The proliferation marker Ki67 has been suggested as a promising cancer biomarker. As Ki67 needs an exact quantification, this marker is a prototype of a new generation of tissue-based biomarkers. In ...this study, we have systematically evaluated different cut points for Ki67 using three different clinical end points in a large neoadjuvant study cohort.
We have evaluated pretherapeutic Ki67 levels by immunohistochemistry in 1166 breast cancer core biopsies from the neoadjuvant GeparTrio trial. We used the standardized cutoff-finder algorithm for three end points response to neoadjuvant chemotherapy (pCR), disease-free (DFS) and overall-survival (OS). The analyses were stratified for hormone receptor (HR) and HER2 status by molecular subtype radar diagrams (MSRDs).
A wide range of Ki67 cut points between 3%–94% (for pCR), 6%–46% (for DFS) and 4%–58% (for OS) were significant. The three groups of Ki67 ≤ 15% versus 15.1%–35% versus >35% had pCR-rates of 4.2%, 12.8%, and 29.0% (P < 0.0005), this effect was also present in six of eight molecular subtypes. In MSRD, Ki67 was significantly linked to prognosis in uni- and multivariate analysis in the complete cohort and in HR-positive, but not triple-negative tumors.
Ki67 is a significant predictive and prognostic marker over a wide range of cut points suggesting that data-derived cut point optimization might not be possible. Ki67 could be used as a continuous marker; in addition, the scientific community could define standardized cut points for Ki67. Our analysis explains the variability observed for Ki67 cut points in previous studies; however, this should not be seen as weakness, but as strength of this marker. MSRDs are an easy new approach for visualization of biomarker effects on outcome across molecular subtypes in breast cancer. The experience with Ki67 could provide important information regarding the development and implementation of other quantitative biomarkers.
Purpose
Most guidelines about fertility preservation are predominantly focused on scientific evidence, but are less practically orientated. Therefore, practically oriented recommendations are needed ...to support the clinician in daily practice.
Methods
A selective literature search was performed based on the clinical and scientific experience of the authors, focussing on the most relevant diseases and gynaecological cancers. This article (Part I) provides information on topics that are essential for the fertility preservation indication, such as disease prognosis, disease therapy and its associated risks to fertility, recommending disease-specific fertility preservation measures. Part II specifically focusses on fertility preservation techniques.
Results
In breast cancer patients, fertility preservation such as ovarian tissue and oocyte cryopreservation is especially recommended in low-stage cancer and in women < 35 years of age. In Hodgkin’s lymphoma, the indication is mainly based on the chemotherapy regime as some therapies have very low, others very high gonadotoxicity. In borderline ovarian tumours, preservation of fertility usually is achieved through fertility sparing surgery, ovarian stimulation may also be considered. In cervical cancer, endometrial cancer, rheumatic diseases and other malignancies such as Ewing sarcoma, colorectal carcinoma, non-Hodgkin lymphoma, leukaemia etc., several other factors must be considered to enable an individual, stage-dependent decision.
Conclusion
The decision for or against fertility preservation depends on the prognosis, the risks to fertility and individual factors such as prospective family planning.
HER2 is one of the predominant therapeutic targets in breast cancer. The metastatic selection process may lead to discrepancies between the HER2 status of the primary tumor and circulating tumor ...cells (CTCs). This study analyzed the HER2 status of CTCs in patients with HER2-positive primary breast cancer at the time of diagnosis. Aim of the study was to assess potential discordance of HER2 status between primary tumor and CTCs, as this may have important implications for the use of HER2-targeted therapy.
The number and HER2 status of CTCs out of 30ml peripheral blood were assessed in 642 patients using the CellSearch System (Janssen Diagnostics, USA). The cutoff for CTC positivity was the presence of at least 1 CTC, and the cutoff for HER2 positivity of CTCs was the presence of at least 1 CTC with a strong HER2 staining.
258 (40.2%) of the 642 patients were positive for CTCs (median 2; range 1-1,689). 149 (57.8%) of these 258 patients had at least 1 CTC with strong HER2 staining. The presence of HER2-positive CTCs was not associated with tumor size (p = 0.335), histopathological grading (p = 0.976), hormone receptor status (ER: p = 0.626, PR: p = 0.263) or axillary lymph node involvement (p = 0.430). Overall, 83 (32.2%) of the CTC-positive patients exclusively had CTCs with strong HER2 staining, whereas 31 (12.0%) had only CTCs with negative HER2 staining. Within-sample variation in the HER2 status of CTCs was found in 86 (57.8%) of the 149 patients with more than 1 CTC.
This study demonstrated that discordance between the HER2 expression of CTCs and that of the primary tumor frequently occurs in early breast cancer. Future follow-up evaluation will assess whether this discrepancy may contribute to trastuzumab resistance.
MicroRNAs (miRNAs) as modulators of gene expression have been described to display both tumor-promoting and tumor-suppressive functions. Although their role has been studied in different tumor types, ...little is known about how they regulate nuclear factor κB (NF-κB) signaling in breast cancer. Here, we performed an unbiased whole genome miRNA (miRome) screen to identify novel modulators of NF-κB pathway in breast cancer. The screen identified 13 miRNA families whose members induced consistent effects on NF-κB activity. Among those, the miR-520/373 family inhibited NF-κB signaling through direct targeting of RELA and thus strongly reduced expression and secretion of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. With a combination of in vitro and in vivo approaches, we propose a metastasis-suppressive role of miR-520/373 family. miR-520c and miR-373 abrogated both in vitro cell invasion and in vivo intravasation of highly invasive MDA-MB-231 cells. However, knockdown of RELA did not affect their metastatic ability. mRNA profiling of MDA-MB-231 cells on overexpression of miR-520/373 members revealed a strong downregulation of transforming growth factor-β (TGF-β) signaling. Mechanistically, the metastasis-suppressive role of miR-520/373 can be attributed to direct suppression of TGFBR2, as the silencing of TGFBR2 phenocopied the effects of miR-520/373 overexpression on suppression of Smad-dependent expression of the metastasis-promoting genes parathyroid hormone-related protein, plasminogen activator inhibitor-1 and angiopoietin-like 4 as well as tumor cell invasion, in vitro and in vivo. A negative correlation between miR-520c and TGFBR2 expression was observed in estrogen receptor negative (ER(-)) breast cancer patients but not in the ER positive (ER(+)) subtype. Remarkably, decreased expression of miR-520c correlated with lymph node metastasis specifically in ER(-) tumors. Taken together, our findings reveal that miR-520/373 family has a tumor-suppressive role in ER(-) breast cancer by acting as a link between the NF-κB and TGF-β pathways and may thus contribute to the interplay of tumor progression, metastasis and inflammation.
The therapeutic effect of tamoxifen depends on active metabolites, e.g., cytochrome P450 2D6 (CYP2D6) mediated formation of endoxifen. To test for additional relationships, 236 breast cancer patients ...were genotyped for CYP2D6, CYP2C9, CYP2B6, CYP2C19, CYP3A5, UGT1A4, UGT2B7, and UGT2B15; also, plasma concentrations of tamoxifen and 22 of its metabolites, including the (E)‐, (Z)‐, 3‐, and 4′‐hydroxymetabolites as well as their glucuronides, were quantified using liquid chromatography–tandem mass spectrometry (MS). The activity levels of the metabolites were measured using an estrogen response element reporter assay; the strongest estrogen receptor inhibition was found for (Z)‐endoxifen and (Z)‐4‐hydroxytamoxifen (inhibitory concentration 50 (IC50) 3 and 7 nmol/l, respectively). CYP2D6 genotypes explained 39 and 9% of the variability of steady‐state concentrations of (Z)‐endoxifen and (Z)‐4‐hydroxytamoxifen, respectively. Among the poor metabolizers, 93% had (Z)‐endoxifen levels below IC90 values, underscoring the role of CYP2D6 deficiency in compromised tamoxifen bioactivation. For other enzymes tested, carriers of reduced‐function CYP2C9 (*2, *3) alleles had lower plasma concentrations of active metabolites (P < 0.004), pointing to the role of additional pathways.
Clinical Pharmacology & Therapeutics (2011) 89 5, 708–717. doi:10.1038/clpt.2011.27
Brain metastases (BMs) have a major impact on life expectancy and quality of life for many breast cancer patients. Knowledge about treatment patterns and outcomes is limited.
We analysed clinical ...data of 1712 patients diagnosed with BMs from breast cancer between January 2000 and December 2016 at 80 institutions.
Median age at diagnosis of BMs was 56 years (22–90 years). About 47.8% (n = 732) of patients had HER2-positive, 21.4% (n = 328) had triple-negative and 30.8% (n = 471) had hormone receptor (HR)–positive, HER2-negative (luminal-like) primary tumours. The proportion of patients with HER2-positive BMs decreased comparing the years 2000–2009 with 2010–2015 (51%–44%), whereas the percentage of patients with luminal-like tumours increased (28%–34%; p = 0.0331). Patients with BMs in the posterior fossa were more often HER2 positive (n = 169/314, 53.8%) than those diagnosed with triple-negative (n = 65/314, 20.7%) or luminal-like primary breast cancer (n = 80/314, 25.5%), (p < 0.0001). Median overall survival (OS) time after development of BMs for the overall cohort was 7.4 months (95% confidence interval CI: 6.7–8.0 months). One-year survival rate was 37.7% (95% CI: 35.2–40.1). Patients with HER2-positive tumours had the longest median OS of 11.6 months (95% CI: 10.0–13.4) compared with 5.9 months (95% CI: 5.0–7.2) for patients with luminal-like and 4.6 months (95% CI: 3.9–5.4) for patients with triple-negative tumours. Patients with HER2-positive tumours who received anti-HER2 treatment had longer median OS than those without (17.1 months versus 7.2 months, p < 0.0001).
Prognosis of patients after developing BMs varies significantly according to the subtype. The outcome in this cohort is similarly poor in triple-negative and HR-positive/HER2-negative patients. Our results underline the high medical need for improvement of treatment and prevention strategies for BMs in breast cancer patients.
•The first analysis of 1712 patients of breast cancer patients with brain metastases (BMs) is presented.•Localisation of BMs was different depending on tumour subtypes.•Survival times differ depending on the subtype and localisation of BMs.•A change in the incidence of BMs over time was observed.
Trastuzumab improves the outcome of women with HER2 positive breast cancer. We aimed to assess whether trastuzumab decreases the detection rate of circulating tumor cells (CTCs) in women with high ...risk, HER2 nonamplified, early breast cancer.
The EORTC 90091-10093 BIG 1–12 Treat CTC is a phase II trial, conducted in 70 hospitals and 6 CTC laboratories across 5 European countries. Patients with centrally confirmed HER2 nonamplified breast cancer and ≥1 centrally confirmed CTC per 15ml of blood by CellSearch® following surgery and (neo)adjuvant chemotherapy were randomized (1:1) to 6 cycles of trastuzumab intravenously versus 18weeks of observation. Randomization was stratified for center, locally confirmed estrogen receptor status and adjuvant versus neoadjuvant chemotherapy. The primary end point was rate of detection of ≥1 CTC per 15ml of blood at week 18. Secondary end points were invasive disease-free survival (iDFS) and cardiac safety.
Between 30 April 2013 and 17 October 2016, 1317 patients were screened; 95 (7.2%) had detectable CTC(s), and 63 (4.8%) were randomized to trastuzumab (n=31) or observation (n=32). Fifty-eight patients were assessable for the primary end point, 29 in each arm. In 9 of the 58 patients, CTC(s) were still detected at week 18:5 in the trastuzumab and 4 in the observation arm (one-sided Fisher’s exact test, P=0.765). An Independent Data Monitoring Committee recommended stopping further accrual for futility for the primary end point. Median follow-up at database lock was 13months (IQR 4–16.5). The 1-year iDFS was 93.8% (95% CI 77.3–98.4) in the observation versus 84.8% (95% CI 63.4–94.2) in the trastuzumab arm. No grade 2–4 cardiac events were observed in the trastuzumab arm.
Trastuzumab does not decrease the detection rate of CTCs in HER2 nonamplified, nonmetastatic breast cancer.
Systemic lupus erythematosus (SLE) is an autoimmune disease which may negatively influence fertility. Treatment with cyclophosphamide can damage the ovaries. The ovarian function can also be reduced ...by autoimmune oophoritis in lupus patients. We analysed the influence of SLE on the ovarian reserve against disease intensity and duration of illness. We determined the ovarian reserve in 33 premenopausal SLE patients without previous cyclophosphamide-treatment by measuring the anti-Muellerian hormone (AMH) and compared these with the AMH values of 33 age-matched healthy controls. Numbers of children and miscarriages were reported. Disease intensity of the SLE patients was determined using SLEDAI and ECLAM and duration of illness was taken into account. We found that the AMH values in the SLE group were significantly lower than in the healthy control group. No significant differences between the groups regarding number of children and miscarriages were noted and no correlation between the AMH value and the duration of illness or the SLEDAI as an indicator of disease activity was found. Despite mild disease activity SLE patients had a significantly lower ovarian reserve than age-matched healthy women. This could be a sign that SLE itself has a negative influence on the ovarian reserve.
Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating ...epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAM
/CK
/CD45
/DAPI
) and subsequent FACS sorting. In contrast to CD45
white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.
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