Direct serum and tissue assay for EGFR mutation in non-small cell lung cancer by high-resolution melting analysis

Biological therapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have noted promising outcomes for patients with non-small cell lung carcinoma (NSCLC), especially those with mutated EGFR. Tissue EGFR gene mutation testing can predict the benefit of taking a first-line EGFR-TKI, thus, allowing the physician to prescribe the most suitable therapy. Unfortunately, most lung cancer patients, especially NSCLC patients present with advanced disease that is surgically unresectable. The goal of this study was to develop high-resolution melting (HRM) assays to detect EGFR mutations in exons 18 to 21, compare their sensitivity and concordance to direct sequencing, and evaluate the feasibility and reliability of serum as a tissue alternate for routine EGFR mutation screening. EGFR mutations of 126 Formalin-Fixed Paraffin-Embedded (FFPE), 47 fresh frozen tissues and from 47 matched pre-operation serum specimens of NSCLC patients were screened by the HRM assays. EGFR mutations by HRM were confirmed through sequencing. We found 78 EGFR mutations in 70 FFPE tissues, 25 EGFR mutations in 24 fresh frozen tissues, with a mutation rate of 55.56% (70/126) and 51.06% (24/47), respectively. Most mutations were correctly identified by sequencing. EGFR mutations were detected in 22 serum samples from 24 tissue EGFR mutation-positive patients. The concordance rate between serum and tissue in EGFR mutation screening was 91.67%. We conclude that the HRM assay can provide convincing and valuable results both for serum and tissues samples, thus, it is suitable for routine serum EGFR mutation screening for NSCLC patients, especially those surgically unresectable.     

The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual’s high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients.     

MicroRNA profiling of gastric cancer patients from formalin-fixed paraffin-embedded samples

MicroRNA (miRNA) is a small non-coding RNA that targets specific mRNA. Recent progress in the extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissues has facilitated miRNA profiling using samples stored in laboratories worldwide. In the present study, miRNA profiling of gastric cancer patients is determined using FFPE samples. First, criteria were established for determining evaluable RNA from the FFPE samples. miRNA profiling was then undertaken using miRNA oligo chips with 885 featured genes. The FFPE samples were obtained from 47 gastric cancer patients who underwent operations between 1997 and 2007. Results showed that out of 47 paired samples, 37 pairs (78.8%) were evaluable by our criteria. A total of 30 miRNAs were significantly up-regulated and 11 miRNAs were down-regulated in gastric cancer compared with those in normal gastric tissue. Among these, 14 miRNAs, including miR-21, were identified as prognostic factors of gastric cancer patients. Furthermore, miR-34a was selected as an independent prognostic factor. In conclusion, we identified miRNAs that are associated with the prognosis of gastric cancer patients. miRNA profiling using FFPE samples is a useful and promising method of evaluation for samples stored in laboratories worldwide, and can generate extremely valuable clinical data.     

Harnessing the potential of reverse-phase protein array technology: Advancing precision oncology strategies

The last few decades have seen remarkable strides in the field of cancer therapy. Precision oncology coupled with comprehensive genomic profiling has become routine clinical practice for solid tumors, the advent of immune checkpoint inhibitors has transformed the landscape of oncology treatment, and the number of cancer drug approvals has continued to increase. Nevertheless, the application of genomics-driven precision oncology has thus far benefited only 10%–20% of cancer patients, leaving the majority without matched treatment options. This limitation underscores the need to explore alternative avenues with regard to selecting patients for targeted therapies. In contrast with genomics-based approaches, proteomics-based strategies offer a more precise understanding of the intricate biological processes driving cancer pathogenesis. This perspective underscores the importance of integrating complementary proteomic analyses into the next phase of precision oncology to establish robust biomarker-drug associations and surmount challenges related to drug resistance. One promising technology in this regard is the reverse-phase protein array (RPPA), which excels in quantitatively detecting protein modifications, even with limited amounts of sample. Its cost–effectiveness and rapid turnaround time further bolster its appeal for application in clinical settings. Here, we review the current status of genomics-driven precision oncology, as well as its limitations, with an emphasis on drug resistance. Subsequently, we explore the application of RPPA technology as a catalyst for advancing precision oncology. Through illustrative examples drawn from clinical trials, we demonstrate its utility for unraveling the molecular mechanisms underlying drug responses and resistance.     

Castration-resistant prostate cancer: new science and therapeutic prospects

There is a growing number of new therapies targeting different pathways that will revolutionize patient management strategies in castration-resistant prostate cancer (CRPC) patients. Today there are more clinical trial options for CRPC treatment than ever before, and there are many promising agents in late-stage clinical testing. The hypothesis that CRPC frequently remains driven by a ligand-activated androgen receptor (AR) and that CRPC tissues exhibit substantial residual androgen levels despite gonadotropin-releasing hormone therapy, has led to the evaluation of new oral compounds such as abiraterone and MDV 3100. Their results, coupled with promising recent findings in immunotherapy (eg sipuleucel-T) and with agents targeting angiogenesis (while awaiting the final results of the CALGB trial 90401) will most probably impact the management of patients with CRPC in the near future. Other new promising agents need further development. With our increased understanding of the biology of this disease, further trial design should incorporate improved patient selection so that patient populations are those who may be most likely to benefit from treatment.     

Technology insight: pharmacoproteomics for cancer--promises of patient-tailored medicine using protein microarrays

Patient-tailored medicine can be defined as the selection of specific therapeutics to treat disease in a particular individual based on genetic, genomic or proteomic information. While individualized treatments have been used in medicine for years, advances in cancer treatment have now generated a need to more precisely define and identify those patients who will derive the most benefit from new-targeted agents. Cellular signaling pathways are a protein-based network, and the intended drug effect is to disrupt aberrant protein phosphorylation-based enzymatic activity and epigenetic phenomena. Pharmacoproteomics, or the tailoring of therapy based on proteomic knowledge, will begin to take a central role in this process. A new type of protein array platform, the reverse-phase protein microarray, shows potential for providing detailed information about the state of the cellular 'circuitry' from small samples such as patient biopsy specimens. Measurements of hundreds of specific phosphorylated proteins that span large classes of important signaling pathways can be obtained at once from only a few thousand cells. Clinical implementation of these new proteomic tools to aid the clinical, medical and surgical oncologist in making decisions about patient care will now require thoughtful communication between practicing clinicians and research scientists.     

Extraction of tamoxifen and its metabolites from formalin-fixed,paraffin-embedded tissues: an innovative quantitation method using liquid chromatography and tandem mass spectrometry

Purpose

Tamoxifen is a key therapeutic option for breast cancer treatment. Understanding its complex metabolism and pharmacokinetics is important for dose optimization. We examined the possibility of utilizing archival formalin-fixed paraffin-embedded (FFPE) tissue as an alternative sample source for quantification since well-annotated retrospective samples were always limited.

Methods

Six 15 μm sections of FFPE tissues were deparaffinized with xylene and purified using solid-phase extraction. Tamoxifen and its metabolites were separated and detected by liquid chromatography–tandem mass spectrometry using multiple-reaction monitoring.

Results

This method was linear between 0.4 and 200 ng/g for 4-hydroxy-tamoxifen and endoxifen, and 4–2,000 ng/g for tamoxifen and N-desmethyl-tamoxifen. Inter- and intra-assay precisions were <9 %, and mean accuracies ranged from 81 to 106 %. Extraction recoveries were between 83 and 88 %. The validated method was applied to FFPE tissues from two groups of patients, who received 20 mg/day of tamoxifen for >6 months, and were classified into breast tumor recurrence and non-recurrence. Our preliminary data show that levels of tamoxifen metabolites were significantly lower in patients with recurrent cancer, suggesting that inter-individual variability in tamoxifen metabolism might partly account for the development of cancer recurrence. Nevertheless, other causes such as non-compliance or stopping therapy of tamoxifen could possibly lead to the concentration differences.

Conclusions

The ability to successfully study tamoxifen metabolism in such tissue samples will rapidly increase our knowledge of how tamoxifen’s action, metabolism and tissue distribution contribute to breast cancer control. However, larger population studies are required to understand the underlying mechanism of tamoxifen metabolism for optimization of its treatment.     

Developments and Current Treatment Standards in Cervical Cancer

The prognosis of patients with cervical cancer has improved dramatically in the last 20 years due to the success of systematic screening programs; however, the disease remains a major health problem worldwide. Patients with advanced, recurrent or metastatic disease still have a poor prognosis. Over the last century, radical surgery and radiotherapy have been used to improve survival rates in patients with primary cervical cancer. Both methods have been extensively modified over the years; however, neither improvements in surgical therapy (e.g. radical lymph node dissection) nor modified radiotherapy methods (e.g. neutron irradiation, hyperthermia, interstitial brachytherapy) have significantly decreased the mortality rates. Recently, cooperative clinical trials have demonstrated the superiority of multimodality strategies for patients with high-risk cervical cancer. In these studies, chemotherapy has been integrated into primary therapy; results have shown the most significant improvement of locally advanced disease in more than three decades. The introduction of modern surgical techniques, such as laparoscopic lymphonodectomy and radical pelvic exenteration, are new developments that will change the treatment of cervical cancer in the near future. This review summarizes different aspects of cervical cancer therapy, with emphasis on invasive disease, and provides a perspective on developments which may improve both local and systemic treatment of the disease.     

Molecular Classification of Triple Negative Breast Cancer and the Emergence of Targeted Therapies

Triple negative breast cancer (TNBC) represents 15% to 20% of all primary breast cancers and is the most aggressive subtype of breast cancer. There has been rapid progress in targeted therapy and biomarker development to identify the optimal treatments for TNBC.To update recent developments, this article comprehensively reviews molecular classification and biomarkers of TNBC and targeted therapy developments in immunotherapy, PARP and AKT pathway inhibitors, antibody-drug conjugates and androgen receptor blockade.The treatment of TNBC has dramatically evolved beyond basic cytotoxic chemotherapy into an expanding domain of targeted therapies tailored to the heterogeneity of this complex and aggressive disease. Progress will continue through the sustained and devoted efforts of our investigators and the patients who dedicatedly enroll in clinical trials. Through a daring persistence to challenge the status quo we now have the opportunity to offer our patients with TNBC a new sense of hope.     

Pancreatic cancer: what is new in 2005?

During the year 2005, many publications were presented about pancreatic tumours concerning the palliative treatment of advanced adenocarcinoma, perioperative strategies (i.e., adjuvant and neoadjuvant), and genetic or environmental factors that predispose to this cancer. Several valuable first lines of chemotherapy for unresectable cancers will be soon available, in view of promising results of biotherapies (i.e., erlotinib) or capecitabine combined with gemcitabine. Perioperative treatments may provide additional chances to cure the disease or to delay the tumor relapse. In addition, it should allow to better select the patients for a surgical resection (neoadjuvant strategy). About basic research, new developments of proteomic and pharmacogenomic are promising. Early detection of pancreatic adenocarcinoma in high-risk patients (about 10% of pancreatic adenocarcinomas), although delicate to perform, may become soon a reality in our practice. The deleterious role of tobacco consumption and way of feeding has been confirmed by several communications this year.     

CAR-T细胞治疗结直肠癌的研究进展

近年来,结直肠癌的发病率、死亡率呈现逐年增长的趋势,严重威胁人类的健康。除了少部分早期发现的结直肠癌可以通过手术切除治疗外,大多数尚缺乏有效的治疗手段。CAR修饰的T细胞疗法（chimeric antigen receptor engineered T cells,CAR-T）作为免疫疗法的一种新模式,无疑成为最令人期待的治疗方法之一。目前,已有多个针对结直肠癌的临床试验正在进行,本文将对CAR-T细胞治疗结直肠癌的研究进展作一综述。     

Molecule based diagnosis

Based on reviews of the concept of diagnostics and in general and in specific tumour areas it was clear that development of diagnostic procedures involving genomics will allow for much better targeted and tailored treatments in the future. This will result in better efficacy and better tolerability of cancer treatments, but will also allow for progress in prediction, diagnosis and dose selection. Large collaborative projects studying the efficacy and safety of drugs on the genome level is promising to bring important benefits to both patients and the national economy by reducing useless drug therapy. In colorectal cancer there are several genetic defects identified that can act as the target for directed therapy in the future. Expressions of tumour specific antigens open the way for immunological targeted therapies. Developments in the understanding of the genomic basis for resistance to anti-tumour therapy is promising to help targeting patients likely to respond and not develop resistance. A Japanese model is being developed to determine the relative risk of breast cancer of Japanese women. Based on this prevention therapies can be instigated. The last four years have seen the introduction of four novel targeted therapies. If this model should become a standard in the future, much stronger collaboration between academic research and pharmaceutical industry need to develop.     

Tissue-based research in kidney cancer: current challenges and future directions.

The past several years have seen unprecedented advances in the application of various therapeutic strategies for the treatment of patients with renal cancer. The availability of active immunotherapy, antiangiogenic therapy, and targeted therapy for this disease has brought front and center issues related to choosing the appropriate treatment for particular patient populations. It is increasingly evident that the most promising treatment selection strategies will incorporate identifying specific features of the tumor itself. To facilitate this move toward personalized medicine, it is critically important to establish some standard principles for renal cancer tissue collection, preparation, and analysis for translational research studies. In this article, we identify and discuss some critical issues related to tissue-based kidney cancer research. We focus on five major areas as follows: (a) surgical and image-guided techniques for tissue collection; (b) quality control of specimen collection, processing, storage, and review; (c) issues related to analysis of paraffin embedded tissues; (d) genomic studies; and (e) assessment of reproducibility of assays across institutions. In addition, some practical implementation strategies are proposed. Although many of the topics discussed are specific for renal cancer, several are also relevant to tissue based biomarker investigations in a broad array of malignancies.     

Effective oral chemotherapy for breast cancer: pillars of strength.

Traditionally, anticancer therapy has been dominated by intravenous drug therapy. However, oral agents provide an attractive approach to chemotherapy and use of oral treatments is increasing. We discuss the benefits and challenges of oral chemotherapy from the perspectives of patients, healthcare providers and healthcare funders. Important issues include patient preference, efficacy, compliance, bioavailability, reimbursement, use in special patient populations, financial and staff time savings and flexibility of dosing. We review data for traditional oral agents (e.g. cyclophosphamide, methotrexate), newer oral chemotherapies (e.g. capecitabine), oral formulations of traditionally intravenous agents (e.g. vinorelbine, idarubicin) and new biologic agents under evaluation in breast cancer (e.g. tyrosine kinase inhibitors). Lastly, we review studies of all-oral combination regimens. The wealth of data available and the increasing use of oral agents in breast cancer suggest that many of the concerns and perceptions about oral therapy, including efficacy and bioavailability, have been overcome, and that oral therapy will play a major role in breast cancer management in the future in both the metastatic and adjuvant settings.     

Chemoprevention of cancer

Chemoprevention is a relatively new area of clinical cancer research. In this article we have presented a general overview of the concepts as well as the status of ongoing clinical trials using the most promising agents in selected populations. The reader is referred to recent excellent reviews presenting more detailed discussions of the various topics discussed, such as the selection process for new agents, in vitro and animal model screening procedures, epidemiologic studies, and other agents now being screened and tested for potential clinical study. Increased understanding of the biology of carcinogenesis will undoubtedly lead to new ideas and approaches. Pathways for conversion of proto-oncogenes to tumor-supporting oncogenes, the mechanisms of action of tumor suppressor genes, and the involvement of abnormal growth factor responses in producing "field cancerization" are some examples of future targets for chemopreventive intervention. Long-term goals of this effort will include application of the results of early promising prevention studies, such as those in oral premalignant lesions, to the design of carefully controlled trials to demonstrate actual inhibition of cancer occurrence. In the laboratory, basic studies need to be conducted to increase our understanding of the molecular events underlying carcinogenesis, in order to identify additional targets for new agents to enhance the clinical efforts. Some of the most exciting developments in the control of cancer over the next decade will very likely result from this chemopreventive approach, with application not only to prevention of the first or primary malignancy, especially in high risk populations, but also to clinical situations traditionally considered to be in the domain of chemotherapeutic strategies, such as adjuvant treatment after definitive therapy of a primary cancer.     

Immunotherapy of lung cancer: an update

In Germany lung cancer is the leading cause of cancer-associated death in men. Surgery, chemotherapy and radiation may enhance survival of patients suffering from lung cancer but the enhancement is typically transient and mostly absent with advanced disease; eventually more than 90% of lung cancer patients will die of disease. New approaches to the treatment of lung cancer are urgently needed. Immunotherapy may represent one new approach with low toxicity and high specificity but implementation has been a challenge because of the poor antigenic characterization of these tumors and their ability to escape immune responses. Several different immunotherapeutic treatment strategies have been developed. This review examines the current state of development and recent advances with respect to non-specific immune stimulation, cellular immunotherapy (specific and non-specific), therapeutic cancer vaccines and gene therapy for lung cancer. The focus is primarily placed on immunotherapeutic cancer treatments that are already in clinical trial or well progressed in preclinical studies. Although there seems to be a promising future for immunotherapy in lung cancer, presently there is not standard immunotherapy available for clinical routine.