High expression of focal adhesion kinase (p125<Superscript>FAK</Superscript>) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome

Introduction

Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy.

Methods

We analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis.

Results

Elevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01).

Conclusions

Immunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.

     

Targeted next-generation sequencing identifies clinically relevant mutations in patients with chronic neutrophilic leukemia at diagnosis and blast crisis

Purpose

Chronic neutrophilic leukemia is a rare form of myeloproliferative neoplasm characterized by mature neutrophil hyperleukocytosis. The majority of patients harbor somatic mutations of CSF3R gene and are potentially amenable to targeted therapy with JAK inhibitors. The incidence and clinical significance of additional mutations requires clarification.

Materials and methods

A next-generation sequencing approach for myeloid malignancy-associated mutations was applied to diagnostic and matched blast crisis samples from four chronic neutrophilic leukemia patients.

Results

Next-generation sequencing confirmed the CSF3R T618I in all patients with identification of concurrent SRSF2, SETBP1, NRAS and CBL mutations at diagnosis. At blast crisis, clonal evolution was evidenced by an increased CSF3R T618I allele frequency and by loss or acquisition of CBL and NRAS mutations.

Conclusion

The diagnostic utility of a targeted next-generation sequencing approach was clearly demonstrated with the identification of additional mutations providing the potential for therapeutic stratification of chronic neutrophilic leukemia patients.

     

Phosphorylated CXCR4 expression has a positive prognostic impact in colorectal cancer

Background

The CXCL12-CXCR4 chemokine axis plays an important role in cell trafficking as well as in tumor progression. In colorectal cancer (CRC), the chemokine receptor CXCR4 has been shown to be an unfavorable prognostic factor in some studies, however, the role of its activated (phosphorylated) form, pCXCR4, has not yet been evaluated. Here, we aimed to investigate the prognostic value of CXCR4 and pCXCR4 in a large cohort of CRC patients.

Patients and methods

A tissue microarray (TMA) of 684 patient specimens of primary CRCs was analyzed by immunohistochemistry (IHC) for the expression of CXCR4 and pCXCR4 by tumor cells and tumor-infiltrating immune cells (TICs).

Results

The combined high expression of CXCR4 and pCXCR4 showed a favorable 5-year overall survival rate (68%; 95%CI = 59–76%) compared to tumors showing a high expression of CXCR4 only (48%; 95%CI = 41–54%). High expression of pCXCR4 was significantly associated with a favorable prognosis in a test and validation group (p = 0.015 and p = 0.0001). Moreover, we found that CRCs with a high density of pCXCR4+ tumor-infiltrating immune cells (TICs) also showed a favorable prognosis in a test and validation group (p = 0.054 and p = 0.004). Univariate Cox regression analysis for TICs revealed that a high density of pCXCR4+ TICs was a favorable prognostic marker for overall survival (HR = 0.97,95%CI = 0.96–1.00; p = 0.01). In multivariate Cox regression survival analyses a high expression of pCXCR4 in tumor cells lost its association with a better overall survival (HR = 0.99; 95%CI = 0.99–1.00, p = 0.098).

Conclusion

Our results show that high densities of CXCR4 and pCXCR4 positive TICs are favorable prognostic factors in CRC.

     

Germline promoter hypermethylation in <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> genes is not present in hereditary breast cancer patients

Purpose

Germline promoter hypermethylation of BRCA1 and BRCA2 genes is an alternative event of gene silencing that has not been widely investigated in hereditary breast and ovarian cancer (HBOC) syndrome.

Methods

We analyzed germline BRCA promoter hypermethylation in HBOC patients with and without BRCA mutations and control subjects, using a recently developed BRCA methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay.

Results

Neither the patients tested nor the control subjects showed germline hypermethylation of the BRCA1 and BRCA2 promoter regions analyzed.

Conclusions

Despite the results achieved at somatic levels by other researchers, these were not confirmed in our study at the germline level. Our results show the need to establish more predictive CpG sites in the BRCA promoter regions to optimize the MS-MLPA assayfor the detection of germline hypermethylation as an effective pre-screening tool for whole-BRCA genetic analysis in HBOC, because we can not rule out the existence of germline promoter hypermethylation in BRCA.

     

Morbidity of local therapy for locally advanced metastatic breast cancer: an analysis of the Surveillance,Epidemiology, and End Results (SEER)–Medicare Registry

Purpose

Cyclin D/cyclin-dependent kinase 4/6 (CDK4/6) complex inhibitors have recently been proven effective when combined with endocrine therapy in clinical trials. However, the clinical benefit from CDK4/6 inhibitor varied from different patients. In order to optimize the clinical application of CDK4/6 inhibitors, this review focuses on the potential biomarkers applicable to identify patients who will benefit the most from CDK4/6 inhibition.

Methods

We have summarized the clinical trials about addition of CDK4/6 inhibitors to endocrine therapy and reviewed literature currently available on the potential biomarkers in predicting efficacy of CDK4/6 inhibitors. The primary objective was to determine the predictors. The secondary objective was to optimize the combination therapeutics for patients with estrogen receptor (ER)-positive breast cancer.

Results

We reviewed clinical trials on antiestrogen agents combined with the CDK4/6 inhibitor (Palbociclib, Ribociclib, or Abemaciclib) in ER-positive breast cancer. It was confirmed that the addition of CDK4/6 inhibitors was associated with an improved efficacy. More importantly, we discussed potential biomarkers for identifying the subpopulations of breast cancer patients who would derive the greatest benefit from CDK4/6 inhibitors.

Conclusions

We have found that although CDK4/6 inhibitors combined with endocrine therapy were potent, the toxicity and financial burden also increased. To maximize the effect of the combinations and select patients that best response to such combinations, further experiments and trials are expected to confirm these molecules as reliable biomarkers.

     

Comparison of plasma ctDNA and tissue/cytology-based techniques for the detection of <Emphasis Type="Italic">EGFR</Emphasis> mutation status in advanced NSCLC: Spanish data subset from ASSESS

Purpose

The analysis of epidermal growth factor receptor (EGFR) mutations in many patients with advanced non-small-cell lung cancer (aNSCLC) has provided the opportunity for successful treatment with specific, targeted EGFR tyrosine kinase inhibitors. However, this therapeutic decision may be challenging when insufficient tumor tissue is available for EGFR mutation testing. Therefore, blood surrogate samples for EGFR mutation analysis have been suggested.

Methods

Data were collected from the Spanish cohort of patients in the large, non-interventional, diagnostic ASSESS study (NCT01785888) evaluating the utility of circulating free tumor-derived DNA from plasma for EGFR mutation testing. The incidence of EGFR mutation in Spain and the level of concordance between matched tissue/cytology and plasma samples were evaluated.

Results

In a cohort of 154 eligible patients, EGFR mutations were identified in 15.1 and 11.0% of tumor and plasma samples, respectively. The most commonly used EGFR mutation testing method for the tumor tissue samples was the QIAGEN Therascreen^® EGFR RGQ PCR kit (52.1%). Fragment Length Analysis?+?PNA LNA Clamp was used for the plasma samples. The concordance rate for EGFR mutation status between the tissue/cytology and plasma samples was 88.8%; the sensitivity was 45.5%, and the specificity was 96.7%.

Conclusions

The high concordance between the different DNA sources for EGFR mutation testing supports the use of plasma samples when tumor tissue is unavailable.

     

Expression Analysis of Survivin and XIAP in Gallbladder Cancer: a Case-control Study in Indo-Gangetic Plain

Purpose

Gallbladder cancer is a highly mortal disease with poor prognosis because of late presentation of disease. Survivin and X-linked inhibitor of apoptosis (XIAP) are one of the two important members of inhibitors of apoptosis. Thus, this study aimed to look at the expression of Survivin and XIAP in gallbladder cancer patients.

Methods

Survivin and XIAP expression were investigated in tissues of gallbladder cancer patients (40 cases) and compared with cholelithiasis as control (40 cases) by using immunohistochemistry. Their expression was correlated with clinicopathological parameters.

Results

Significantly higher (p < 0.05), Survivin protein was expressed in gallbladder cancer (n = 67.5%) than control (n = 35%). But it did not show any significant association with any of the clinicopathological parameter while XIAP was not expressed in the GBC patients (p > 0.05).

Conclusion

Overexpression of Survivin in gallbladder cancer suggests its possible role and association with poor prognosis. But XIAP has not been found to be associated with gallbladder carcinogenesis.

     

Assessing the relationship between fear of cancer recurrence and health care utilization in early-stage breast cancer survivors

Purpose

The purpose of this study was to determine whether fear of cancer recurrence (FCR) is associated with greater health care utilization (HCU) in early-stage breast cancer survivors.

Methods

Three hundred early-stage breast cancer survivors diagnosed within the past 7 years reported on FCR as well as calls and visits to oncology providers and primary care providers during the preceding 3 months. Participants also reported on use of mental health services and psychotropic medications since diagnosis. Structural equation modeling was used to create a latent FCR factor and evaluate this factor as a predictor of various HCU outcomes controlling for age at diagnosis, years since diagnosis, generalized anxiety, objective risk of recurrence, and number of comorbidities.

Results

FCR predicted more visits to both oncology providers (RR?=?1.53, p?=?.002) and primary care providers (RR?=?1.31, p?=?.013), as well as more phone calls to oncology providers (RR?=?2.08, p?=?.007). FCR was not a significant predictor of phone calls to primary care providers (RR?=?1.39, p?=?.054), utilization of mental health treatment (OR?=?1.27, p?=?.362), or use of psychotropic medications (OR?=?1.37, p?=?.178).

Conclusions

FCR was associated with increases in some types of HCU, which may reflect excessive medical reassurance-seeking and lead to unnecessary medical costs.

Implications for Cancer Survivors

FCR is a serious concern that warrants greater attention to reduce distress-related health care utilization. Utilization of mental health services to address FCR may represent higher-value health care.

     

The CXCR4-CXCL12 axis in Ewing sarcoma: promotion of tumor growth rather than metastatic disease

Background

Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources.

Methods

To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions.

Results

Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p?<?0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p?=?0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p?=?0.027). Evaluation of in vivo hypoxia-inducible factor-1α expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression.

Conclusions

Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.

     

Efficacy of doxorubicin-transferrin conjugate in apoptosis induction in human leukemia cells through reactive oxygen species generation

Background

Doxorubicin (DOX) is a small molecular cytotoxic agent that can be transferred efficiently to cancer cells by nanocarriers. This anthracycline antibiotic serves as an effective anti-neoplastic drug against both hematological and solid malignancies. Here, we set out to assess the capacity of a novel doxorubicin - transferrin conjugate (DOX-TRF) to provoke apoptosis in human normal and leukemia cells through free radicals produced via a redox cycle of doxorubicin (DOX) when released from its conjugate.

Methods

After DOX-TRF exposure, we determined the time-course of apoptotic and necrotic events, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, as well as alterations in cytochrome c levels and intracellular calcium concentrations in human leukemia-derived cell lines (CCRF-CEM, K562 and its doxorubicin-resistant derivative K562/DOX) and normal peripheral blood-derived mononuclear cells (PBMC).

Results

We found that DOX-TRF can induce apoptosis in all leukemia-derived cell lines tested, which was associated with morphological changes and decreases in mitochondrial membrane potential. In comparison to free DOX treated cells, we observed a time-dependency between a higher level of ROS generation and a higher drop in mitochondrial membrane potential, particularly in the doxorubicin-resistant cell line. In addition, we found that the apoptotic cell death induced by DOX-TRF was directly associated with a release of cytochrome c from the mitochondria and an increase in intracellular calcium level in all human leukemia-derived cell lines tested.

Conclusions

Our data indicate that DOX-TRF is considerably more cytotoxic to human leukemia cells than free DOX. In addition, we show that DOX-TRF can effectively produce free radicals, which are directly involved in apoptosis induction.

     

Anti-tumor effect of CDK inhibitors on <Emphasis Type="Italic">CDKN2A</Emphasis>-defective squamous cell lung cancer cells

Background

Squamous cell lung cancer (SqCLC) is a distinct histologic subtype of non-small cell lung cancer (NSCLC). Although the discovery of driver mutations and their targeted drugs has remarkably improved the treatment outcomes for lung adenocarcinoma, currently no such molecular target is clinically available for SqCLC. The CDKN2A locus at 9p21 encodes two alternatively spliced proteins, p16^INK4a (p16) and p14^ARF (p14), which function as cell cycle inhibitors. The Cancer Genome Atlas (TCGA) project revealed that CDKN2A is inactivated in 72% of SqCLC cases. In addition, it was found that CDKN2A mutations are significantly more common in SqCLC than in adenocarcinoma. Down-regulation of p16 and p14 by CDKN2A gene inactivation leads to activation of cyclin-dependent kinases (CDKs), thereby permitting constitutive phosphorylation of Rb and subsequent cell cycle progression. Here, we hypothesized that CDK inhibition may serve as an attractive strategy for the treatment of CDKN2A-defective SqCLC.

Methods

We investigated whether the CDK inhibitors flavopiridol and dinaciclib may exhibit antitumor activity in CDKN2A-defective SqCLC cells compared to control cells. The cytotoxic effect of the CDK inhibitors was evaluated using cell viability assays, and the induction of apoptosis was assessed using TUNEL assays and Western blot analyses. Finally, anti-tumor effects of the CDK inhibitors on xenografted cells were investigated in vivo.

Results

We found that flavopiridol and dinaciclib induced cytotoxicity by enhancing apoptosis in CDKN2A-defective SqCLC cells, and that epithelial to mesenchymal transition (EMT) decreased and autophagy increased during this process. In addition, we found that autophagy had a cytoprotective role.

Conclusion

Our data suggest a potential role of CDK inhibitors in managing CDKN2A-defective SqCLC.

     

The Role of HER2 Testing in Advanced Colorectal Cancer

Purpose of Review

About 1/3 of all metastatic colorectal cancer (mCRC) patients may harbor a mutation in the KRAS or NRAS gene suggesting inefficacy of EGFR inhibitors cetuximab and panitumumab. In spite of tailoring treatment in RAS wild-type patients to receive EGFR inhibitors, not all show response.

Recent Findings

Studies have shown that HER2-neu amplification/alteration in addition to alteration in BRAF and PI3KA may explain resistance to EGFR inhibitors. Several pre-clinical studies have identified that HER2-neu amplification can result in both de novo and acquired resistance to EGFR inhibitors. Recently, several clinical studies have highlighted the use of single or combination HER2-neu directed therapies in HER2-neu amplified/overexpressed mCRC.

Summary

About 5% mCRC patients will demonstrate HER2-neu overexpression and response to HER2-neu-directed therapies can be in the range of 30–38%. Patients not responding to EGFR-inhibitors warrant testing for HER2-neu testing to explain resistance. In the near future, HER2-neu testing is likely to be integrated into our routine clinical practice for management of metastatic colorectal cancer patients.

     

A high frequency of PALB2 mutations in Jamaican patients with breast cancer

Purpose

Jamaica is an island nation with one of the highest breast cancer incidence rates in the Caribbean (40/100,000 per year). The contribution of cancer susceptibility gene mutations to the burden of breast cancer in Jamaica has not yet been explored. We sought to determine the prevalence of germline mutations in BRCA1, BRCA2, and PALB2 in 179 unselected Jamaican women with breast cancer.

Methods

We sequenced the entire coding regions of BRCA1, BRCA2, and PALB2 for all the study subjects.

Results

Overall, 8 of 179 patients (4.5%) had a mutation in one of the three genes: one in BRCA1, two in BRCA2, and five in PALB2.

Conclusions

These data suggest that in addition to BRCA1 and BRCA2, PALB2 should be included in genetic testing for breast cancer patients in Jamaica.

     

Workplace experiences and turnover intention among adult survivors of childhood cancer

Purpose

The purpose of this study was to investigate workplace experiences and turnover intention (consideration of leaving or changing a job) and to examine factors associated with turnover intention among survivors.

Methods

Adult survivors of childhood cancer with a history of employment (n?=?289) completed measures of workplace experiences (n?=?50, 18–29 years; n?=?183, 30–44 years; n?=?56; >?45 years of age at follow-up). Turnover intention was assessed using three items from the Job Satisfaction Scale. Responses were dichotomized as reflecting high vs. low turnover intention. Path analysis was used to estimate the influence of demographic characteristics, treatment exposures (cranial radiation therapy [CRT]), and workplace experiences on turnover intention.

Results

Thirty percent of survivors reported high turnover intention (95% CL, 25 to 36%). Exposure to CRT (P?=?0.003), older attained age (P?<?0.001), experiencing formal workplace discrimination (P?=?0.008), and having lower continuance (P?<?0.001) or affective commitment (P?<?0.001) were associated with high turnover intention among survivors. Informal discrimination, mediated through job satisfaction, also influenced survivors’ reported intent to leave their jobs.

Conclusions

One third of adult survivors of childhood cancer report turnover intention, which is related to their cancer treatment, but more temporally proximal, workplace discrimination. Additional research is needed to understand the consequences of turnover intention among survivors.

Implications for Cancer Survivors

Survivors and their health care providers should be aware of legislative policies related to workplace discrimination (e.g., American with Disabilities Act) and related implications for job turnover.

     

Prevalence of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Infection in Patients with Squamous Cell Carcinoma of the Oesophagus. A Descriptive Case Series Study

Introduction

Helicobacter pylori is an important causative factor in gastric carcinogenesis; its role in extra-gastric gastrointestinal malignancies such as oesophageal cancer is controversial. H. pylori is thought to cause extensive gastric atrophy associated with squamous cell carcinoma of the oesophagus. We conducted a study to determine the prevalence of H. pylori infection in patients with squamous cell carcinoma of the oesophagus.

Method

We collected biopsies from the antrum and corpus of 59 patients with confirmed squamous cell carcinoma of the oesophagus, two from each area. These were then examined by an experienced histopathologist using methylene blue staining for the presence of H. pylori.

Results

H. pylori was found in 30 (51 %) of the patients, a prevalence similar to that of the general population in South Africa. Five patients were found to have associated intestinal metaplasia, and all but two had chronic inflammation.

Conclusion

The prevalence of H. pylori in our patients with squamous cell carcinoma of the oesophagus is 51 %, similar to that previously reported in the general population.

     

Genetic mutation analysis of the malignant transformation of sinonasal inverted papilloma by targeted amplicon sequencing

Background

The mechanism underlying the malignant transformation of inverted papilloma (IP) has not yet been elucidated.

Methods

To clarify the genes responsible for the malignant transformation, we analyzed 10 cases of IP, 8 of IP with dysplasia, and 11 of squamous cell carcinoma (SCC) by targeted amplicon sequencing.

Results

The number of mutant genes increased in the order of IP?<?dysplasia?<?SCC. Significant differences were observed in the mutation rates of three genes (KRAS, APC and STK11) in particular. TP53 was altered frequently in each group and might be involved in malignant transformation based on to the site of the mutation. A comparison of the genetic variants by region of IP tissue among patients with IP alone, and those with dysplasia or SCC revealed significant differences in the mutation rate of the KRAS gene.

Conclusion

Identification of genetic mutations in KRAS is effective for predicting the malignant transformation of IP.

     

Altered expression of major immune regulatory molecules in peripheral blood immune cells associated with breast cancer

Background

The purpose of this study was to clarify the alterations of major immune regulators in peripheral blood mononuclear cells (PBMCs) of cancer patients and to analyze the association with the disease progression in breast cancer patients.

Methods

The study included 6 healthy volunteers (HVs), 12 primary breast cancer (PBC) patients, and 30 metastatic breast cancer (MBC) patients. The expression of immune regulators such as, CCR6, CD4, CD8, CD14, CD40, CD56, CD80, CTLA4, CXCR4, FOXP3, IDO-1, IDO-2, NKG2D, NRP-1, PD-1, and PD-L1 mRNA in PBMCs was measured by quantitative RT-PCR. Analysis of variance with contrasts was performed to find expression patterns of the three groups (HVs, PBC, MBC).

Results

We clarified the alterations of mRNA of major immune regulators PD-L1, FOXP3, CD80, CD40, and CD14 in PBMCs of cancer patients and the association of these alternations with disease progression. Furthermore, PD-L1 expression was correlated with serum interferon-γ production.

Conclusion

Our data suggested that mRNA expressions of PD-L1, FOXP3, CD80, CD40 and CD14 in PBMCs are affected by disease progression. Understanding the roles of these various interactions will be of importance to future studies aiming to uncover biomarkers for predicting response to immune therapy.