Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56hi/GD2+/CD81hi), primitive neuroectodermal tumors (CD271hi/CD99+), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1+/numyogenin+), carcinomas (CD45−/EpCAM+), germ cell tumors (CD56+/CD45−/NG2+/CD10+) and eventually also hemangiopericytomas (CD45−/CD34+). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.
Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients.
The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (Ptrend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC.
Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection of OSCC.
In breast carcinoma, the stromal loss of CD34 expression and acquisition of SMA myofibroblastic features may constitute a prerequisite for tumor invasiveness. However, this hypothesis remains controversial, with some authors describing the loss of CD34 fibrocytes in the absence of SMA myofibroblastic-like cells in the stroma of invasive carcinoma. Others have also described the disappearance of CD34 fibrocytes from in situ carcinoma. To clarify this issue, we compared the distribution of CD34 fibrocytes and SMA reactive myofibroblasts between stromal areas of tumor-free mammary tissue, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). In addition to 28 IDC, 300 normal duct–lobular units and 600 ducts with DCIS (158 low-grade, 266 intermediate, and 176 high-grade) were scored. The relationships between staining patterns and different histological features (grade of DCIS and presence or absence of necrosis) were compared. Loss of CD34 expression and acquisition of SMA expression were more frequent in high-grade in situ lesions than in intermediate and low-grade lesions (p<0.001). When necrosis was found in association with grade 2 or 3 DCIS, the decrease in CD34 expression was higher than in lesions without necrosis and that independently of the grade of DCIS (p<0.05). Necrosis did not appear to play a significant role in the expression of SMA (p = 0.35). In all cases, the stroma of invasive carcinomas showed a complete loss of CD34 fibrocytes. Future research on both CD34 fibrocytes and mechanisms stromal changes are essential in the future and may potentially lead to new treatment approaches.
To assess whether the value of CYFRA21-1 in the aspirates of ultrasonography-guided fine-needle aspiration biopsy (US-FNAB) can contribute to improving the performances of US-FNAB in the diagnosis of axillary lymph node (LN) metastasis in breast cancer patients.
US-FNAB was performed in 156 axillary LNs in 152 breast cancer patients (mean age: 51.4 years, range: 17–92 years). Concentrations of CYFRA21-1 were measured from washouts of the syringe used during US-FNAB. Tumor marker concentrations, US-FNAB, intraoperative sentinel node biopsy (SNB), and surgical pathology results were reviewed and analyzed. For comparison, the values of CEA and CA15-3 were also measured from washouts.
Among the 156 LNs, 75 (48.1%) were benign, and 81 (51.9%) were metastases. Mean concentrations of CYFRA21-1 were significantly higher in metastasis compared to benign LNs (P<0.001). US-FNAB combined to CYFRA21-1 showed significantly higher sensitivity, NPV, and accuracy compared to US-FNAB alone (all values P<0.05). All diagnostic indices of US-FNAB combined to CYFRA21-1 were significantly higher compared to US-FNAB combined with CEA or CA15-3 (all P<0.001). Of the 28 metastatic LNs which showed metastasis on SNB, CYFRA21-1 showed higher positive rate of 75.0% (CEA or CA15-3∶60.7%, P = 0.076).
Measuring CYFRA 21-1 concentrations from US-FNAB aspirates improves sensitivity, NPV, and accuracy of US-FNAB alone, and may contribute to reducing up to 75.0% of unnecessary intraoperative SNB. Compared to CEA or CA15-3, CYFRA21-1 shows significantly higher performances when combined to US-FNAB in the preoperative diagnosis of LN metastasis in breast cancer patients.
Lysophosphatidic acid (LPA) is a phospholipid growth factor involved in cell proliferation, differentiation, migration, inflammation, angiogenesis, wound healing, cancer invasion, and survival. This study was directed to evaluate the immunoexpression of LPA-1, cell proliferation, apoptosis, and angiogenesis markers in preneoplastic lesions induced with cadmium chloride in rat prostate.
The following parameters were calculated in ventral prostate of normal rats and rats that received Cd in drinking water during 24 months: percentages of cells immunoreactive to LPA-1 (LILPA1), PCNA (LIPCNA), MCM7 (LIMCM7), ubiquitin (LIUBI), apoptotic cells (LIAPO), and p53 (LIp53); volume fraction of Bcl-2 (VFBcl-2); and length of microvessels per unit of volume (LVMV/mm3). Data were analyzed using Student's t-test and Pearson correlation test.
The LILPA1 in dysplastic lesions and normal epithelium of Cd-treated rats was significantly higher than those in the control group. Markers of proliferation were significantly increased in dysplastic lesions, whereas some apoptotic markers were significantly decreased. No significant differences between groups were found in VFBcl-2. Dysplastic lesions showed a significant increase of LIp53. The length of microvessels per unit of volume was elevated in dysplastic acini. Statistically significant correlations were found only between LILPA1 and LIUBI.
Our results suggest that LPA-1 might be implicated in dysplastic lesions induced by cadmium chloride development. More studies are needed to confirm its potential contribution to the disease.
Colon carcinogenesis is a multistep process and it emanates from a series of molecular and histopathological alterations. Glycyrrhizic acid (GA) is a natural and major pentacyclic triterpenoid glycoside of licorice roots extracts. It has several pharmacological and biological properties such as anti-inflammatory, anti-viral, and anti-cancer. In the present study, we investigated the chemopreventive potential of GA against 1,2-dimethyhydrazine (DMH)-induced precancerous lesions i.e., aberrant crypt foci (ACF) and mucin depleted foci (MDF), and its role in regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of Wistar rats.
Animals were divided into 5 groups. In group III, IV and V, GA was administered at the dose of 15 mg/kg b. wt. orally while in group II, III and IV, DMH was administered subcutaneously in the groin at the dose of 20 mg/kg b.wt once a week for first 5 weeks and animals were euthanized after 9 weeks.
GA supplementation suppressed the development of precancerous lesions and it also reduced the infiltration of mast cells, suppressed the immunostaining of Ki-67, NF-kB-p65, COX-2, iNOS and VEGF while enhanced the immunostaining of p53, connexin-43, caspase-9 and cleaved caspase-3. GA treatment significantly attenuated the level of TNF-α and it also reduced the depletion of the mucous layer as well as attenuated the shifting of sialomucin to sulphomucin.
Our findings suggest that GA has strong chemopreventive potential against DMH-induced colon carcinogenesis but further studies are warranted to elucidate the precise mechanism of action of GA.
Expression of embryonic stem cells (ESCs) markers (SOX2, OCT4, Nanog and Nestin) is crucial for progression of various human malignancies. The purpose of this study was to investigate the expression and prognostic impact of these molecules in nasopharyngeal carcinoma (NPC) patients by immunohistochemistry and immunofluorescence. In the present study, we found that the expression levels of SOX2, OCT4 and Nanog were highly expressed in NPC compared with the non-tumorous tissues. Furthermore, these proteins correlated significantly with several clinicalpathological factors and epithelial-mesenchymal transition (EMT)-associated indicators (E-cadherin/N-cadherin and Snail). In multivariate analyses, high expression of OCT4 (P = 0.013) and Nanog (P = 0.040), but not that of SOX2, was associated with worse survival and had strongly independent prognostic effects. Of note, OCT4 and Nanog were more frequently located at the invasive front of tumors, and correlated significantly with various aggressive behaviors including T classification, N classification, M classification and clinical stage. Furthermore, patients with co-expression of OCT4 and Nanog in the invasive front had significantly worse survival (P = 0.005). Interestingly, at the invasive front, these molecules correlated significantly with Nestin expression in endothelial cells (P<0.001). These findings provide evidence that ESCs biomarkers OCT4 and Nanog serves as independent prognostic factors for NPC. Additionally, cancer cells in the invasive front of NPC acquiring ESCs-like features should be maintained by vascular niches.
Over the last decade, telomere length (TL) has gained attention as a potential biomarker in cancer disease. We previously reported that long blood TL was associated with a poorer outcome in patients with breast cancer and renal cell carcinoma. Based on these findings, we hypothesized that certain immunological components may have an impact on TL dynamics in cancer patients. One aim of the present study was to investigate a possible association between serum cytokines and TL of peripheral blood cells, tumors and corresponding kidney cortex, in patients with clear cell renal cell carcinoma. For this purpose, a multiplex cytokine assay was used. Correlation analysis revealed significant positive correlations between tumor TL and peripheral levels of three cytokines (IL-7, IL-8 and IL-10). In a parallel patient group with various kidney tumors, TL was investigated in whole blood and in immune cell subsets in relation to peripheral levels of regulatory T cells (Tregs). A significant positive association was found between whole blood TL and Treg levels. However, the strongest correlation was found between Tregs and TL of the T lymphocyte fraction. Thus, patients with higher Treg levels displayed longer T cell telomeres, which might reflect a suppressed immune system with fewer cell divisions and hence less telomere shortening. These results are in line with our earlier observation that long blood TL is an unfavorable prognostic factor for cancer-specific survival. In summary, we here show that immunological components are associated with TL in patients with renal cell carcinoma, providing further insight into the field of telomere biology in cancer.
While post-treatment breast cancer survivors face up to twice the cancer risk of the general population, modifiable health behaviors may somewhat reduce this risk. We sought to better understand health behaviors that early stage breast cancer survivors engage in to reduce recurrence risk.
Data came from a cross-sectional multi-site survey of 186 early-stage breast cancer survivors who received genomic testing for breast cancer recurrence risk (Oncotype DX) during their clinical care. Study outcomes were meeting health behavior recommendations (daily fruit and vegetable intake, regular physical activity, and having a healthy body mass index (BMI)).
Approximately three-quarters of survivors we surveyed believed the 3 behaviors might reduce their cancer risk but many did not engage in these behaviors for this purpose: 62% for BMI, 36% for fruit and vegetable consumption, and 37% for physical activity. Survivors with higher recurrence risk, as indicated by their genomic test results, were no more likely to meet any of the three health behavior recommendations. Adherence to health behavior recommendations was higher for women who were white, college-educated, and had higher incomes.
Many nonadherent breast cancer survivors wish to use these behavioral strategies to reduce their risk for recurrence, suggesting an important opportunity for intervention. Improving BMI, which has the largest association with cancer risk, is an especially promising target.
S-phase kinase protein 2 (Skp2), an oncogenic protein, is a key regulator in different cellular and molecular processes, through ubiquitin-proteasome degradation pathway. Increased levels of Skp2 are observed in various types of cancer and associated with poor prognosis. However, in human breast carcinomas, the underlying mechanism and prognostic significance of cytoplasmic Skp2 is still undefined.
To investigate the role of cytoplasmic Skp2 expression in human breast carcinomas, we immnohistochemically assessed cytoplasmic Skp2, p-Akt1, and p27 expression in 251 patients with invasive ductal carcinomas of the breast. Association of cytoplasmic Skp2 expression with p-Akt1 and p27 was analyzed as well as correspondence with other clinicopathological parameters. Disease-free survival and overall survival were determined based on the Kaplan-Meier method and Cox regression models.
Cytoplasmic of Skp2 was detected in 165 out of 251 (65.7%) patients. Cytoplasmic Skp2 expression was associated with larger tumor size, more advanced histological grade, and positive HER2 expression. Increased cytoplasmic Skp2 expression correlated with p-Akt1 expression, with 54.2% (51/94) of low p-Akt1-expressing breast carcinomas, but 72.6% (114/157) of high p-Akt1-expressing breast carcinomas exhibiting cytoplasmic Skp2 expression. Elevated cytoplasmic Skp2 expression with low p-Akt1 expression was associated with poor disease-free and overall survival (DFS and OS), and Cox regression models demonstrated that cytoplasmic Skp2 expression was an independent prognostic marker for invasive breast carcinomas.
Cytoplasmic Skp2 expression is associated with aggressive prognostic factors, such as larger tumor size, and advanced histological grade of the breast cancers. Results demonstrate that combined cytoplasmic Skp2 and p-Akt1 expression may be prognostic for patients with invasive breast carcinomas, and cytoplasmic Skp2 may serve as a potential therapeutic target.
Tumors are infiltrated by macrophages, T and B-lymphocytes, which may favor tumor development by promoting angiogenesis, growth and invasion. The aim of this study was to investigate the clinical relevance of the relative amount of macrophages (CD68+), T-cells (CD3+) and B-cells (CD20+) at the invasive front of breast carcinomas, and the expression of matrix metalloproteases (MMPs) and their inhibitors (TIMPs) either at the invasive front or at the tumor center. We performed an immunohistochemical study counting CD3, CD20 and CD68 positive cells at the invasive front, in 102 breast carcinomas. Also, tissue sections were stained with MMP-2, -9, -11, -14 and TIMP-2 antibodies, and immunoreactivity location, percentage of reactive area and intensity were determined at the invasive front and at the tumor center. The results showed that an increased CD68 count and CD68/(CD3+CD20) ratio were directly associated with both MMP-11 and TIMP-2 expression by mononuclear inflammatory cells at the tumor center (p = 0.041 and p = 0.025 for CD68 count and p = 0.001 and p = 0.045 for ratio, respectively for MMP-11 and TIMP-2). In addition, a high CD68/(CD3+CD20) ratio (>0.05) was directly associated with a higher probability of shortened relapse-free survival. Multivariate analysis revealed that CD68/(CD3+CD20) ratio was an independent factor associated with distant relapse-free survival (RR: 2.54, CI: (1.23–5.24), p<0.01). Therefore, CD68/(CD3+CD20) ratio at the invasive front could be used as an important prognostic marker.
The androgen receptor (AR) expression and the CAG repeat length within the AR gene appear to be involved in the carcinogenesis of male breast carcinoma (MBC). Although phenotypic differences have been observed between MBC and normal control group in AR gene, there is lack of correlation analysis between AR expression and CAG repeat length in MBC. The purpose of the study was to investigate the prognostic value of CAG repeat lengths and AR protein expression.
81 tumor tissues were used for immunostaining for AR expression and CAG repeat length determination and 80 normal controls were analyzed with CAG repeat length in AR gene. The CAG repeat length and AR expression were analyzed in relation to clinicopathological factors and prognostic indicators.
AR gene in many MBCs has long CAG repeat sequence compared with that in control group (P = 0.001) and controls are more likely to exhibit short CAG repeat sequence than MBCs. There was statistically significant difference in long CAG repeat sequence between AR status for MBC patients (P = 0.004). The presence of long CAG repeat sequence and AR-positive expression were associated with shorter survival of MBC patients (CAG repeat: P = 0.050 for 5y-OS; P = 0.035 for 5y-DFS AR status: P = 0.048 for 5y-OS; P = 0.029 for 5y-DFS, respectively).
The CAG repeat length within the AR gene might be one useful molecular biomarker to identify males at increased risk of breast cancer development. The presence of long CAG repeat sequence and AR protein expression were in relation to survival of MBC patients. The CAG repeat length and AR expression were two independent prognostic indicators in MBC patients.
Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients’ treatment in oncology.
Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2–5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22–43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range18–38%) or between different tumor zones (CV 24%, range 17–38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18–34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29–98%) and lymph node metastases (CV 65%, range 40–146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.
Progranulin (PGRN) is considered to play an important role in breast cancer tumorigenesis and in inhibiting tamoxifen-induced apoptosis. We aimed to determine whether PGRN levels are associated with breast cancer recurrence after curative surgery.
We evaluated the associations between preoperative serum PGRN levels and breast cancer recurrence in a cohort of 697 newly diagnosed breast cancer patients who underwent curative surgery between April 2001 and December 2004. The mean age ± standard deviation (SD) was 46±9.8 years, and all patients with hormone receptor (HR)-positive tumors received adjuvant tamoxifen therapy. At a median follow-up of 62.2 months (range, 2.9–98.2), 89 patients (12.8%) had experienced a recurrence and 51 patients (7.3%) had died. In the HR-positive group, serum PGRN levels were associated with recurrence according to the log-rank test for trend (p for trend = 0.049). There was no association between PGRN levels and recurrence in the HR-negative group (p for trend = 0.658). Adjusted hazard ratios, including possible confounders, revealed a linear relationship between serum PGRN levels and recurrence in the HR-positive group (p for trend = 0.049), and this association was further strengthened after excluding patients who had no lymph node metastasis (p for trend = 0.038).
Serum PGRN levels were clinically significant for predicting recurrence in patients with HR-positive breast cancer during adjuvant tamoxifen therapy.
First-generation gene signatures that identify breast cancer patients at risk of recurrence are confined to estrogen-positive cases and are driven by genes involved in the cell cycle and proliferation. Previously we induced sets of stromal genes that are prognostic for both estrogen-positive and estrogen-negative samples. Creating risk-management tools that incorporate these stromal signatures, along with existing proliferation-based signatures and established clinicopathological measures such as lymph node status and tumor size, should better identify women at greatest risk for metastasis and death.
To investigate the strength and independence of the stromal and proliferation factors in estrogen-positive and estrogen-negative patients we constructed multivariate Cox proportional hazards models along with tree-based partitions of cancer cases for four breast cancer cohorts. Two sets of stromal genes, one consisting of DCN and FBLN1, and the other containing LAMA2, add substantial prognostic value to the proliferation signal and to clinical measures. For estrogen receptor-positive patients, the stromal-decorin set adds prognostic value independent of proliferation for three of the four datasets. For estrogen receptor-negative patients, the stromal-laminin set significantly adds prognostic value in two datasets, and marginally in a third. The stromal sets are most prognostic for the unselected population studies and may depend on the age distribution of the cohorts.
The addition of stromal genes would measurably improve the performance of proliferation-based first-generation gene signatures, especially for older women. Incorporating indicators of the state of stromal cell types would mark a conceptual shift from epithelial-centric risk assessment to assessment based on the multiple cell types in the cancer-altered tissue.
The aim of the present study was to investigate the efficacy of adjuvant dose-dense sequential chemotherapy with epirubicin, paclitaxel, and CMF in subgroups of patients with high-risk operable breast cancer, according to tumor subtypes defined by immunohistochemistry (IHC).
Materials and Methods
Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 1,039 patients participating in two adjuvant dose-dense sequential chemotherapy phase III trials were centrally assessed in tissue micro-arrays by IHC for 6 biological markers, that is, estrogen receptor (ER), progesterone receptor (PgR), HER2, Ki67, cytokeratin 5 (CK5), and EGFR. The majority of the cases were further evaluated for HER2 amplification by FISH. Patients were classified as: luminal A (ER/PgR-positive, HER2-negative, Ki67low); luminal B (ER/PgR-positive, HER2-negative, Ki67high); luminal-HER2 (ER/PgR-positive, HER2-positive); HER2-enriched (ER-negative, PgR-negative, HER2-positive); triple-negative (TNBC) (ER-negative, PgR-negative, HER2-negative); and basal core phenotype (BCP) (TNBC, CK5-positive and/or EGFR-positive).
After a median follow-up time of 105.4 months the 5-year disease-free survival (DFS) and overall survival (OS) rates were 73.1% and 86.1%, respectively. Among patients with HER2-enriched tumors there was a significant benefit in both DFS and OS (log-rank test; p = 0.021 and p = 0.006, respectively) for those treated with paclitaxel. The subtype classification was found to be of both predictive and prognostic value. Setting luminal A as the referent category, the adjusted for prognostic factors HR for relapse for patients with TNBC was 1.91 (95% CI: 1.31–2.80, Wald's p = 0.001) and for death 2.53 (95% CI: 1.62–3.60, p<0.001). Site of and time to first relapse differed according to subtype. Locoregional relapses and brain metastases were more frequent in patients with TNBC, while liver metastases were more often seen in patients with HER2-enriched tumors.
Triple-negative phenotype is of adverse prognostic value for DFS and OS in patients treated with adjuvant dose-dense sequential chemotherapy. In the pre-trastuzumab era, the HER2-enriched subtype predicts favorable outcome following paclitaxel-containing treatment.
Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC) (p = 0.007), while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC) (p = 0.006). Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023), ERα negativity (p = 0.001), and the HER2-driven and basal/triple-negative breast cancers (p = 0.018). Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001) and invasive breast cancer overexpressing EGFR (p = 0.019). We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120) (p<0.01). In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005). We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC.
A germline microRNA binding site-disrupting variant, the KRAS-variant (rs61764370), is associated with an increased risk of developing several cancers. Because this variant is most strongly associated with ovarian cancer risk in patients from hereditary breast and ovarian families (HBOC), and with the risk of premenopausal triple negative breast cancer, we evaluated the association of the KRAS-variant with women with personal histories of both breast and ovarian cancer, referred to as double primary patients.
Germline DNA from double primary patients was tested for the KRAS-variant (n = 232). Confirmation of pathologic diagnoses, age of diagnoses, interval between ovarian cancer diagnosis and sample collection, additional cancer diagnoses, and family history were obtained when available. All patients were tested for deleterious BRCA mutations.
The KRAS-variant was significantly enriched in uninformative (BRCA negative) double primary patients, being found in 39% of patients accrued within two years of their ovarian cancer diagnosis. Furthermore, the KRAS-variant was found in 35% of uninformative double primary patients diagnosed with ovarian cancer post-menopausally, and was significantly associated with uninformative double primary patients with a positive family history. The KRAS-variant was also significantly enriched in uninformative patients who developed more then two primary cancers, being found in 48% of women with two breast primaries plus ovarian cancer or with triple primary cancers.
These findings further validate the importance of the KRAS-variant in breast and ovarian cancer risk, and support the association of this variant as a genetic marker for HBOC families previously considered uninformative.
Background and Objectives
Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well.
We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples.
Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma.
Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study.
Previous studies suggested that the molecular subtypes were strongly associated with sentinel lymph node (SLN) status. The purpose of this study was to determine whether molecular subtype classification was associated with non-sentinel lymph nodes (NSLN) metastasis in patients with a positive SLN.
Methodology and Principal Findings
Between January 2001 and March 2011, a total of 130 patients with a positive SLN were recruited. All these patients underwent a complete axillary lymph node dissection. The univariate and multivariate analyses of NSLN metastasis were performed. In univariate and multivariate analyses, large tumor size, macrometastasis and high tumor grade were all significant risk factors of NSLN metastasis in patients with a positive SLN. In univariate analysis, luminal B subgroup showed higher rate of NSLN metastasis than other subgroup (P = 0.027). When other variables were adjusted in multivariate analysis, the molecular subtype classification was a determinant of NSLN metastasis. Relative to triple negative subgroup, both luminal A (P = 0.047) and luminal B (P = 0.010) subgroups showed a higher risk of NSLN metastasis. Otherwise, HER2 over-expression subgroup did not have a higher risk than triple negative subgroup (P = 0.183). The area under the curve (AUC) value was 0.8095 for the Cambridge model. When molecular subtype classification was added to the Cambridge model, the AUC value was 0.8475.
Except for other factors, molecular subtype classification was a determinant of NSLN metastasis in patients with a positive SLN. The predictive accuracy of mathematical models including molecular subtype should be determined in the future.
Genomic grade (GG) is a 97-gene signature which improves the accuracy and prognostic value of histological grade (HG) in invasive breast carcinoma. Since most of the genes included in the GG are involved in cell proliferation, we performed a retrospective study to compare the prognostic value of GG, Mitotic Index and Ki67 score.
A series of 163 consecutive breast cancers was retained (pT1–2, pN0, pM0, 10-yr follow-up). GG was computed using MapQuant Dx(R).
GG was low (GG-1) in 48%, high (GG-3) in 31% and equivocal in 21% of cases. For HG-2 tumors, 50% were classified as GG-1, 18% as GG-3 whereas 31% remained equivocal. In a subgroup of 132 ER+/HER2− tumors GG was the most significant prognostic factor in multivariate Cox regression analysis adjusted for age and tumor size (HR = 5.23, p = 0.02).
In a reference comprehensive cancer center setting, compared to histological grade, GG added significant information on cell proliferation in breast cancers. In patients with HG-2 carcinoma, applying the GG to guide the treatment scheme could lead to a reduction in adjuvant therapy prescription. However, based on the results observed and considering (i) the relatively close prognostic values of GG and Ki67, (ii) the reclassification of about 30% of HG-2 tumors as Equivocal GG and (iii) the economical and technical requirements of the MapQuant micro-array GG test, the availability in the near future of a PCR-based Genomic Grade test with improved performances may lead to an introduction in clinical routine of this test for histological grade 2, ER positive, HER2 negative breast carcinoma.
Long-term colonic inflammation promotes carcinogenesis and histological abnormalities of the liver, and colorectal tumours frequently arise in a background of dysplasia, a precursor of adenomas. Altered colonic microbiota with an increased proportion of bacteria with pro-inflammatory characteristics, have been implicated in neoplastic progression. The composition of the microbiota can be modified by dietary components such as probiotics, polyphenols and dietary fibres. In the present study, the influence of probiotics in combination with blueberry husks on colorectal carcinogenesis and subsequent liver damage was evaluated.
Colorectal tumours were induced in rats by cyclic treatment with dextran sulphate sodium (DSS). Blueberry husks and a mixture of three probiotic strains (Bifidobacterium infantis DSM 15159, Lactobacillus gasseri, DSM 16737 and Lactobacillus plantarum DSM 15313) supplemented a basic diet fortified with oats. The condition of the rats was monitored using a disease activity index (DAI). A qualitative and quantitative histological judgement was performed on segments of distal colon and rectum and the caudate lobe of the liver. The formation of short-chain fatty acids, bacterial translocation, the inflammatory reaction and viable count of lactobacilli and Enterobaceriaceae were addressed.
Blueberry husks with or without probiotics significantly decreased DAI, and significantly reduced the number of colonic ulcers and dysplastic lesions. With a decreased proportion of blueberry husk in the diet, the probiotic supplement was needed to achieve a significant decrease in numbers of dysplastic lesions. Probiotics decreased faecal viable count of Enterobacteriaceae and increased that of lactobacilli. Blueberry husks with or without probiotics lowered the proportion of butyric acid in distal colon, and decreased the haptoglobin levels. Probiotics mitigated hepatic injuries by decreasing parenchymal infiltration and the incidence of stasis and translocation. The results demonstrate a dietary option for use of blueberry husks and probiotics to delay colonic carcinogenesis and hepatic injuries in the rat model.
The burden of breast cancer in Asia is escalating. We evaluated the impact of ethnicity on survival after breast cancer in the multi-ethnic region of South East Asia.
Using the Singapore-Malaysia hospital-based breast cancer registry, we analyzed the association between ethnicity and mortality following breast cancer in 5,264 patients diagnosed between 1990 and 2007 (Chinese: 71.6%, Malay: 18.4%, Indian: 10.0%). We compared survival rates between ethnic groups and calculated adjusted hazard ratios (HR) to estimate the independent effect of ethnicity on survival. Malays (n = 968) presented at a significantly younger age, with larger tumors, and at later stages than the Chinese and Indians. Malays were also more likely to have axillary lymph node metastasis at similar tumor sizes and to have hormone receptor negative and poorly differentiated tumors. Five year overall survival was highest in the Chinese women (75.8%; 95%CI: 74.4%–77.3%) followed by Indians (68.0%; 95%CI: 63.8%–72.2%), and Malays (58.5%; 95%CI: 55.2%–61.7%). Compared to the Chinese, Malay ethnicity was associated with significantly higher risk of all-cause mortality (HR: 1.34; 95%CI: 1.19–1.51), independent of age, stage, tumor characteristics and treatment. Indian ethnicity was not significantly associated with risk of mortality after breast cancer compared to the Chinese (HR: 1.14; 95%CI: 0.98–1.34).
In South East Asia, Malay ethnicity is independently associated with poorer survival after breast cancer. Research into underlying reasons, potentially including variations in tumor biology, psychosocial factors, treatment responsiveness and lifestyle after diagnosis, is warranted.
A large number of epidemiological studies have evaluated the association between Epstein-Barr virus infection and breast carcinoma risk but results have been inconsistent.
Research using the polymerase chain reaction technique for detecting the Epstein-Barr virus was selected; 24 studies and 1535 cases were reviewed. Information on the study populations, sample types, publication calendar period and histological types of breast carcinoma were collected. An unconditional logistic regression model was used to analyze potential parameters related to the Epstein-Barr virus prevalence. A Kappa test was used to evaluate the consistency in detecting different Epstein-Barr virus DNA regions. Nine studies that included control groups and 1045 breast cancer cases were adopted in this meta-analysis.
We found that 29.32% of the patients with breast carcinoma were infected with the Epstein-Barr virus. The prevalence of Epstein-Barr was highest in Asia (35.25%) and lowest in the USA (18.27%). Statistical analysis revealed a trend that showed lobular breast carcinoma might have the strongest association with Epstein-Barr virus infection. This meta-analysis showed a significant increase in breast malignancy risk in patients testing positive for the Epstein-Barr virus (OR = 6.29, 95% CI = 2.13–18.59). This result suggests that an Epstein-Barr virus infection is statistically associated with increased breast carcinoma risk.