Ulrika Axcrona

Probability of Metachronous Testicular Cancer in Patients With Biopsy-Proven Intratubular Germ Cell Neoplasia Depends on First-Time Treatment of Germ Cell Cancer

PURPOSE To evaluate the probability of subsequent testicular cancer (STC) in patients with intratubular germ cell neoplasia unclassified (IGCNU) treated for first-time invasive germ cell cancer. PATIENTS AND METHODS Sixty-one patients with germ cell testicular cancer or extragonadal germ cell cancer received follow-up from diagnosis of IGCNU to development of STC, initiation of IGCNU-definitive treatment (orchiectomy/radiotherapy), emigration, death, or end of follow-up. The probability of STC was assessed in subgroups according to chemotherapy burden. RESULTS The probability of STC in the nonexposed patients was significantly increased compared with those exposed to chemotherapy (P = .05; 5-year probability of 54% [95% CI, 33% to 78%] and 23% [95% CI, 11% to 45%], respectively). In the group of patients treated with one to three cycles or no chemotherapy, the probability of STC was significantly increased compared with those exposed to four or more cycles (P = .03; 5-year probability of 42% [95% CI, 27% to 62%] and 22% [95% CI, 8% to 54%], respectively). Twenty-two of 22 patients were tumor-free and alive at a median of 56 months (range, 2 to 184 months) after diagnosis of STC. CONCLUSION Platinum-based chemotherapy may reduce the probability of STC in patients with IGCNU, particularly in those treated with four or more cycles of chemotherapy. A watch-and-wait strategy for patients with IGCNU may be justified in selected patients with future plans for paternity.

The prognostic value of reactive stroma on prostate needle biopsy: A population-based study

Reactive tumor stroma has been shown to play an active role in prostatic carcinogenesis. A grading system for reactive stroma in prostate cancer (PC) has recently been established and found to predict biochemical recurrence and prostate cancer‐specific mortality (PCSM) in prostatectomized patients. To the best of our knowledge, there has been no study investigating the prognostic value of reactive stromal grading (RSG) with regard to PCSM when evaluated in diagnostic prostate needle biopsies.

The prognostic value of reactive stroma on prostate needle biopsy

Reactive tumor stroma has been shown to play an active role in prostatic carcinogenesis. A grading system for reactive stroma in prostate cancer (PC) has recently been established and found to predict biochemical recurrence and prostate cancer‐specific mortality (PCSM) in prostatectomized patients. To the best of our knowledge, there has been no study investigating the prognostic value of reactive stromal grading (RSG) with regard to PCSM when evaluated in diagnostic prostate needle biopsies.

Exome Sequencing of Bilateral Testicular Germ Cell Tumors Suggests Independent Development Lineages

Intratubular germ cell neoplasia, the precursor of testicular germ cell tumors (TGCTs), is hypothesized to arise during embryogenesis from developmentally arrested primordial germ cells (PGCs) or gonocytes. In early embryonal life, the PGCs migrate from the yolk sac to the dorsal body wall where the cell population separates before colonizing the genital ridges. However, whether the malignant transformation takes place before or after this separation is controversial. We have explored the somatic exome-wide mutational spectra of bilateral TGCT to provide novel insight into the in utero critical time frame of malignant transformation and TGCT pathogenesis. Exome sequencing was performed in five patients with bilateral TGCT (eight tumors), of these three patients in whom both tumors were available (six tumors) and two patients each with only one available tumor (two tumors). Selected loci were explored by Sanger sequencing in 71 patients with bilateral TGCT. From the exome-wide mutational spectra, no identical mutations in any of the three bilateral tumor pairs were identified. Exome sequencing of all eight tumors revealed 87 somatic non-synonymous mutations (median 10 per tumor; range 5-21), some in already known cancer genes such as CIITA, NEB, platelet-derived growth factor receptor α (PDGFRA), and WHSC1. SUPT6H was found recurrently mutated in two tumors. We suggest independent development lineages of bilateral TGCT. Thus, malignant transformation into intratubular germ cell neoplasia is likely to occur after the migration of PGCs. We reveal possible drivers of TGCT pathogenesis, such as mutated PDGFRA, potentially with therapeutic implications for TGCT patients.

SHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas

Androgen plays a vital role in prostate cancer development. However, it is not clear whether androgens influence stem-like properties of prostate cancer, a feature important for prostate cancer progression. In this study, we show that upon DHT treatment in vitro, prostate cancer cell lines LNCaP and PC-3 were revealed with higher clonogenic potential and higher expression levels of stemness related factors CD44, CD90, Oct3/4 and Nanog. Moreover, sex hormone binding globulin (SHBG) was also simultaneously upregulated in these cells. When the SHBG gene was blocked by SHBG siRNA knock-down, the induction of Oct3/4, Nanog, CD44 and CD90 by DHT was also correspondingly blocked in these cells. Immunohistochemical evaluation of clinical samples disclosed weakly positive, and areas negative for SHBG expression in the benign prostate tissues, while most of the prostate carcinomas were strongly positive for SHBG. In addition, higher levels of SHBG expression were significantly associated with higher Gleason score, more seminal vesicle invasions and lymph node metastases. Collectively, our results show a role of SHBG in upregulating stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples is significantly associated with poor clinicopathological features, indicating a role of SHBG in prostate cancer progression.

The relationship between perineural invasion, tumor grade, reactive stroma and prostate cancer-specific mortality: A clinicopathologic study on a population-based cohort

In vitro and in vivo studies have shown that nerves, tumor epithelium, and stroma interact and promote prostate cancer (PC) progression. Perineural invasion (PNI) is established amidst these interactions and may therefore indicate an aggressive PC phenotype. The purpose of the present study was to determine the relationship between PNI, tumor grade, reactive stroma, and PC‐specific mortality.

The Length of a Positive Surgical Margin Is of Prognostic Significance in Patients with Clinically Localized Prostate Cancer Treated with Radical Prostatectomy

Objective: To establish predictors of clinical failure in patients operated with radical prostatectomy (RP) for clinically localized prostate cancer (PC) by analyzing the pathological characteristics of positive surgical margins (PSM). Patients and Methods: The RP specimens of 303 consecutive patients operated with RP between 1985 and 2009 were reviewed. PSM were analyzed with regard to the PSM length, location and multifocality and the Gleason score (GS) at the PSM. Results: Of the 163 patients with PSM, 79 (48%) progressed to clinical failure compared to 30 (22%) in the negative-margin-status group. In univariate analysis, a GS at the PSM ≥4 + 3 = 7 (p = 0. 013) and a PSM length >3.0 mm (p < 0.005) were significantly associated with higher clinical failure rates compared to a GS at the PSM ≤3 + 4 = 7 and ≤3.0 mm in extent, respectively. A linear extent of the PSM ≤3.0 mm appeared to have the same clinical outcome as in the group with a negative margin status. In multivariate analysis, a PSM length >3.0 mm remained an independent predictor of clinical failure. Conclusions: PSM length is an independent predictor of clinical failure following RP.

White Blood Cell BRCA1 Promoter Methylation Status and Ovarian Cancer Risk

Women carrying germline BRCA1 mutations are at high risk for epithelial ovarian cancer, especially high-grade serous ovarian cancer (HGSOC) (1, 2). Thus, 4% to 10% of all women with ovarian cancer may carry BRCA1 germline mutations (3, 4). Recently, germline mutations in other genes, including PALB2, BRIP1, RAD51C, and RAD51D, all acting in the same DNA repair pathway as BRCA1 and BRCA2, have been associated with familial risk for ovarian and breast cancer (58). These findings are consistent with the hypothesis that disturbances in DNA repair by homologous recombination are important for cancer development in these organs. Promoter methylation represents an alternative mechanism of gene inactivation, and promoter methylation of the MLH1 mismatch repair gene, as well as BRCA1 methylation, has been observed in normal tissues in some families with a high risk for colorectal or breast cancer who do not have germline mutations in these genes (912). For breast and ovarian cancer risk, associations with BRCA1 methylation in white blood cells (WBCs) have not been consistent (10, 1316), and most studies have been small, with limited statistical power to detect any clear differences. We hypothesized that normal tissue BRCA1 methylation may be associated with an increased risk for ovarian cancer, with a particular propensity for HGSOC, analogous to observations for germline BRCA1 mutations. Here, we determined WBC BRCA1 promoter methylation status in a large casecontrol study and then attempted to replicate the findings in a similarly designed validation study. To determine whether normal tissue BRCA1 methylation may be established early in life, we also assessed WBC BRCA1 methylation in samples of newborn girls and healthy young women. Methods Study Design Overview We compared WBC BRCA1 promoter methylation status between patients with ovarian cancer and population control participants. The initial study was followed by a similarly designed casecontrol study to validate our results. In addition, we performed extensive sensitivity analyses to test the robustness of our findings. Initial Study White blood cell DNA was available from 934 patients with epithelial ovarian cancer treated at Oslo University Hospital, Norwegian Radium Hospital, between 1993 and 2011 (Figure 1, A). All samples collected in the biobank during that period were included; however, borderline ovarian tumors were excluded. Also, all patients had been tested for pathogenic BRCA1 or BRCA2 germline mutations, and patients carrying such mutation were excluded. Samples were collected before any systemic chemotherapy, with 583 samples collected from patients after surgery and 351 collected from patients before surgery or from those who did not have surgery. As a control, we used random samples of women from CONOR (Cohort of Norway), a large collection of population studies in Norway with similar questionnaire data, clinical measurements, and blood samples (17). The CONOR participants were recruited between 1994 and 2003 (for details, see Supplement Table 1). Thus, in the initial casecontrol study, 1698 women without cancer were frequency matched by 5-year age categories (at blood sampling) to the 934 patients with ovarian cancer. Among participants with successful BRCA1 methylation analysis, the patients (age range, 15 to 90 years; median, 62 years) were somewhat older than the control participants (range, 20 to 93 years; median, 57 years); therefore, we adjusted for age at blood sampling in the casecontrol comparisons. Figure 1. Flow chart of patients with ovarian cancer and healthy control participants included and successfully analyzed in the initial (A) and validation (B) studies. HGSOC = high-grade serous ovarian cancer; LGSOC = low-grade serous ovarian cancer; qPCR = quantitative polymerase chain reaction. Supplement. Supplementary Material and Methods To assess whether the percentage of methylated alleles would display a doserisk association with ovarian cancer, we analyzed methylation-positive samples by pyrosequencing. Validation Study We estimated sample size for the validation study on the basis of the strength of association found for the HGSOC group in our initial study (Supplement). Thus, we analyzed WBC DNA from 607 patients with ovarian cancer (274 from Oslo University Hospital and 333 from Haukeland University Hospital, Bergen), including 286 with HGSOC, and 1984 population control participants randomly selected from CONOR, with frequency matching in 5-year age increments. The Oslo patients with ovarian cancer had blood collected in 2011 to 2015, and the Bergen patients in 2001 to 2015 (Figure 1, B). Among the patients with ovarian cancer, 433 had blood collected before and 174 after surgery. All the Oslo patients had been tested for pathogenic BRCA1 and BRCA2 germline mutations, with negative results, whereas the Bergen patients had not been tested. As in the initial casecontrol study, samples were collected before chemotherapy began. The control group drawn from the CONOR study (17) did not overlap with that of the initial study. We adjusted for age at blood sampling by using a procedure similar to that of the initial study. Newborns and Healthy Young Persons To assess whether normal tissue BRCA1 methylation might be established early in life, we determined WBC methylation status in umbilical cord blood from a sample of newborn girls (n= 611) from MoBa (the Norwegian Mother and Child Cohort Study) (18). In addition, WBC methylation was determined in a group of healthy women aged 20 to 25 years (n= 292) selected from the CONOR study (17). These 2 groups were not part of the control groups used in the casecontrol comparisons (Supplement). Tumor and Normal Tissue BRCA1 Methylation We hypothesized that WBC BRCA1 methylation may be a surrogate marker of BRCA1 tissue methylation in general. If correct, one would expect that many women with ovarian cancer and positive BRCA1 methylation status would have BRCA1 methylation in their tumor tissue. To address these questions, we analyzed normal and ovarian cancer tissue samples from patients with and without positive WBC methylation status (Supplement). Methylation Status Among Persons Carrying BRCA1/2 Germline Mutations To explore a potential relationship between WBC BRCA1 methylation and BRCA1/2 germline mutation, we determined BRCA1 methylation status in 251 patients with ovarian cancer and germline BRCA1 mutations, 100 with BRCA2 mutations, and 15 with familial ovarian cancer who had negative results on BRCA1/2 mutation testing. Sensitivity Analysis Previous studies suggested that cancer burden influences WBC global DNA methylation patterns (19). To evaluate whether tumor load affects BRCA1 promoter methylation in blood, we compared BRCA1 methylation frequency between samples obtained before and after surgery; we also compared methylation status among FIGO (International Federation of Gynecology and Obstetrics) stages across the pooled groups of patients with ovarian cancer and those with HGSOC. We also assessed the potential effect of previous breast cancer treatment as well as of sample storage time. Further, we measured WBC BRCA1 methylation status in a separate cohort of 658 patients with ovarian cancer, from whom blood samples were collected at various time points after chemotherapy, when most patients had limited or no detectable residual disease. Previously, global DNA methylation assessments suggested that methylation of some CpG (cytosineguanine) nucleotide sequence sites across the genome might vary among different WBC fractions (20). Thus, we examined published data sets for potential variation among WBC fractions in adults (21) as well as newborns (18) to identify any variation with respect to methylation of the BRCA1 promoter CpGs of relevance to the present study (Supplement). Laboratory Analysis In brief, DNA was isolated from the samples and bisulfite converted (>99.5% validated conversion rate) before BRCA1 promoter methylation status was determined by quantitative polymerase chain reaction (qPCR). Two researchers (E.O.B. and L.M.), who were blinded to sample identity, independently scored each sample as methylation positive or negative. Further details, including assessment of individual CpG methylation across the promoter area, methylation quantification by pyrosequencing, and haplotype analysis, are given in the Supplement. Data Analysis We compared BRCA1 methylation status between patients with ovarian cancer and population control participants by using logistic regression models adjusted for age, and we reported odds ratios (ORs) from these models. In addition, we looked for heterogeneity by using a likelihood ratio test to compare results between the initial and validation studies. Because germline BRCA1 mutation carriers seem to have a propensity for HGSOC, we analyzed this subgroup separately. In a separate analysis pooling cases and controls from the 2 studies (initial and validation), we assessed the frequency of BRCA1 methylation within 10-year age groups by using logistic regression, stratified for the 2 studies. The patient and control samples from the initial study analyzed by pyrosequencing were dichotomized according to the median value of methylation, and ORs were calculated separately for each group (Supplement). We used chi-square tests to compare the proportions of persons with BRCA1 methylation between groups. The precision of the estimates of the proportion with BRCA1 methylation was presented with 95% CIs. Potential confounding by other unknown covariates was assessed by using the method described by VanderWeele (22) (Supplement). The statistical analyses were performed in Stata, version 12.1 (StataCorp), and SPSS, version 19 (IBM). The study was conducted according to the STREGA (Strengthening the Reporting of Genetic Association Studies) and GRIPS (Strengthening the Reporting of Genetic Risk Prediction Studies) statements (23, 24). Ethical Considerations

Multifocal Primary Prostate Cancer Exhibits High Degree of Genomic Heterogeneity

BACKGROUND Most primary prostate cancers are multifocal with individual tumors harboring different aggressiveness; however, the genomic heterogeneity among these tumors is poorly understood. OBJECTIVE To better understand the biological basis for clinical variability among different lesions, we sought to comprehensively characterize the heterogeneity of somatic gene mutations in multifocal prostate cancer. DESIGN, SETTING, AND PARTICIPANTS High-coverage whole-exome sequencing of 153 frozen tissue samples, taken from two to three distinct tumor foci and one non-cancerous area from each of 41 patients, covering a total of 89 tumor foci. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS State-of-the-art bioinformatics tools for mutation calling and copy number determination from whole-exome sequencing data. RESULTS AND LIMITATIONS We found a very high degree of interfocal heterogeneity among tumors, that is, 76% of pairwise-compared tumor foci from the same prostatectomy specimen had no point mutations in common and DNA copy number changes were rarely shared across cancer foci. The few point mutations shared across tumor foci were seldom in cancer-critical genes. CONCLUSIONS In this first large genomic heterogeneity study of primary prostate cancer, we observe that different tumor foci within the same patient are genetically distinct, only rarely sharing any somatic gene mutations, including those in cancer driver genes. This heterogeneity affects how genomics-based management of prostate cancer can be implemented, as information from all tumor foci is necessary to draw valid conclusions about the cancers genomic alterations. PATIENT SUMMARY Most primary prostate cancers consist of multiple tumors within the same organ, but little is known about their relationships. We have compared the sets of gene mutations among such tumors and found that they only exceptionally have any in common. This will influence treatment decisions in the future as each tumors mutations will render it unique and have to be considered to gain the best treatment results.

Intraductal Carcinoma of the Prostate on Diagnostic Needle Biopsy Predicts Prostate Cancer Mortality: A Population-Based Study

Intraductal carcinoma of the prostate (IDC‐P) is a distinct histopathologic feature associated with high‐grade, advanced prostate cancer. Although studies have shown that IDC‐P is a predictor of progression following surgical or radiation treatment for prostate cancer, there are sparse data regarding IDC‐P on diagnostic needle biopsy as a prognosticator of prostate cancer mortality.