetic diagnosis and counseling. However, several authors have proposed the two‐hit model as one of the underlying mechanisms for this phenotypic heterogeneity. Our data could also support this two‐hit model in which the 16p13.11‐p12.3 duplication might contribute to the phenotype, not only as a single event but also in association with the SOX5 deletion. The SOX5 gene plays important roles in various developmental processes and has been associated with several neurodevelopmental disorders, mainly intellectual disability, developmental delay and language and/or speech delay as well as with behavior problems and dysmorphic features. However, many of the physical features and behavioral manifestations as well as language deficiencies present in our patient are consistent with those previously reported for SOX5 deletions. Patients carrying multiple genomic variants, as the one presented here, illustrate the difficulty in analyzing genotypes when the contribution of each variant results in overlapping phenotypes and/or, alternatively, in the modification of the clinical manifestations defined by the coexisting variant.

Background: Milroy and Milroy-like disease are rare disorders characterised by congenital lymphoedema caused by dysfunctional lymphatic vessel formation. Loss of extracellular response mediated by vascular endothelial growth factor receptor 3 (VEGFR-3) is associated with Milroy disease, and VEGFR-3 gene is mutated in around 70% of the cases diagnosed. The only genetic alteration known to be associated with Milroy-like disease was recently identified in a family with a frameshift mutation in vascular endothelial growth factor C (VEGFC) gene, which encodes a VEGFR3 ligand. Methods and results: We report a newborn patient with an external phenotype consistent with Milroy disease and a truncating mutation (p.R210X) in the VEGFC gene detected by exome sequence analysis. Subsequent analysis, by lymphoscintigraphic scan, performed for research purposes, allowed us to correct the diagnosis, confirming patient’s disease as Milroy-like. The mutation segregates with the phenotype in the family according to a dominant model with full penetrance. Conclusions: The clinical presentation, similar to Milroy disease, indicates an overlapping of the external phenotype of both diseases, suggesting that genetic analysis of VEGFC would be useful in diagnosing patients that present with Milroy features but have no mutation in VEGFR-3. Establishing a well-defined genetic pattern would help with differential diagnosis.

Summary: Objective  Mutations in SLC16A2, the gene encoding the thyroid hormone (TH)‐specific transporter monocarboxylate transporter 8 (MCT8), result in a thyroid phenotype and severe mental retardation caused by neuronal TH deficiency. These mutational effects raise the question of whether polymorphic variation in SLC16A2 may also be associated with differences in serum levels of TH and/or TSH.Design  This is the first major study of the frequency of the SLC16A2 rs6647476 single nucleotide polymorphism (SNP) (amino acid change Ser107Pro). We also studied the relationships of SLC16A2 genetic variants with serum levels of TSH, T4 and T3, with their mRNA expression and with expression of the TH‐responsive genes ZAKI‐4 and BTEB in white blood cells. Experiments in cultured fibroblasts were carried out to ascertain the dynamics of the T3 response.Methods  A total of 276 men were studied. Genotyping of the S107P SNP was carried out using polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP); serum hormone levels were determined by chemiluminescence; expression of mRNA was quantified by real‐time PCR.Results  The SLC16A2 S107P SNP was found in 36% of Galician males. With the present sample size we did not find any association of this polymorphism with variability in serum levels of TSH, free T4 (fT4) or fT3, or with basal expression of mRNA for SLC16A2 or the two TH‐responsive genes ZAKI‐4 and BTEB, either in white blood cells or in cultured human fibroblasts from either Ser107 or Pro107 genotypes under T3 stimulation.Conclusions  The S107P change in MCT8 is frequent in the male population in Galicia. In the population studied in this report an association with a thyroid phenotype was not demonstrated, even though the S107P SNP causes an important amino acid change.

Hypogonadotrophic hypogonadism is associated with uncontrolled diabetes mellitus. Hyperglycaemia is a unique metabolic abnormality of the hyperglycaemic hyperosmolar nonketotic state (HHNKS) and, as glucose availability regulates gonadotrophin release, we investigated whether gonadotrophin release is inhibited in diabetic women with HHNKS, and whether hyperglycaemia, hypernatraemia or both inhibit in vitro gonadotrophin-releasing hormone (GnRH) expression in GT1-7 neurones. Three groups of postmenopausal women were studied: nine diabetics with HHNKS, nine hospitalised ill nondiabetics and 15 healthy women. In addition, the effects of glucose (5.55, 33.3, 66.6 mmol/l) and sodium chloride (150 and 170 mmol/l) on GnRH expression were investigated using GT1-7 neurones. Postmenopausal diabetics with HHNKS showed a decrease in serum levels of luteinising hormone (diabetic HHNKS 2.2 +/- 0.9 IU/l versus ill nondiabetic 21.0 +/- 2.3 IU/l and healthy controls 20.9 +/- 2.8 IU/l, P < 0.01), follicle-stimulating hormone (diabetic HHNKS 8.2 +/- 2.1 IU/l versus ill nondiabetic 50.4 +/- 9.1 IU/l and controls 60.2 +/- 6.9 IU/l, P < 0.01) and free 3,5,3'-triiodothyronine (diabetic HHNKS 1.48 +/- 0.57 pmol/l versus ill nondiabetic 4.28 +/- 0.26 pmol/l and controls 3.88 +/- 0.11 pmol/l, P < 0.01). The plasma cortisol level was higher in both diabetic (985 +/- 130 nmol/l) and ill nondiabetic (726 +/- 52 nmol/l) women than in healthy women (512 +/- 47 nmol/l), but no differences were observed in plasma oestradiol, thyroid-stimulating hormone or free thyroxine. In vitro GT1-7 neurones expressed three-fold less GnRH at 170 mmol/l than at 150 mmol/l NaCl, whereas changing glucose concentrations in the culture medium did not affect GnRH expression. In conclusion, postmenopausal diabetic women with HHNKS show decreased serum gonadotrophin levels, and severe hypernatraemia may participate in the hypogonadotropism observed in HHNKS.