High frequency of complex TP53 mutations in CNS metastases from breast cancer

Background:

Brain metastasis from breast cancer is usually associated with a poor prognosis and early death. Alteration of p53 may contribute to malignant progression by abrogation of apoptosis induced by oncogene activation and by acquisition of gain-of-function properties, which promote tumour aggression. Mutation in TP53 occurs at high frequency in carcinomas of the lung and gastro-intestinal tract, but is much less frequent, at 25%, in primary breast cancer. The frequency of TP53 alteration in the central nervous system (CNS) metastatic breast cancer is not known.

Methods:

In all, 23 cases of histologically confirmed CNS metastatic breast cancer were identified and the coding sequence of TP53 determined. TP53 was also sequenced in two control series of primary breast carcinomas from independent clinical centres.

Results:

We demonstrate a strikingly high frequency of TP53 mutation in the CNS metastatic lesions with an over-representation of complex mutations (non-sense/deletions/insertions). Complex mutations occur in metastatic lesions in both triple-negative breast cancer and hormone receptor/HER2-positive cases. Analysis of paired primary carcinomas and brain metastatic lesions revealed evidence for both clonal selection and generation of new mutations (missense and complex) in progression from a primary breast carcinoma to brain metastasis.

Conclusion:

Mutation in TP53 is the most common genetic alteration reported during metastasis to the brain in breast cancer.     

Detection of HER2 amplification in circulating free DNA in patients with breast cancer

Background:

Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20–25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer.

Methods:

Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control.

Results:

We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry.

Conclusions:

The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer.     

Breast lesions in reduction mammaplasty specimens: a histopathological pattern in 534 patients

Background:

The prevalence of breast lesions (benign, precancerous and cancer lesions) in reduction mammaplasty (RM) specimens has rarely been reported in Europe and never in the Swiss population.

Methods:

Personal and histopathological data from 534 female patients who underwent RM were reviewed.

Results:

Benign and/or malignant lesions were detected in 76.2% of all patients. Benign breast lesions associated with an increased risk of developing breast cancer represented 2.8% of all lesions. Breast cancer in situ was identified in 5 (0.9%) patients. Patient age and previous history of breast cancer were risk factors for incidental breast cancer.

Conclusion:

The rate of incidental carcinoma in situ was higher for patients with breast cancer history. Probably due to preoperative breast cancer investigation, no occult invasive breast cancer was found in reduction mammary specimens. Therefore before RM, breast cancer evaluation should be considered for all patients, especially for those with breast cancer risk factors (e.g., patient age, personal history of breast cancer).     

Monitoring gastric cancer progression with circulating tumour DNA

Background:

Circulating tumour DNA (ctDNA) is an emerging candidate biomarker for malignancies and may be useful for monitoring the disease status of gastric cancer.

Methods:

We performed targeted deep sequencing of plasma cell-free DNA (cfDNA) by massively parallel sequencing in patients with tumours harbouring TP53 mutations. The quantitative values of TP53-ctDNA during the clinical course were compared with the tumour status.

Results:

Three out of ten patients with TP53 mutations in primary tumours showed detectable TP53 mutation levels in preoperative cfDNA. Although the cfDNA concentrations were not always reflective of the disease course, the ctDNA fraction correlated with the disease status.

Conclusions:

ctDNA may serve as a useful biomarker to monitor gastric cancer progression and residual disease.     

Low-level constitutional mosaicism of a de novoBRCA1 gene mutation

Background:

Pathogenic BRCA1 mutations are usually inherited. Constitutional low-level BRCA1 mosaicism has never been reported.

Methods:

Next-generation sequencing (NGS) of cancer gene panel of germline and tumour DNA in a patient with early onset, triple-negative breast cancer.

Results:

Constitutional de novo mosaicism (5%) for a pathogenic (c.1953dupG; p.Lys652Glufs*21) BRCA1mutation was detected in leukocytes, buccal tissue and normal breast tissue DNA, with ∼50% mutation in tumorous breast tissue.

Conclusion:

This is the first reported case of low-level, multiple tissue, constitutional mosaicism in BRCA1, and highlights the need to consider deep sequencing in affected individuals clinically suspected of having cancer predisposition whose tumours display a BRCA mutation.     

Paired related homoeobox 1, a new EMT inducer,is involved in metastasis and poor prognosis in colorectal cancer

Background:

Paired related homoeobox 1 (PRRX1) has been identified as a new epithelial-mesenchymal transition (EMT) inducer in breast cancer. However, the function of PRRX1 in colorectal cancer (CRC) has not been elucidated.

Methods:

We utilised ectopic PRRX1-expressing cell lines to analyse the function of PRRX1 in CRC. The clinical significance of PRRX1 was also examined on three independent CRC case sets.

Results:

PRRX1 induced EMT and the stem-like phenotype in CRC cells. In contrast to studies of breast cancer, abundant expression of PRRX1 was significantly associated with metastasis and poor prognosis in CRC.

Conclusion:

PRRX1 is an indicator of metastasis and poor prognosis in CRC cases. Further investigation is required to uncover the signalling network regulating PRRX1.     

Reduced association of anti-apoptotic protein Mcl-1 with E3 ligase Mule increases the stability of Mcl-1 in breast cancer cells

Background:

Mechanisms that increase resistance to apoptosis help promote cellular transformation. Cancer cells have deregulated apoptotic pathways, where increased expression and stability of anti-apoptotic proteins Mcl-1 and Bcl-2 increases resistance to apoptosis. Pathways that increase the stability of proteins in cancer cells remain poorly understood.

Methods:

Using human mammary epithelial and established breast cancer cell lines, we assessed the mechanisms that increase the stability of anti-apoptotic proteins in breast cancer cells by caspase assay, western blot, small-inhibitory RNA treatment and immunoprecipitation.

Results:

While breast cancer cells were resistant to de novo inhibition of protein synthesis, a rapid proteosome-mediated degradation of Mcl-1 and Bcl-2 induced apoptosis in mammary epithelial cells. Although Mule, an E3 ligase that targets Mcl-1 for degradation was expressed in mammary epithelial and breast cancer cell lines, rapid increase of polyubiquitinated Mcl-1 and Bcl-2 was detected only in mammary epithelial cells. Only transient formation of the Mule–Mcl-1 complex was detected in breast cancer cells. Downregulation of pERK1/2 in breast cancer cells reduced Mcl-1 levels and increased Mcl-1/Mule complex.

Conclusion:

Our findings suggest that reduced Mule/Mcl-1 complex has a significant role in increasing the stability of Mcl-1 in breast cancer cells and increased resistance to apoptosis.     

Targeting focal adhesion turnover in invasive breast cancer cells by the purine derivative reversine

Background:

The dynamics of focal adhesion (FA) turnover is a key determinant for the regulation of cancer cell migration. Here we investigated FA turnover in a panel of breast cancer models with distinct invasive properties and evaluated the impact of reversine on this turnover in relation to cancer cell invasion in in vitro and in vivo conditions.

Methods:

Live imaging and immunofluorescence assays were used to investigate FA turnover in breast cancer cells. Biochemical studies were used to investigate the impact of reversine on FA signalling and turnover. In vivo activity was investigated using orthotopic breast cancer mouse models.

Results:

Accelerated FA disassembly from plasma membrane protrusions was observed in invasive compared with non-invasive breast cancer cells or non-immortalised mammary epithelial cells. Reversine significantly inhibited FA disassembly leading to stable FAs, which was associated with reduced cell motility and invasion. The inhibitory effect of reversine on FA turnover accounted for a large part on its capacity to interfere with FAK function on regulating its downstream targets. In orthotopic breast cancer mouse models, reversine revealed a potent inhibitory activity on tumour progression to metastasis.

Conclusion:

These results support the utility of targeting FA turnover as a therapeutic approach for invasive breast cancer.     

Evaluation of a candidate breast cancer associated SNP in ERCC4 as a risk modifier in BRCA1 and BRCA2 mutation carriers. Results from the Consortium of Investigators of Modifiers of BRCA1/BRCA2 (CIMBA)

Background:

In this study we aimed to evaluate the role of a SNP in intron 1 of the ERCC4 gene (rs744154), previously reported to be associated with a reduced risk of breast cancer in the general population, as a breast cancer risk modifier in BRCA1 and BRCA2 mutation carriers.

Methods:

We have genotyped rs744154 in 9408 BRCA1 and 5632 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and assessed its association with breast cancer risk using a retrospective weighted cohort approach.

Results:

We found no evidence of association with breast cancer risk for BRCA1 (per-allele HR: 0.98, 95% CI: 0.93–1.04, P=0.5) or BRCA2 (per-allele HR: 0.97, 95% CI: 0.89–1.06, P=0.5) mutation carriers.

Conclusion:

This SNP is not a significant modifier of breast cancer risk for mutation carriers, though weak associations cannot be ruled out.     

Nuclear CSPP1 expression defined subtypes of basal-like breast cancer

Background:

The multi-exon CSPP1 gene, encoding for centrosome and microtubule-associated proteins involved in ciliogenesis and cell division, is a candidate oncogene in luminal breast cancer but expression of CSPP1 proteins remained unexplored.

Methods:

CSPP1 gene and protein expression was examined in normal mammary tissue, human breast cancer cell lines, and primary breast cancer biopsies from two patient cohorts. Cell type and epitope-dependent subcellular-specific CSPP1 staining pattern in normal mammary gland epithelium and cancer biopsies were correlated to molecular and clinical parameters.

Results:

A novel, nuclear localised CSPP1 isoform was exclusively detected in luminal epithelial cells, whereas cytoplasmic CSPP-L was generally expressed in normal mammary epithelium. Luminal cell-related nuclear CSPP1 expression was preserved in type-matched cell lines and carcinomas, and correlated to gene copy number and mRNA expression. In contrast, basal-like carcinomas displayed generally lower CSPP1 mRNA expression. Yet, a subgroup of basal-like breast carcinomas depicted nuclear CSPP1 expression, displayed luminal traits, and differed from nuclear CSPP1 devoid counterparts in expression of eight genes. Eight-gene signature defined groups of basal-like tumours from an independent cohort showed significant differences in survival.

Conclusions:

Differential expression of a nuclear CSPP1 isoform identified biologically and clinically distinct subgroups of basal-like breast carcinoma.     

Mutational analysis of Polycomb genes in solid tumours identifies PHC3 amplification as a possible cancer-driving genetic alteration

Background:

Polycomb group genes (PcGs) are epigenetic effectors implicated in most cancer hallmarks. The mutational status of all PcGs has never been systematically assessed in solid tumours.

Methods:

We conducted a multi-step analysis on publically available databases and patient samples to identify somatic aberrations of PcGs.

Results:

Data from more than 1000 cancer patients show for the first time that the PcG member PHC3 is amplified in three epithelial neoplasms (rate: 8–35%). This aberration predicts poorer prognosis in lung and uterine carcinomas (P<0.01). Gene amplification correlates with mRNA overexpression (P<0.01), suggesting a functional role of this aberration.

Conclusion:

PHC3 amplification may emerge as a biomarker and potential therapeutic target in a relevant fraction of epithelial tumours.     

Epstein-Barr virus as a marker of biological aggressiveness in breast cancer

Purpose:

Although a potential role of the Epstein-Barr virus (EBV) in the pathogenesis of breast cancer (BC) has been underlined, results remain conflicting. Particularly, the impact of EBV infection on biological markers of BC has received little investigation.

Methods:

In this study, we established the frequency of EBV-infected BC using real-time quantitative PCR (RT–PCR) in 196 BC specimens. Biological and pathological characteristics according to EBV status were evaluated.

Results:

EBV DNA was present in 65 of the 196 (33.2%) cases studied. EBV-positive BCs tended to be tumours with a more aggressive phenotype, more frequently oestrogen receptor negative (P=0.05) and with high histological grade (P=0.01). Overexpression of thymidine kinase activity was higher in EBV-infected BC (P=0.007). The presence of EBV was weakly associated with HER2 gene amplification (P=0.08).

Conclusion:

Our study provides evidence for EBV-associated BC undergoing distinct carcinogenic processes, with more aggressive features.     

p53 mutation, deprivation and poor prognosis in primary breast cancer

Background:

The deprivation gap for breast cancer survival remains unexplained by stage at presentation, treatment, or co-morbidities. We hypothesised that p53 mutation might contribute to the impaired outcome observed in patients from deprived communities.

Methods:

p53 mutation status was determined using the Roche Amplichip research test in 246 women with primary breast cancer attending a single cancer centre and related to deprivation, pathology, overall, and disease-free survival.

Results:

p53 mutation, identified in 64/246 (26%) of cancers, was most common in 10 out of 17 (58.8%) of the lowest (10th) deprivation decile. Those patients with p53 mutation in the 10th decile had a significantly worse disease-free survival of only 20% at 5 years (Kaplan–Meier logrank χ²=6.050, P=0.014) and worse overall survival of 24% at 5 years (Kaplan–Meier logrank χ²=6.791, P=0.009) than women of deciles 1–9 with p53 mutation (c.f. 56% and 72%, respectively) or patients in the 10th decile with wild-type p53 (no disease relapse or deaths).

Conclusion:

p53 mutation in breast cancer is associated with socio-economic deprivation and may provide a molecular basis, with therapeutic implications, for the poorer outcome in women from deprived communities.     

Absence of the DNA repair enzyme human 8-oxoguanine glycosylase is associated with an aggressive breast cancer phenotype

Background:

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is the most abundant marker of DNA damage and it reflects oxidative stress. Human 8-oxoguanine glycosylase (hOGG1) is a DNA-repair enzyme that participates in 8-oxodG removal.

Methods:

hOGG1 protein expression was immunohistochemically studied in 96 patients with local or locally advanced breast cancer and in 20 lesions of non-malignant breast disease. 8-OxodG levels had been previously determined in all patients.

Results:

hOGG1 was overexpressed in invasive vs non-invasive lesions (P=0.006). 8-OxodG and hOGG1 had a significant inverse association (P=0.046). Lack of hOGG1 expression was associated with the most poor prognostic factors of breast cancer. In addition, all triple-negative breast carcinomas (TNBCs) were hOGG1 negative (P=0.027 vs non-TNBCs). Patients with a lack of both hOGG1- and 8-oxodG immunostaining showed extremely poor breast cancer-specific survival compared with those with either 8-oxodG- or hOGG1-positive tumours (P<0.000005).

Conclusion:

The current results imply that absence of hOGG1 expression is associated with features of aggressive breast cancer. Tumours lacking both 8-oxodG and hOGG1 seem to indicate especially poor prognosis.     

Carbonic anhydrase IX is a predictive marker of doxorubicin resistance in early-stage breast cancer independent of HER2 and TOP2A amplification

Background:

In early-stage breast cancer, adjuvant chemotherapy is associated with significant systemic toxicity with only a modest survival benefit. Therefore, there is considerable interest in identifying predictive markers of response to therapy. Doxorubicin, one of the most common drugs used to treat breast cancer, is an anthracycline chemotherapeutic agent, a class of drugs known to be affected by hypoxia. Accordingly, we examined whether expression of the endogenous hypoxia marker carbonic anhydrase IX (CA IX) is predictive of outcome in early-stage breast cancer patients treated with doxorubicin.

Methods:

We obtained 209 early-stage pre-treatment surgically-resected breast tumours from patients, who received doxorubicin in their chemotherapeutic regimen and had >10 years of follow-up. Immunohistochemistry was used to detect CA IX, and we used fluorescence in situ hybridisation to detect both human epidermal growth factor receptor (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification.

Results:

Carbonic anhydrase IX intensity was significantly correlated with progression-free survival (PFS) and overall survival (OS) in patients receiving 300 mg m⁻² of doxorubicin (HR=1.82 and 3.77; P=0.0014 and 0.010, respectively). There was a significant, inverse correlation between CA IX score and oestrogen receptor expression, but no significant correlations were seen with either HER2 or TOP2A ratio.

Conclusion:

We demonstrate that CA IX expression is correlated with worse PFS and OS for breast cancer patients treated with doxorubicin, independent of HER2 or TOP2A gene amplification. This study provides evidence that using CA IX to detect hypoxia in surgically-resected breast tumours may be of clinical use in choosing an appropriate chemotherapy regimen.     

Mammographic density and its interaction with other breast cancer risk factors in an Asian population

Background:

Joint effects of mammographic density and other risk factors on breast cancer risk remain unclear.

Methods:

From The Singapore Breast Screening Project, we selected 491 cases and 982 controls. Mammographic density was measured quantitatively. Data analysis was by conditional logistic regression.

Results:

Density was a significant risk factor, adjusting for other factors. Density of 76–100% had an odds ratio of 5.54 (95% CI 2.38–12.90) compared with 0–10%. Density had significant interactions with body mass index and oral contraceptive use (P=0.02).

Conclusions:

Percent density increases breast cancer risk in addition to effects of other risk factors, and modifies the effects of BMI and OCs.     

BRCA1 testing should be offered to individuals with triple-negative breast cancer diagnosed below 50 years

Background:

Triple-negative (TN) tumours are the predominant breast cancer subtype in BRCA1 mutation carriers. Recently, it was proposed that all individuals below 50 years of age with TN breast cancer should be offered BRCA testing. We have evaluated the BRCA1 mutation frequency and the implications for clinical practice of undertaking genetic testing in women with TN breast cancer.

Methods:

We undertook BRCA1 mutation analysis in 308 individuals with TN breast cancer, 159 individuals from unselected series of breast cancer and 149 individuals from series ascertained on the basis of young age and/or family history.

Results:

BRCA1 mutations were present in 45 out of 308 individuals. Individuals with TN cancer <50 years had >10% likelihood of carrying a BRCA1 mutation in both the unselected (11 out of 58, 19%) and selected (26 out of 111, 23%) series. However, over a third would not have been offered testing using existing criteria. We estimate that testing all individuals with TN breast cancer <50 years would generate an extra 1200 tests annually in England.

Conclusion:

Women with TN breast cancer diagnosed below 50 years have >10% likelihood of carrying a BRCA1 mutation and are therefore eligible for testing in most centres. However, implementation may place short-term logistical and financial burdens on genetic services.     

A promoting early presentation intervention increases breast cancer awareness in older women after 2 years: a randomised controlled trial

Background:

We have developed the Promoting Early Presentation (PEP) Intervention to equip older women with the knowledge, skills, confidence and motivation to present promptly with breast symptoms, and thereby improve survival from breast cancer. The PEP Intervention consists of a 10-min interaction between a radiographer and an older woman, supported by a booklet. Our previous report showed that at 1 year, the PEP intervention increased the proportion who were breast cancer aware compared with usual care.

Methods:

We randomised 867 women aged 67–70 years attending for their final routine appointment on the National Health Service Breast Screening Programme to receive the PEP Intervention, a booklet alone or usual care. The primary outcome was breast cancer awareness measured using a validated questionnaire asking about knowledge of breast cancer symptoms, knowledge that the risk of breast cancer increases with age and breast checking behaviour.

Results:

At 2 years, the PEP Intervention increased the proportion who were breast cancer aware compared with usual care (21 vs 6% odds ratio 8.1, 95% confidence interval 2.7–25.0).

Conclusions:

The uniquely large and sustained effect of the PEP Intervention on breast cancer awareness increases the likelihood that a woman will present promptly should she develop breast cancer symptoms up to many years later.     

The proline TP53 variant stimulates likely lymphangiogenesis in an orthotopic mouse model of pancreatic cancer

Background:

Pancreatic cancer is a deadly disease characterised by high incidence of TP53 mutations. Restoration of TP53 function is perceived as a highly attractive therapeutic strategy, whose effects are not well characterised.

Methods:

The current work adapted an inducible strategy of stage-specific reexpression of wild-type (wt) TP53 in an in vivo orthotopic mouse model of pancreatic cancer.

Results:

The reconstitution of wt TP53 function in TP53-mutant DanG and MiaPaCa-2 cells caused G1 cell cycle arrest but no evidence of apoptosis induction. Consistent with subcutaneous xenograft models, we found that wt TP53 reduced primary tumour growth. Wt TP53 reexpression during early tumour growth led to significant increase in vascularisation. This correlated with an unexpectedly high rate of micro-metastases in lymph nodes of animals with wt TP53 induction, despite the 90% decrease in median primary tumour weight. Reexpression of wt TP53 later in tumour development did not significantly affect the number of CD31-reactive vessels, but increased lymphatic vessel density.

Conclusion:

The increased number of lymphatic vessels and micro-metastases suggests that wt TP53 induction complexly affected the biology of different tumour constituents of pancreatic cancer. Our observation suggests that combination of the inducible system with an orthotopic model can yield important insights into in vivo pancreatic cancer biology.     

Loss of partner and breast cancer prognosis - a population-based study, Denmark, 1994-2010

Background:

The extent to which experiencing a stressful life event influences breast cancer prognosis remains unknown, as the findings of the few previous epidemiological studies are inconsistent. This large population-based study examines the association between a common major life event, loss of a partner and breast cancer recurrence and all-cause mortality.

Methods:

N=21 213 women diagnosed with a first primary breast cancer 1994–2006, who had a cohabiting partner in the 4 years before their breast cancer diagnosis, were followed for death and recurrence in population-based registers and clinical databases. Information on education, disposable income, comorbidity and prognostic risk factors were included in Cox regression analyses.

Results:

Women who had lost a partner either before diagnosis or in subsequent years were not at significantly higher risk of recurrence or dying than women who had not lost a partner.

Conclusion:

Our results do not support the concern that experiencing a stressful life event, the loss of a partner, negatively affects prognosis of breast cancer.