Immunohistochemical markers as predictors of prognosis in multifocal prostate cancer.

The impact of tumor focality on prostate cancer (PCa) prognosis has been addressed in several studies with conflicting results. Tumor foci from multifocal (MF) PCa can show highly heterogeneous molecular features. Our aim was to analyze the protein expression of PTEN, SPOP, SLC45A3, ETV1, ERG and the "triple hit" (ERG overexpression, PTEN plus SLC45A3 loss) in unifocal (UF) and MF PCa, to evaluate their value as prognostic markers according to focality, and the role of tumor heterogeneity in MF disease. PTEN, SPOP, SLC45A3, ETV1 and ERG immunohistochemical expression was evaluated in 185 PCa from 9 TMAs, 51 UF and 134 MF. In a subset of 69 MF cases, the dominant and secondary foci (DF and SF) were compared. Heterogeneity was considered when both tumor foci presented different expression patterns. Relationship with clinicopathological features was also analyzed. MF PCa was diagnosed in significantly younger patients when compared to UF ones (p = 0.007). ETV1 overexpression was associated with UF disease (p = 0.028). A shorter time to PSA recurrence was related to SLC45A3 wt expression in UF PCa (p = 0.052), and to SPOP expression loss (p = 0.043) or "triple hit" phenotype in MF PCa (p = 0.041). In MF cases, PTEN loss, SLC45A3 loss and "triple hit" phenotype were associated with the DF and had significant heterogeneity. In conclusion, our results indicate that UF and MF PCa have relevant and consistent molecular differences. The analysis of an immunohistochemical panel, composed by PTEN, SPOP, SLC45A3, ETV1 and ERG, could be useful to predict outcome in MF cases.

Effects of Mediterranean diet or mindfulness-based stress reduction on fetal and neonatal brain development: a secondary analysis of a randomized clinical trial.

BACKGROUND: Maternal suboptimal nutrition and high stress levels are associated with adverse fetal and infant neurodevelopment. OBJECTIVE: This study aimed to investigate if structured lifestyle interventions involving a Mediterranean diet or mindfulness-based stress reduction during pregnancy are associated with differences in fetal and neonatal brain development. STUDY DESIGN: This was a secondary analysis of the randomized clinical trial Improving Mothers for a Better Prenatal Care Trial Barcelona that was conducted in Barcelona, Spain, from 2017 to 2020. Participants with singleton pregnancies were randomly allocated into 3 groups, namely Mediterranean diet intervention, stress reduction program, or usual care. Participants in the Mediterranean diet group received monthly individual sessions and free provision of extra-virgin olive oil and walnuts. Pregnant women in the stress reduction group underwent an 8-week mindfulness-based stress reduction program adapted for pregnancy. Magnetic resonance imaging of 90 fetal brains was performed at 36 to 39 weeks of gestation and the Neonatal Neurobehavioral Assessment Scale was completed for 692 newborns at 1 to 3 months. Fetal outcomes were the total brain volume and lobular or regional volumes obtained from a 3-dimensional reconstruction and semiautomatic segmentation of magnetic resonance images. Neonatal outcomes were the 6 clusters scores of the Neonatal Neurobehavioral Assessment Scale. Multiple regression analyses were conducted to assess the association between the interventions and the fetal and neonatal outcomes. RESULTS: When compared with the usual care group, the offspring exposed to a maternal Mediterranean diet had a larger total fetal brain volume (mean, 284.11 cm3; standard deviation, 23.92 cm3 vs 294.01 cm3; standard deviation, 26.29 cm3; P=.04), corpus callosum (mean, 1.16 cm3; standard deviation, 0.19 cm3 vs 1.26 cm3; standard deviation, 0.22 cm3; P=.03), and right frontal lobe (44.20; standard deviation, 4.09 cm3 vs 46.60; standard deviation, 4.69 cm3; P=.02) volumes based on magnetic resonance imaging measures and higher scores in the Neonatal Neurobehavioral Assessment Scale clusters of autonomic stability (mean, 7.4; standard deviation, 0.9 vs 7.6; standard deviation, 0.7; P=.04), social interaction (mean, 7.5; standard deviation, 1.5 vs 7.8; standard deviation, 1.3; P=.03), and range of state (mean, 4.3; standard deviation, 1.3 vs 4.5; standard deviation, 1.0; P=.04). When compared with the usual care group, offspring from the stress reduction group had larger fetal left anterior cingulate gyri volume (1.63; standard deviation, 0.32 m3 vs 1.79; standard deviation, 0.30 cm3; P=.03) based on magnetic resonance imaging and higher scores in the Neonatal Neurobehavioral Assessment Scale for regulation of state (mean, 6.0; standard deviation, 1.8 vs 6.5; standard deviation, 1.5; P<.01). CONCLUSION: Maternal structured lifestyle interventions involving the promotion of a Mediterranean diet or stress reduction during pregnancy were associated with changes in fetal and neonatal brain development.

An automatic pipeline for atlas-based fetal and neonatal brain segmentation and analysis.

BACKGROUND AND OBJECTIVE: The automatic segmentation of perinatal brain structures in magnetic resonance imaging (MRI) is of utmost importance for the study of brain growth and related complications. While different methods exist for adult and pediatric MRI data, there is a lack for automatic tools for the analysis of perinatal imaging. METHODS: In this work, a new pipeline for fetal and neonatal segmentation has been developed. We also report the creation of two new fetal atlases, and their use within the pipeline for atlas-based segmentation, based on novel registration methods. The pipeline is also able to extract cortical and pial surfaces and compute features, such as curvature, local gyrification index, sulcal depth, and thickness. RESULTS: Results show that the introduction of the new templates together with our segmentation strategy leads to accurate results when compared to expert annotations, as well as better performances when compared to a reference pipeline (developing Human Connectome Project (dHCP)), for both early and late-onset fetal brains. CONCLUSIONS: These findings show the potential of the presented atlases and the whole pipeline for application in both fetal, neonatal, and longitudinal studies, which could lead to dramatic improvements in the understanding of perinatal brain development.

Maternal Dietary Inflammatory Index during Pregnancy Is Associated with Perinatal Outcomes: Results from the IMPACT BCN Trial.

The information available on the effects of maternal dietary habits on systemic inflammation and adverse maternal outcomes is limited. We aimed to evaluate whether Dietary Inflammatory Index (DII) score during pregnancy is associated with maternal body mass index (BMI), Mediterranean diet (MD) adherence, and perinatal outcomes. At 19−23 weeks' gestation, 1028 pregnant women were recruited. Dietary information was assessed using a 17-item dietary score to evaluate MD adherence and a validated 151-item food frequency questionnaire. DII score was established according to 33 food and nutritional proinflammatory and anti-inflammatory items. Participants were distributed into tertiles according to the DII score, where a lower DII score (first tertile) represented an anti-inflammatory diet and the third tertile represented the more proinflammatory diet. Maternal characteristics and perinatal outcomes were collected, and newborns' birthweight percentiles were calculated. Adjusted logistic regression models were used to assess the association of the DII score with maternal and perinatal characteristics, setting the third tertile as the reference group. Women in the third tertile showed lower adherence to MD score compared to the first tertile: median (25th to 75th percentile) 9 (7 to 11) vs. 6 (4.25 to 8), p < 0.001. The proinflammatory diet was significantly associated with a higher maternal pre-pregnancy BMI (adjusted ß = 0.88; 95% CI: 0.31 to 1.45) and lower newborn's birthweight percentile (adjusted ß = −9.84th; 95% CI: −19.6 to −0.12). These data show that a proinflammatory diet profile may be associated with maternal overweight and fetal undergrowth.

SPOP and CHD1 alterations in prostate cancer: Relationship with PTEN loss, tumor grade, perineural infiltration, and PSA recurrence.

BACKGROUND: In the non-ETS fusion of prostate cancer (PCa) pathway, SPOP mutations emerge as a distinct oncogenic driver subclass. Both SPOP downregulation and mutation can lead to SPOP target stabilization promoting dysregulation of key regulatory pathways. CHD1 gene is commonly deleted in PCa. CHD1 loss significantly co-occurs with SPOP mutations, resulting in a PCa subclass with increased AR transcriptional activity and with a specific epigenetic pattern. METHODS: In this study, SPOP alterations at mutational and protein levels and CHD1 copy number alterations have been analyzed and correlated with ERG and PTEN protein expression and with the clinical pathological features of the patients. RESULTS: SPOP protein loss has been detected in 42.9% of the cases, and it has been strongly associated with PTEN protein loss (p < .001). CHD1 gene loss has been detected in 24.5% and SPOP mutations in 5.9% of the cases. Loss of CHD1 has been strongly associated with SPOP mutations (p = .003) and has shown a trend to be associated with ERG wt cancers (p = .08). The loss of SPOP protein (p = .01) and the combination of PTEN and SPOP protein loss (p = .002) were both statistically more common in grade group 5 cancers, with a prevalence of 60% and 37.5%, respectively. Furthermore, SPOP loss/PTEN loss and SPOP wt/PTEN loss phenotypes were strongly associated with extraprostatic perineural infiltration (p = .007). Strong CHD1 loss was associated with a shorter time to PSA recurrence in the univariate (p = .04), and showed a trend to be associated with the PSA recurrence risk in the multivariate analysis (p = .058). CONCLUSIONS: The results of the present study suggest that the loss of SPOP protein expression, either alone or in combination with loss of PTEN and, on the other hand, a marked loss of the CHD1 gene are very promising prognostic biomarkers in PCa.

Immunohistochemical expression of mismatch repair proteins (MSH2, MSH6, MLH1, and PMS2) in prostate cancer: correlation with grade groups (WHO 2016) and ERG and PTEN status.

The role of DNA MMR genes in prostate cancer (PrCa) is controversial, as genetic alterations leading to microsatellite instability are incompletely defined in these tumors. ERG rearrangements and PTEN loss are concomitant events in PrCa. The aim of this study has been to analyze the immunohistochemical (IHC) expression of MSH2, MSH6, MLH1, PMS2, ERG, and PTEN and their potential association with the grade group (GG) grading system (WHO 2016) and PSA recurrence in a series of 200 PrCa (PSMAR-Biobank, Barcelona, Spain). MSH2, MLH1, PMS2, and PTEN losses were documented in 8%, 5%, 2%, and 36.5%, respectively. ERG expression was found in 48%. MSH6 showed an increase of expression with respect to basal levels in 42.1% of the cases. A statistical association between MSH6 overexpression and GG5 was found (p = 0.0281). ERG-wild-type cases were associated with single MSH2 loss (p = 0.024), and MSH2 and/or MLH1 loss (p = 0.019). The percentage of cases with PTEN loss was 20.5% (8/39) in GG1, 37.6% (53/141) of clustered GG2 to 4, and 60% (12/20) of GG5 (chi-square test, p = 0.01). Thus, PTEN expression loss was statistically more frequent in the upper-grade tumors. PMS2 loss was an infrequent event, but it was statistically associated with shorter time to PSA recurrence (p = 0.011). These results suggest the existence of an alternative non-ERG pathway associated with MSH2 or MLH1 expression loss. MSH6 overexpression could be a marker of aggressiveness in PrCa. The IHC assessment of DNA MMR proteins, ERG and PTEN, could identify different altered PrCa pathways, which could aid patient stratification.

SPOP and FOXA1 mutations are associated with PSA recurrence in ERG wt tumors, and SPOP downregulation with ERG-rearranged prostate cancer.

BACKGROUND: ERG fusion-related prostate cancer (PrCa) is the most prevalent oncogenic driver subclass. SPOP, FOXA1, and IDH1 mutations are other three main oncogenic driver subclasses in non-ETS-fusion PrCa. ERG protein levels seem to be increased in SPOP-mutated cases, and different studies reported that SPOP mutations and ERG fusions are mutually exclusive. The aim of this study has been to analyze the alterations in non-ETS-oncogenic drivers in PrCa. METHODS: SPOP, FOXA1, and IDH mutations were investigated by polymerase chain reaction (PCR) and Sanger direct sequencing. ERG, SPOP, and TMPRSS2-ERG messenger RNA expression was assessed by quantitative real-time PCR from complementary DNA, and the presence of the fusion was also analyzed by nonquantitative PCR. The clinical pathological features were retrieved from the charts of the 111 patients included in the study (MARBiobanc, Barcelona, Spain). RESULTS: Loss of SPOP expression (25.2%) was associated with ERG overexpression (P = 0.0036). SPOP mutations were found in 5.4% cases, all with wild-type (wt) ERG (P = 0.007). FOXA1 mutations were found in 8.2% cases, most of them ERG wt (P = 0.06). No IDH1 mutations were found. SPOP or FOXA1 mutations were found in 1.7% of ERG-rearranged, and 34.2% of non-ERG-rearranged cases (P < 0.0001). SPOP or FOXA1 alterations (mutations or expression loss) were significantly more common in GG5, while isolated ERG overexpression was more common in GG1 tumors (P = 0.042). SPOP-or FOXA1-mutated cases were associated with a shorter time to prostate-specific antigen (PSA) recurrence in the univariate (P = 0.0009), and with the PSA recurrence risk in the multivariate (P = 0.023) analysis. CONCLUSIONS: In conclusion, SPOP and FOXA1 mutations may have prognostic value in ERG wt tumors. Interestingly, in absence of SPOP mutations, downregulation of this gene is a feature of many ERG-rearranged prostate tumors.

Strong cytoplasmic ETV1 expression has a negative impact on prostate cancer outcome.

Overexpression of ETS genes is involved in prostate cancer (PrCa), but there is little information on the non-ERG components of this family. We have investigated ETV1, ETV4, and ETV5 overexpression, with or without PTEN loss, and their association with grade group (GG), pathological stage, focality, and PSA recurrence in PrCa. ETS gene expression was analyzed by qPCR in 104 cases. ETV1 and PTEN immunohistochemistry was assessed in TMA sections from 194 additional cases (PSMAR-Biobank, Barcelona, Spain). ETS mRNA overexpression was found in 23.1%, being ETV1 the most frequently overexpressed (18.3%). ETV1 protein overexpression was detected in 30.4% cases (moderate in 19.6%, strong in 10.8%). PTEN protein expression loss was detected in 36.1% cases and was not associated with ETV1. Strong-moderate ETV1 protein overexpression reaches its highest values in GG3-4, whereas its negativity was significantly more common in GG1 tumors (p = 0.034). ETV1-overexpressing tumors were more often unifocal (p = 0.0007) and high stage (p = 0.032). PTEN loss was less common in GG1 (p = 0.012) and showed a trend to be less frequent in pT2 (p = 0.062) tumors. Strong ETV1 immunostaining (histoscore > 177) was associated with shorter time to PSA recurrence in the univariate (p = 0.002) and in the multivariate analysis (p = 0.018). Moreover, when strong ETV1 overexpression was not combined with PTEN loss, its association with PSA recurrence was even stronger (p = 0.0004). In conclusion, non-ERG ETS overexpression, particularly ETV1 overexpression, has a non-negligible role in PrCa. Strong ETV1 protein expression has a negative impact on prostate cancer outcome that seems to be independent of PTEN status.

ERG overexpression plus SLC45A3 (prostein) and PTEN expression loss: Strong association of the triple hit phenotype with an aggressive pathway of prostate cancer progression.

TMPRSS2 and SLC45A3 rearrangements may coexist in the same tumor. ERG rearrangements and PTEN loss are concomitant events in prostate cancer (PrCa), and can cooperate in progression. We have reported that mRNA expression of TMPRSS2-ERG and SLC45A3-ERG rearrangements plus PTEN loss define an aggressive tumor subset. The aim of this study has been to validate these results by immunohistochemistry in a large cohort of tumors. ERG, SLC45A3 and PTEN immunostaining and their association with pathological features and PSA progression-free survival were analyzed in 220 PrCa (PSMAR-Biobank, Barcelona, Spain). ERG protein expression was found in 46.8% and SLC45A3 and PTEN loss in 30% and 34% tumors, respectively. Single ERG positive immunostaining was associated with GS = 6 tumors (p = 0.016), double ERG+/PTEN loss with GS = 7 (p = 0.008) and Grade Group 2 (GG) or GG3 cases (p = 0.042), ERG+/SLC45A3 loss/PTEN loss ("triple hit") with GS ≥ 8 (p < 0.0001) and GG4 or GG5 tumors (p = 0.0003). None of GS = 6 nor = GG1 cases showed this combination. In the GS ≥ 8 group, ERG+ (p = 0.002), PTEN loss (p = 0.009) and "triple hit" (p = 0.003) were associated with Gleason pattern 3 component, and single SLC45A3 loss (p = 0.036) with GS ≥ 8 without pattern 3. The number of aberrant events and the triple hit were strongly associated with shorter PSA progression-free survival. In GS = 6 PrCa, single ERG+ was also associated with progression. ERG+ identifies a distinct pathway of PrCa. Additional assessment of PTEN and SLC45A3 adds relevant prognostic information. The triple hit phenotype (ERG+/SLC45A3 loss/PTEN loss) is associated with progression and could be used for patient stratification, treatment and follow-up.