Generation and characterisation of a semi-synthetic siderophore-immunogen conjugate and a derivative recombinant triacetylfusarinine C-specific monoclonal antibody with fungal diagnostic application.

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.

Polyclonal Immunoglobulin G N-Glycosylation in the Pathogenesis of Plasma Cell Disorders.

The pathological progression from benign monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to active myeloma (MM) is poorly understood. Abnormal immunoglobulin G (IgG) glycosylation in myeloma has been reported. Using a glycomic platform composed of hydrophilic interaction UPLC, exoglycosidase digestions, weak anion-exchange chromatography, and mass spectrometry, polyclonal IgG N-glycosylation profiles from 35 patients [MGUS (n = 8), SMM (n = 5), MM (n = 8), complete-response (CR) post-treatment (n = 5), relapse (n = 4), healthy age-matched control (n = 5)] were characterized to map glycan structures in distinct disease phases of multiple myeloma. N-Glycan profiles from MGUS resembled normal control. The abundance of neutral glycans containing terminal galactose was highest in SMM, while agalactosylated glycans and fucosylated glycans were lowest in MM. Three afucosyl-biantennary-digalactosylated-sialylated species (A2G2S1, A2BG2S1, and A2BG2S2) decreased 2.38-, 2.4-, and 4.25-fold, respectively, from benign to active myeloma. Increased light chain sialylation was observed in a longitudinal case of transformation from MGUS to MM. Bisecting N-acetylglucosamine was lowest in the CR group, while highest in relapsed disease. Gene expression levels of FUT 8, ST6GAL1, B4GALT1, RECK, and BACH2 identified from publicly available GEP data supported the glycomic changes seen in MM compared to control. The observed differential glycosylation underlined the heterogeneity of the myeloma spectrum. This study demonstrates the feasibility of mapping glycan modifications on the IgG molecule and provides proof of principle that differential IgG glycosylation patterns can be successfully identified in plasma cell disorders.

A novel inhibitory anti-invasive MAb isolated using phenotypic screening highlights AnxA6 as a functionally relevant target protein in pancreatic cancer.

BACKGROUND: Discovery and validation of new antibody tractable targets is critical for the development of new antibody therapeutics to address unmet needs in oncology. METHODS: A highly invasive clonal variant of the MDA-MB-435S cell line was used to generate monoclonal antibodies (MAbs), which were screened for anti-invasive activity against aggressive cancer cells in vitro. The molecular target of selected inhibitory MAb 9E1 was identified using immunoprecipitation/liquid chromatography-tandem mass spectrometry. The potential anti-tumour effects of MAb 9E1 were investigated in vitro together with immunohistochemical analysis of the 9E1 target antigen in normal and cancer tissues. RESULTS: MAb 9E1 significantly decreases invasion in pancreatic, lung squamous and breast cancer cells and silencing of its target antigen, which was revealed as AnxA6, leads to markedly reduced invasive capacity of pancreatic and lung squamous cancer in vitro. IHC using MAb 9E1 revealed that AnxA6 exhibits a high prevalence of membrane immunoreactivity across aggressive tumour types with restricted expression observed in the majority of normal tissues. In pancreatic ductal adenocarcinoma, high AnxA6 IHC score correlated with the presence of tumour budding at the invasive front of tumours (P=0.082), the presence of perineural invasion (P= <0.0001) and showed a weak correlation with reduced survival (P=0.2242). CONCLUSIONS: This study highlights the use of phenotypic hybridoma screening as an effective strategy to select a novel function-blocking MAb, 9E1 with anti-cancer activity in vitro. Moreover, through characterisation of the 9E1 target antigen, AnxA6, our findings support further investigation of AnxA6 as a potential candidate target for antibody-mediated inhibition of pancreatic cancer.

Identification and functional validation of RAD23B as a potential protein in human breast cancer progression.

Identification of protein targets that play a role in breast cancer invasion may help to understand the rapid progression of cancer and may lead to the development of new biomarkers for the disease. In this study, we compared two highly invasive and two poorly invasive breast cancer cell lines using comparative label-free LC-MS profiling in order to identify differentially expressed proteins that may be linked to the invasive phenotype in vitro. Forty-five proteins were found to be upregulated, and 34 proteins, downregulated. UV excision repair protein RAD23 homologue B (RAD23B) was found among the downregulated proteins in highly invasive breast cancer cell lines. In poorly invasive breast cancer cell lines, siRNA-mediated downregulation of RAD23B subsequently led to an increase in invasion and adhesion in vitro. Immunohistochemistry analysis of 164 specimens of invasive breast cancer showed that having a high percentage (>80%) of RAD23B positive nuclei was significantly associated with histopathological grades 1 and 2 breast cancer and with low mitotic activity. In addition, a high staining intensity for RAD23B in the cytoplasm was significantly associated with histopathological grade 3 breast cancer. This study suggests a potential role of RAD23B in breast cancer progression and may further imply a tumor suppressor role of nuclear RAD23B in breast cancer.

7B7: a novel antibody directed against the Ku70/Ku80 heterodimer blocks invasion in pancreatic and lung cancer cells.

Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.

EGFR and HER2 inhibition in pancreatic cancer.

The aim of this study was to investigate the effect of lapatinib, a selective inhibitor of EGFR/HER2 tyrosine kinases, on pancreatic cancer cell lines both alone and in combination with chemotherapy. Two cell lines, BxPc-3 and HPAC, displayed the greatest sensitivity to lapatinib (IC(50)<2 µM). Lapatinib also demonstrated some activity in three K-Ras mutated pancreatic cancer cell lines which displayed resistance to erlotinib. Drug effect/combination index (CI) isobologram analysis was used to study the interactions of lapatinib with gemcitabine, cisplatin and 5'deoxy-5'fluorouridine. Concentration-dependent anti-proliferative effects of lapatinib in combination with chemotherapy were observed. To evaluate the potential effect of lapatinib in pancreatic cancer tumours, and to identify a subset of patient most likely to benefit from lapatinib, expression of EGFR and HER2 were investigated in 72 pancreatic cancer tumour specimens by immunohistochemistry. HER2 membrane expression was observed in only 1 % of cases, whereas 44 % of pancreatic tumours expressed EGFR. Based on our in vitro results, lapatinib may provide clinical benefit in EGFR positive pancreatic ductal adenocarcinoma.

Evaluation of IGF1R and phosphorylated IGF1R as targets in HER2-positive breast cancer cell lines and tumours.

Insulin-like growth factor-1 receptor (IGF1R) signalling is implicated in resistance to trastuzumab. However, the benefit of co-targeting HER2 and IGF1R has not been extensively studied, and the relationship between activated IGF1R and clinical response to trastuzumab has not been reported. This study aimed to evaluate the combination of trastuzumab with IGF1R tyrosine kinase inhibitors (TKIs) in a panel of HER2-positive breast cancer cell lines, and to examine the relationship between IGF1R expression and activation and response to trastuzumab in HER2-positive breast cancer patients. The anti-proliferative effects of trastuzumab combined with IGF1R TKIs BMS-536924 or NVP-AEW541 were measured in nine HER2-positive cell lines. IGF1R and phosphorylated IGF1R/insulin receptor (pIGF1R/IR) were measured by immunohistochemistry in 160 tumour samples from trastuzumab-treated patients (ICORG 06-22). The HER2-positive cell lines displayed varying sensitivity to IGF1R TKIs alone (IC(50)s: 0.7 to >10 µM). However, when combined with trastuzumab, a significantly enhanced effect was observed in five cell lines treated with BMS-536924, and three with NVP-AEW541. While IGF1R levels correlated with reduced response to NVP-AEW541 alone, neither IGF1R nor pIGF1R were predictive of response to BMS-536924 or NVP-AEW541 in combination with trastuzumab. Low HER2 levels correlated with response to BMS-536924 in combination with trastuzumab. Akt levels correlated with improved response to trastuzumab and NVP-AEW541 (P = 0.039). Cytoplasmic IGF1R staining was observed in all tumours, membrane IGF1R was detected in 13.8 %, and pIGF1R/IR was detected in 48.8 %. Although membrane IGF1R staining was associated with larger tumour size (P = 0.041), and lower tumour grade (P = 0.024), no association between IGF1R or pIGF1R/IR and patient survival was observed. In conclusion, while neither IGF1R expression nor activation was predictive of response to trastuzumab, these pre-clinical data provide evidence that co-targeting HER2 and IGF1R may be beneficial in some HER2-amplified breast cancers.

Clinical Impact of Immune Checkpoint Inhibitor (ICI) Response, DNA Damage Repair (DDR) Gene Mutations and Immune-Cell Infiltration in Metastatic Melanoma Subtypes.

Molecular and histopathological analysis of melanoma subtypes has revealed distinct epidemiological, genetic, and clinical features. However, immunotherapy for advanced metastatic melanoma patients does not differ based on subtype. Response to immune checkpoint inhibitors (ICI) has been shown to vary, therefore, predictive biomarkers are needed in the design of precision treatments. Targeted sequencing and histopathological analysis (CD8 and CD20 immunohistochemistry) were performed on subtypes of metastatic melanoma (cutaneous melanoma (CM, n = 10); head and neck melanoma (HNM, n = 7); uveal melanoma (UM, n = 4); acral lentiginous melanoma (AM, n = 1) and mucosal melanoma (MM, n = 1) treated with ICI). Progression-free survival (PFS) was significantly associated with high CD8 expression (p = 0.025) and mutations in DNA damage repair (DDR) pathway genes (p = 0.012) in all subtypes but not with CD20 expression. Our study identified that immune cell infiltration and DDR gene mutations may have an impact in response to ICI treatment in metastatic melanoma but differs among subtypes. Therefore, a comprehensive understanding of the immune infiltration cells' role and DDR gene mutations in metastatic melanoma may identify prognostic biomarkers.

New ligament healing model based on tissue-engineered collagen scaffolds.

The anterior cruciate ligament (ACL) is often the target of knee trauma. This ligament does not heal very well, leading to joint instability. Long-term instability of the knee can lead to early arthritis and loss of function. To develop efficient strategies to stimulate posttraumatic ACL regeneration in vivo, a good healing model is needed in vitro. Such a model must remain as simple as possible, but should include key features to provide relevant answers to precise questions about the clinical problem addressed. Here, we report tissue-engineered type I collagen scaffolds developed to establish an ACL healing model in vitro and a potential ACL substitute in vivo. Such scaffolds were used to evaluate ACL cell growth, migration, and the capacity to synthesize and assemble collagen fibers for up to 40 days in vitro and up to 180 days in vivo. They were anchored with two bone plugs to allow their static stretching in culture and to facilitate their surgical implantation in knee joints. Our results have shown that living ACL fibroblasts can attach, migrate, and colonize this type of scaffold. In vitro, the cells populated the scaffolds and expressed mRNAs coding for the prolyl-4-hydroxylase, involved in collagen fibers' assembly. In vivo, acellular implants were vascularized and populated with caprine cells that migrated from the osseous insertions toward the center of the grafts. This model is a very good tool to study ACL repair and identify the factors that could accelerate its healing postsurgery.

Modelling of pancreatic cancer biology: transcriptomic signature for 3D PDX-derived organoids and primary cell line organoid development.

With a five-year survival rate of 9%, pancreatic ductal adenocarcinoma (PDAC) is the deadliest of all cancers. The rapid mortality makes PDAC difficult to research, and inspires a resolve to create reliable, tractable cellular models for preclinical cancer research. Organoids are increasingly used to model PDAC as they maintain the differentiation status, molecular and genomic signatures of the original tumour. In this paper, we present novel methodologies and experimental approaches to develop PDAC organoids from PDX tumours, and the simultaneous development of matched primary cell lines. Moreover, we also present a method of recapitulating primary cell line cultures to organoids (CLOs). We highlight the usefulness of CLOs as PDAC organoid models, as they maintain similar transcriptomic signatures as their matched patient-derived organoids and patient derived xenografts (PDX)s. These models provide a manageable, expandable in vitro resource for downstream applications such as high throughput screening, functional genomics, and tumour microenvironment studies.

Genomic Profiling and Functional Analysis of let-7c miRNA-mRNA Interactions Identify SOX13 to Be Involved in Invasion and Progression of Pancreatic Cancer.

BACKGROUND: Pancreatic cancer is a devastating disease; its lethality is related to rapid growth and tendency to invade adjacent organs and metastasize at an early stage. OBJECTIVE: The aim of this study was to identify miRNAs and their gene targets involved in the invasive phenotype in pancreatic cancer to better understand the biological behaviour and the rapid progression of this disease. METHODS: miRNA profiling was performed in isogenic matched high invasive and low-invasive subclones derived from the MiaPaCa-2 cell line and validated in a panel of pancreatic cancer cell lines, tumour, and normal pancreas. Online miRNA target prediction algorithms and gene expression arrays were used to predict the target genes of the differentially expressed miRNAs. miRNAs and potential target genes were subjected to overexpression and knockdown approaches and downstream functional assays to determine their pathological role in pancreatic cancer. RESULTS: Differential expression analysis revealed 10 significantly dysregulated miRNAs associated with invasive capacity (Student's t-tests; P value <0.05; fold change = ±2). The expression of top upregulated miR-135b and downregulated let-7c miRNAs correlated with the invasive abilities of eight pancreatic cancer cell lines and displayed differential expression in pancreatic cancer and adjacent normal tissue specimens. Ectopic overexpression of let-7c decreased proliferation, invasion, and colony formation. Integrated analysis of miRNA-mRNA using in silico algorithms and experimental validation databases identified four putative gene targets of let-7c. One of these targets, SOX13, was found to be upregulated in PDAC tumour compared with normal tissue in TCGA and an independent data set by qPCR and immunohistochemistry. RNAi knockdown of SOX13 reduced the invasion and colony formation ability of pancreatic cancer cells. CONCLUSION: The identification of key miRNA-mRNA gene interactions and networks provide potential diagnostic and therapeutic strategies for better treatment options for pancreatic cancer patients.

PRDX3 is associated with metastasis and poor survival in uveal melanoma.

AIMS: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and 40% develop fatal metastatic disease. Overexpression of thioredoxin-dependent peroxidase reductase (PRDX3) has been implicated in several cancers, including prostate, breast, colorectal and lung cancer. The aim of this study was to compare the immunohistochemical expression of PRDX3 in formalin-fixed, paraffin-embedded (FFPE) primary UM tissues of patients who did and did not develop metastatic disease. METHODS: Immunohistochemical staining of PRDX3 was performed on FFPE tissue microarray samples of 92 primary UM tumours from patients who did and did not develop metastatic disease. The immunohistochemical staining was assessed by two observers who were blinded to all clinicopathological and cytogenetic details including metastatic/non-metastatic information. Based on a scoring system, expression of PRDX3 was graded as high or low. RESULTS: There were 55 tumours (59.8%) from patients who developed metastatic disease, while 37 (40.2%) were from patients who did not develop metastasis. A statistically significant difference in PRDX3 expression was observed in patients who did and did not develop metastasis (p=0.001). A significant positive correlation between high PRDX3 expression and metastasis was observed (p=0.001). A significant negative correlation between PRDX3 expression and survival was found (p=0.005). Kaplan-Meier survival analysis showed a statistically significant difference in overall survival between tumours that demonstrated low and high expression of PRDX3 (67.61 vs 130.64 months, respectively, p=0.013). CONCLUSIONS: High immunohistochemical expression of PRDX3 in primary UM tissue is associated with metastasis and poor survival.

Expression of multidrug resistance markers ABCB1 (MDR-1/P-gp) and ABCC1 (MRP-1) in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma patients respond poorly to conventional chemotherapy, this unresponsiveness may be attributable to multidrug resistance (MDR). The mechanisms of MDR in renal cancer are not fully understood and the specific contribution of ABC transporter proteins which have been implicated in the chemoresistance of various cancers has not been fully defined in this disease. METHODS: In this retrospective study the expression of two of these transporter efflux pumps, namely MDR-1 P-gp (ABCB1) and MRP-1 (ABCC1) were studied by immunohistochemistry in archival material from 95 renal cell carcinoma patients. RESULTS: In the first study investigating MDR-1 P-gp and MRP-1 protein expression patterns in renal cell carcinoma patients, high levels of expression of both efflux pumps are observed with 100% of tumours studied showing MDR-1 P-gp and MRP-1 positivity. CONCLUSION: Although these findings do not prove a causal role, the high frequency of tumours expressing these efflux pumps suggests that they may be important contributors to the chemoresistance of this tumour type.

Current and future biomarkers for risk-stratification and treatment personalisation in multiple myeloma.

Multiple myeloma, an incurable malignancy of the plasma cells in the bone marrow, has a complex pathogenesis due to clonal heterogeneity. Over the years, many clinical trials and researches have led to the development of effective myeloma treatments, resulting in survival prolongation. Molecular prognostic markers for risk-stratification to predict survival, and predictive markers for treatment response are being extensively explored. This review discusses the current risk-adaptive strategies based on genetic and molecular risk signatures that are in practice to predict survival and describes the future prognostic and predictive biomarkers across the fields of genomics, proteomics, and glycomics in myeloma. Gene expression profiling and next generation sequencing are coming to the forefront of risk-stratification and therapeutic-response prediction. Similarly, proteomic and glycomic-based platforms are gaining momentum in biomarker discovery to predict drug resistance and disease progression.

An insight into the molecular genetics of a uveal melanoma patient cohort.

BACKGROUND/AIMS: Uveal melanoma (UM) is a highly aggressive malignancy and presents a clinically significant unmet need in cancer therapeutics. The aim of this study was to identify previously unreported mutations in UM among an Irish cohort of patients which may have potential clinical relevance. METHODS: DNA was extracted from 36 intraocular melanoma patient samples and 4 metastatic melanoma samples among the patient cohort by microdissection from formalin-fixed paraffin embedded tissue blocks and underwent genotyping to test for known single nucleotide polymorphisms in 42 cancer associated genes. These mutations were analysed using a custom-designed sequenom panel. RESULTS: Using high-throughput genotyping, mutually exclusive GNAQ and GNA11 mutations were detected in 31 of 34 UM patients together with a number of non-synonymous changes in established cancer driver genes, PHLPP2, MET, PIK3R1 and IDH-1, variants which have not been previously associated with UM. CONCLUSION: Given the lack of knowledge regarding the clinical relevance of the variants identified in this UM cohort and their likely pathogenic nature in other cancers, further studies of the functional impact of these variant mutations are warranted to establish possible previously, undescribed roles in UM pathogenesis, which may provide additional targets for future therapies.

Novel panel of protein biomarkers to predict response to bortezomib-containing induction regimens in multiple myeloma patients.

BACKGROUND: Multiple myeloma (MM) is a complex heterogeneous disease. Various risk stratification models have been recommended including cytogenetic and FISH analysis to identify high-risk patients who may benefit from novel treatments, but such facilities are not widely available. The International Scoring System (ISS) using beta-2-microglobulin and albumin remains a widely used prognostic scoring system in many clinical practices; however it is not useful in predicting response to treatment in MM. The aim of this study is to identify clinically useful biomarkers to predict response to treatment containing bortezomib. METHODS: 17 MM patient serum samples (9 responders/8 non-responders) were used for the discovery phase (label-free mass spectrometry) and an additional 20 MM patient serum samples were used for the ELISA-based validation phase (14 responders/6 non-responders). RESULTS: CLU and ANG mean levels were higher in the responders group, while Complement C1q had lower concentrations. The combination of all standard biomarkers (albumin, beta-2-microglobulin (ß2M), paraprotein and kappa/lambda (K/L) ratio) had an AUC value of 0.71 with 65% correct classification, while an overall combination of new candidate protein biomarkers with standard biomarkers had an AUC value of 0.89 with 85.3% correct classification. CONCLUSIONS: A combination of new and standard biomarkers consisting of CLU, ANG, C1Q, albumin, ß2M, paraprotein and K/L ratio may have potential as a novel panel of biomarkers to predict MM response to treatment containing bortezomib. GENERAL SIGNIFICANCE: Use of this biomarker panel could facilitate a more personalized therapy approach and to minimize unnecessary side effects from ineffective drugs.

MDR-1, but not MDR-3 gene expression, is associated with unmutated IgVH genes and poor prognosis chromosomal aberrations in chronic lymphocytic leukemia.

Two P-glycoprotein (P-gp) genes, MDR-1 (ABCB1) and MDR-3 (ABCB4), have been identified in humans. This study was designed to investigate whether associations exist between expression of MDR-1 and MDR-3 P-gp and other markers of poor prognosis and/or prior exposure to therapeutic agents in chronic lymphocytic leukemia (CLL). IgVH mutational status, gene usage, CD38 positivity, FISH analysis and clinical information were available on all patients. Twenty-one of 101 patients tested showed MDR-3 P-gp positivity. Associations with markers of poor prognosis or prior chemotherapy did not reach statistical significance, but MDR-3 P-gp positive patients had significantly shorter survivals than MDR-3 P-gp negative patients. MDR-1 P-gp expression (18/25) showed a strong association with unmutated IgVH genes and adverse prognosis cytogenetics (p = 0.015, p = 0.014, respectively), but was independent of prior exposure to chemotherapeutic agents. These results suggest a role for MDR-1 and MDR-3 in chemoresistant disease. This study highlights the value of determining MDR phenotype in CLL patients prior to treatment, to allow the design of novel drug regimens containing agents that reverse MDR function.

Quantitative label-free mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue representing the invasive cutaneous malignant melanoma proteome.

Understanding the events at a protein level that govern the progression from melanoma in situ to invasive melanoma are important areas of current research to be developed. Recent advances in the analysis of formalin-fixed, paraffin-embedded tissue by proteomics, particularly using the filter-aided sample preparation protocol, has opened up the possibility of studying vast archives of clinical material and associated medical records. In the present study, quantitative protein profiling was performed using tandem mass spectrometry, and the proteome differences between melanoma in situ and invasive melanoma were compared. Biological pathway analyses revealed several signalling pathways differing between melanoma in situ and invasive melanoma, including metabolic pathways and the phosphoinositide 3-kinase-Akt signalling pathway. Selected proteins of interest (14-3-3ε and fatty acid synthase) were subsequently investigated using immunohistochemical analysis of tissue microarrays. Identifying the key proteins that play significant roles in the establishment of a more invasive phenotype in melanoma may ultimately aid diagnosis and treatment decisions.

Monoclonal antibody 5C3 raised against formalin fixed paraffin-embedded invasive breast tumour tissue: characterisation of its reactive antigen via immunoprecipitation and internal sequencing.

Monoclonal antibodies (MAbs) provide a powerful tool for the identification of novel tumour associated antigens. In an attempt to identify such an antigen, MAbs were generated by immunization with paraffin wax-embedded formalin-fixed invasive ductal breast tumour tissue from a patient who relapsed following an initial response to adjuvant chemotherapy. Extensive immunocytochemical and Western blot analysis of a range of cell lines and tissues including a series of pre- and post-chemotherapy treated invasive ductal breast carcinomas, with one of these MAbs, antibody 5C3, indicated that the 5C3 reactive antigen displayed a wide spectrum of reactivity amongst various human tumours. A reduced level of 5C3 expression was observed in non-cancerous archival breast tissues and breast cell lines and normal murine tissues compared to the expression observed in infiltrating breast tumour cells. Immunoprecipitation studies using the human ductal breast carcinoma cell line, ZR-75-1 resulted in the isolation of a 175 kDa reactive band which was excised from an SDS-PAGE gel and subjected to internal sequencing. Sequencing analysis and database searching revealed that this 175 kDa band represented a cytokeratin heteropolymer, composed of type I cytokeratin 9 and type II cytokeratin 6. Further studies confirmed that antibody 5C3 recognised this heteropolymer of cytokeratin 9 and 6 but not the individual cytokeratins. This novel method of MAb generation may facilitate the isolation of further potentially interesting cellular antigens. Characterisation of these novel antigens may identify specific disease targets with possible prognostic or predictive significance.

Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

INTRODUCTION: Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. AREAS COVERED: ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. EXPERT OPINION: The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.