Analytical comparability to evaluate impact of manufacturing changes of ARX788, an Anti-HER2 ADC in late-stage clinical development.

ARX788 is an anti-HER2 antibody drug conjugate (ADC) developed using Ambrx proprietary Engineered Precision Biologics technology. The manufacturing process of ARX788 has been optimized during the course of early to late-phase clinical development. A comprehensive evaluation of side-by-side comparability between pre- and post-change process for ARX788 drug substance and drug product from a quality perspective was conducted based on ICH Q5E guidelines consisting of batch release assays, physicochemical and biophysical characterization, biological characterization, and forced degradation studies. All results have substantiated a high degree of similarity between the pre- and post-change ARX788 drug substance batches and drug product lots, demonstrating that the process manufacturing changes did not impact product quality.

Health-related quality of life assessment for head-and-neck cancer patients during and at 3 months after radiotherapy - A prospective, analytical questionnaire-based study.

INTRODUCTION: Health-related quality of life (HRQoL) is a more specific area of QoL that deals with the evaluation and assessment of the impact of the disease and its treatment-related morbidities on a patient's physical, psychological, and social aspects. The aim of the present study was to assess the HRQoL of patients with head-and-neck cancer (HNCs) during and at 3 months after completion of radiotherapy (RT) by intensity-modulated RT. MATERIALS AND METHODS: This study was a prospective, longitudinal, observational, and self-completed questionnaire-based study that included 120 patients with HNC who underwent intensity-modulated RT. The questionnaire had adequate internal consistency. The questionnaires were given to each patient at the beginning of treatment (pretreatment), weekly visits during the course of RT (at the end of 1st, 2nd, 3rd, 4th, 5th, and 6th week), on the day of completion of RT, and then finally at 3 months after completion of RT. Thus, a total of successive nine time points were assessed. RESULTS AND CONCLUSIONS: One hundred and eleven patients completed the questionnaires at all nine time points. HRQoL usually decreases during treatment and then increases to pretreatment levels by 3 months after treatment. The Quality of Life Questionnaire, Core Module and Quality of Life Questionnaire, Head and Neck Module were found to be both valid and reliable. There was a significant QoL reduction for the patients throughout treatment in relation to functions and symptoms in the treatment of HNC. However, all the functions and most of the symptoms returned to baseline at the 3-month follow-up.

The use of adaptive intensity-modulated radiotherapy in the treatment of small-cell carcinoma lung refractory to chemotherapy in a patient with preexisting interstitial lung disease.

This is a case report of a 60-year-old diabetic, hypertensive male with a good performance status and a history of bilateral interstitial lung disease with a left upper lobe lung mass diagnosed to be a Stage IIB mixed small-cell/squamous cell carcinoma which was refractory to carboplatin- and etoposide-based chemotherapy. The patient was then taken up for adaptive intensity-modulated radiotherapy with tighter margin under image guidance with a mid-treatment replanning done at 25#. Acute toxicities were assessed weekly and showed no Grade 3 or more reactions. Pulmonary function test showed no detrimental changes during or after radiation. Response assessment at 12 and 20 weeks showed a partial response with decrease in metabolic activity on serial scans.

Dosimetric Comparison of Pencil-Beam Scanning and Photon-Based Radiation Therapy as a Boost in Carcinoma of Cervix.

PURPOSE: Brachytherapy is essential for local treatment in cervical carcinoma, but some patients are not suitable for it. Presently, for these patients, the authors prefer a boost by using intensity-modulated radiation therapy (IMRT). The authors evaluated the dosimetric comparison of proton-modulated radiation therapy versus IMRT and volumetric-modulated arc therapy (VMAT) as a boost to know whether protons can replace photons. PATIENTS AND METHODS: Five patients who received external beam radiation therapy to the pelvis by IMRT were reviewed. Three different plans were made, including pencil beam scanning (PBS), IMRT, and VMAT. The prescribed planning target volume (PTV) was 20 Gy in 4 fractions. The dose to 95% PTV (D95%), the conformity index, and the homogeneity index were evaluated for PTV. The Dmax, D2cc, and Dmean were evaluated for organs at risk along with the integral dose of normal tissue and organs at risk. RESULTS: The PTV coverage was optimal and homogeneous with modulated protons and photons. For PBS, coverage D95% was 20.01 ± 0.02 Gy (IMRT, 20.08 ± 0.06 Gy; VMAT, 20.1 ± 0.04 Gy). For the organs at risk, Dmax of the bladder for PBS was 21.05 ± 0.05 Gy (IMRT, 20.8 ± 0.21 Gy; VMAT, 21.65 ± 0.41 Gy) while the Dmax for the rectum for PBS was 21.04 ± 0.03 Gy (IMRT, 20.81 ± 0.12 Gy; VMAT, 21.66 ± 0.38 Gy). Integral dose to normal tissues in PBS was 14.17 ± 2.65 Gy (IMRT, 25.29 ± 6.35 Gy; VMAT, 25.24 ± 6.24 Gy). CONCLUSIONS: Compared with photons, modulated protons provide comparable conformal plans. However, PBS reduces the integral dose to critical structures significantly compared with IMRT and VMAT. Although PBS may be a better alternative for such cases, further research is required to substantiate such findings.

Dosimetric influence of filtered and flattening filter free photon beam on rapid arc (RA) radiotherapy planning in case of cervix carcinoma.

AIM: To investigate the dosimetric influence of filtered and flattening filter free (FFF) photon beam of 6 and 10 MV energies on cervix RA radiotherapy planning and to find possibilities to develop the clinically acceptable RA plans with FFFB photon beam and explore their potential benefits to cervix cancer patients. BACKGROUND: FFF photon beams enhances the treatment delivery by increased dose rate which results in shorter treatment time, this shorter treatment time reduces intrafraction motion and enhance comfort to the patients. MATERIALS AND METHODS: RA plans were generated for filtered and flattening filter free photon beams of 6 and 10 MV energies using same dose-volumes constraints. RA plans were generated to deliver a dose of 50.4 Gy in 28 fractions, for a cohort of eleven patients reported with cervix carcinoma. RA plans were evaluated in terms of PTV coverage, dose to OAR's, CI, HI, total no. of monitor units (MUs) and NTID and low dose volume of normal tissues. RESULTS: Clinically acceptable and similar plans were generated for filtered and flattening filter free photon beams. FFFB delivered slightly higher mean target dose (52.28 Gy vs. 52.0 Gy, p = 0.000 for 6 MV and 52.42 Gy vs. 52.0 Gy, p = 0.000 for 10 MV) less homogeneous (1.062 vs. 1.052, p = 0.000 for 6 MV and 1.066 vs. 1.051, p = 0.000 for 10 MV) and less conformal (1.007 vs. 1.004, p = 0.104 for 6 MV and 1.012 vs. 1.003, p = 0.010 for 10 MV) RA plans compared to FB. FFFB delivered more doses to the bladder and rectum, also required more numbers of MUs in comparison to FB. CONCLUSIONS: This study concludes that FB is more beneficial for cervix RA planning in comparison to FFFB, as FB generates more conformal and homogenous rapid arc plans and offers better OAR's sparing.

Comparison between weekly cisplatin-enhanced radiotherapy and cetuximab-enhanced radiotherapy in locally advanced head and neck cancer: first retrospective study in Asian population.

AIM: To present a direct comparison between chemotherapy-enhanced radiotherapy (CERT) and biotherapy-enhanced radiotherapy (BERT) in locally advanced head and neck cancer. METHODS: It is a retrospective analysis of 53 patients with locally advanced head and neck cancer treated from August 2006 to December 2008. For CERT, patients received weekly cisplatin (40 mg/m2 ) and for BERT, a loading dose of 400 mg/m2 of cetuximab given one week prior to radiotherapy followed by 250 mg/m2 given weekly along with radiotherapy. Disease-free survival (DFS) and overall survival (OS) were computed with Kaplan-Meier curve with log-rank test for comparison between the two groups. Multivariate Cox proportional hazards regression analysis was performed to estimate the impact of known relevant prognostic factors on DFS and OS. RESULTS: The median DFS was significantly better with CERT than BERT group (50.82 vs 11.66 months; P = 0.031). The 3 years DFS was significantly higher in CERT group than in BERT group (60.0% vs 14.3%; P = 0.022). The median OS was significantly better with CERT than BERT group (53.61 vs 32.55 months; P = 0.044). The 3 years OS was also significantly higher in CERT group than in BERT group (74.0% vs 42.1%; P = 0.032). There were no significant differences in acute toxicities of all grade and grade ≥3 between the two groups. The compliance to treatment and assisted feeding dependency for more than 6 months duration were also not significantly different. CONCLUSION: CERT is associated with better outcome with no significantly increased acute toxicities compared to BERT.

Tumour volumes: Predictors of early treatment response in locally advanced head and neck cancers treated with definitive chemoradiation.

AIM: To analyse and predict early response 3 months post definitive chemoradiation (CCRT) utilising tumour volume (TV) measurement in locally advanced head and neck cancers (LAHNC). BACKGROUND: LAHNC are 3-dimentional lesions. The largest diameter of these tumours measured for T-classification may not necessarily reflect the true tumour dimensions. TV accurately reflects the tumour burden because it is a measurement of tumour burden in all three dimensions. MATERIALS AND METHODS: It is a single institutional prospective study including 101 patients with LAHNC treated with definitive CCRT. TV data noted were primary tumour volume (PTV), total nodal volume (TNV) and total tumour volume (TTV). Response evaluation was done at 3 months after the completion of definitive CCRT and patients were categorised either having achieved complete response (CR) or residual disease. RESULTS: Patients who had not achieved CR were found to have larger TV compared with those who had achieved CR. There were significant inverse correlations between PTV and response (median 16.37 cm(3) vs. 45.2 cm(3); p = 0.001), and between TTV and response (median 36.14 cm(3) vs. 66.06 cm(3); p < 0.001). Receiver operating characteristic (ROC) analysis identified an "optimal cut-off" value of 41 cm(3) for PTV and 42 cm(3) for TTV above and below which the magnitude of difference in response was the greatest. CONCLUSIONS: If response evaluation 3 months post CCRT is to be predicted it is simply not enough to measure the largest single dimension of the tumour. TV seems to be a better and more accurate reflection of the true total tumour burden or extent of the disease.

Multiplexed magnetic nanoparticle-antibody conjugates (MNPs-ABS) based prognostic detection of ovarian cancer biomarkers, CA-125, ß-2M and ApoA1 using fluorescence spectroscopy with comparison of surface plasmon resonance (SPR) analysis.

A multiplexed MNPs-Abs based fluorescence spectroscopic system in analysis of serum biomarkers; CA-125, ß2-M and ApoA1 for the early detection of ovarian cancer was first time proposed. The lowest detection limits measured in multiplexed setup were 0.26 U/mL, 0.55 ng/mL and 7.7 ng/mL respectively for CA-125, ß2-M and ApoA1. A comparative real sample analysis of healthy normal (Control), benign and ovarian cancer patients with SPR has also been done to validate the process. Moreover CA-125 detection only confirms 50-60% of early stage disease. This multiplexed system achieved sensitivity and specificity up to 94% and 98% respectively to distinguish early stage ovarian cancer patients from healthy individuals.

Exploring signet-ring cells in pregnant female.

INTRODUCTION: Primary signet-ring cell carcinoma (SRCC) of the colon and rectum are rare form, which present at an advanced stage and have poor prognosis. Different treatment policies of SRCC during different gestational age of pregnancy are explored from the literature. CASE STUDY: A 26-year-old young pregnant female with 10-week gestation presented with constipation and blood in stools and on per rectal examination a tender circumferential stricture was present 2 cm above the anal verge. Magnetic resonant imaging (MRI) pelvis of the patient revealed rectal thickening, the biopsy of which revealed characteristic appearance of "linitis plastica" and diagnosed as poorly differentiated adenocarcinoma with signet ring morphology with wide spread positivity for cytokeratin & p53. With this diagnosis, patient underwent medical termination of pregnancy (MTP). DISCUSSION: SRCC of the colon comprises about only 1% of all cases of colon cancer. When compared with other types of adenocarcinoma, patients with SRCC in the colon are younger and more likely to experience lymph node metastasis. Its incidence in pregnancy is estimated to be less than 0.1%. Certainly, any pregnant patient who reports rectal bleeding or has hemoccult positive stool on examination deserves careful evaluation to rule out cancer. The complex treatment of colorectal cancer in pregnancy is based on the gestational age of the fetus, tumor stage and need for emergent vs. elective management.

Radiation-induced dental caries, prevention and treatment - A systematic review.

Treatment of head and neck cancers (HNCs) involves radiotherapy. Patients undergoing radiotherapy for HNCs are prone to dental complications. Radiotherapy to the head and neck region causes xerostomia and salivary gland dysfunction which dramatically increases the risk of dental caries and its sequelae. Radiation therapy (RT) also affects the dental hard tissues increasing their susceptibility to demineralization following RT. Postradiation caries is a rapidly progressing and highly destructive type of dental caries. Radiation-related caries and other dental hard tissue changes can appear within the first 3 months following RT. Hence, every effort should be focused on prevention to manage patients with severe caries. This can be accomplished through good preoperative dental treatment, frequent dental evaluation and treatment after RT (with the exception of extractions), and consistent home care that includes self-applied fluoride. Restorative management of radiation caries can be challenging. The restorative dentist must consider the altered dental substrate and a hostile oral environment when selecting restorative materials. Radiation-induced changes in enamel and dentine may compromise bonding of adhesive materials. Consequently, glass ionomer cements have proved to be a better alternative to composite resins in irradiated patients. Counseling of patients before and after radiotherapy can be done to make them aware of the complications of radiotherapy and thus can help in preventing them.

The dosimetric impact of different photon beam energy on RapidArc radiotherapy planning for cervix carcinoma.

The main purpose of this study is to know the effect of three different photon energies viz., 6, 10, and 15 mega voltage (MV) on RapidArc (RA) planning for deep-seated cervix tumor and to develop clinically acceptable RA plans with suitable photon energy. RA plans were generated for 6, 10, and 15 MV photon energies for twenty patients reported with cervix carcinoma. RA plans were evaluated in terms of planning target volume (PTV) coverage, dose to organs at risk (OARs), conformity index (CI), homogeneity index (HI), gradient measure, external volume index of dose distribution produced, total number of monitor units (MUs), nontumor integral dose (ID), and low dose volume of normal tissue. A two-sample paired t-test was performed to compare the dosimetric parameters of RA plans. Irrespective of photon energy used for RA planning, plans were dosimetrically similar in terms of PTV coverage, OARs sparing, CI and HI. The numbers of MUs were 13.4 ± 1.4% and 18.2 ± 1.5% higher and IDs were 2.7 ± 0.8% and 3.7 ± 0.9% higher in 6 MV plans in comparison to that in the 10 and 15 MV plans, respectively. V1Gy, V2Gy, V3Gy, and V4Gy were higher in 6 MV plans in comparison to that in 10 and 15 MV plans. Based on this study, 6 MV photon beam is a good choice for RA planning in case of cervix carcinoma, as it does not deliver additional exposure to patients caused by photoneutrons produced in high energy beams.

Improving the accuracy of target volume delineation by combined use of computed tomography, magnetic resonance imaging and positron emission tomography in head and neck carcinomas.

OBJECTIVE: Conformal radiation therapy mandates accurate delineation of target volumes, which requires incorporation of modern imaging modalities like magnetic resonance imaging (MRI) and positron emission tomography (PET) in addition to conventionally used computed tomography (CT). This can resolve discrepancies in target delineation in head and neck carcinomas resulting in better local control. We hereby report the comparison of Gross Tumor Volumes (GTVs) (primary) drawn using PET, CT and MRI and their concordance indices. METHODS AND MATERIAL: Twenty five patients with head and neck cancer were taken into this study. MRI, PET and CT planning scans were done as per standard guidelines. Three sets of primary GTVs namely GTV- PET, GTV-CT and GTV-MRI were contoured on fused images. All the three volumes and concordances among the volumes were analyzed. RESULT: The mean GTV-CT, GTV-PET and GTV-MRI volumes were 29.65 cc ± 31.27, 32.05 cc ± 33.75 and 24.85 cc ± 25.28 respectively. There was a significant difference in the GTV-MRI & GTV-CT volumes (P = 0.023) and GTV-PET & GTV-MRI volumes (P = 0.049). However, there was no significant difference in the GTV-PET & GTV-CT volume (P = 0.468). The mean CI (PET-MRI), CI (CT-MRI) and CI (PET-CT) was 0.42, 0.46 and 0.47 respectively, which depicts a moderate concordance. CONCLUSION: PET and MRI are useful imaging tools in head and neck malignancies and should be used in conjunction with CT scan for improved target volume delineation.

A journey into insidious world of MALT lymphoma of the ileum: from the beginning to the end.

INTRODUCTION: Lymphoma of the mucosa-associated lymphoid tissue (MALT) has been used to describe a marginal zone B-cell lymphoma derived from gastrointestinal lymphoid tissue. mucosa-associated lymphoid tissue lymphoma (MALToma) of the ileum are extremely rare and only few reports with endoscopic features have been reported. CASE STUDY: We present a 55-year-old male patient with history of abdominal pain and loss of appetite since 2½ years. Abdomino-pelvic ultrasonography was normal, but computed tomography (CT) scan of the abdomen showed, dilated segment of ileum containing both contrast and debris. He underwent segmental resection of ileum associated with stricture site, histopathology of which revealed MALToma of ileum. Patient was subsequently treated with low dose chemotherapy and strictly followed up. DISCUSSION: Primary treatment possibility should be considered as the treatment of H. pylori infection while surgical resection for superficial lesions followed by low dose chemotherapy is recommended. The present case report explore MALToma of the GI tract, its diagnostic criterions, role of radiological and pathological tools, various investigative techniques and role of surgery and chemotherapy in such cases.

The identification of auto-antibodies in pancreatic cancer patient sera using a naturally fractionated Panc-1 cell line.

The immunogenic nature of cancer can be explored to distinguish pancreatic cancer from related non-cancer conditions. We describe a liquid-based microarray approach followed by statistical analysis and confirmation for discovery of auto-immune biomarkers for pancreatic cancer. Proteins from the Panc-1 pancreatic cancer cell line were fractionated using a 2-D liquid separation method into over 1052 fractions and spotted onto nitrocellulose coated glass slides. The slides were hybridized with 37 pancreatic cancer sera, 24 chronic pancreatitis sera and 23 normal sera to detect elevated levels of reactivity against the proteins in spotted fractions. The response data obtained from protein microarrays was first analyzed by Wilcoxon Rank-Sum Tests to generate two lists of fractions that positively responded to the cancer sera and showed p-values less than 0.02 in the pairwise comparison between cancer specimens and normal and chronic pancreatitis specimens. The top 3 fractions with the lowest correlations were combined in receiver operating characteristic analyses. The area-under-the-curve (AUC) values are 0.813 and 0.792 for cancer vs. normal and cancer vs. pancreatitis respectively. Outlier-Sum statistics were then applied to the microarray data to determine the existence of outliers exclusive in cancer sera. The selected fractions were identified by LC-MS/MS. We further confirmed the occurrence of outliers with three proteins among cancer samples in a confirmation experiment using a separate dataset of 165 serum samples containing 48 cancer sera and 117 non-cancer controls. Phosphoglycerate kinase 1 (PGK1) elicited greater reactivity in 20.9% (10 in 48) of the samples in the cancer group, while no outlier was present in the non-cancer groups.

The identification of phosphoglycerate kinase-1 and histone H4 autoantibodies in pancreatic cancer patient serum using a natural protein microarray.

Protein microarrays have been used to explore whether a humoral response to pancreatic cancer-specific tumor antigens has utility as a biomarker of pancreatic cancer. To determine if such arrays can be used to identify novel autoantibodies in the sera from pancreatic cancer patients, proteins from a pancreatic adenocarcinoma cell line (MIAPACA) were resolved by 2-D liquid-based separations, and then arrayed on nitrocellulose slides. The slides were probed with serum from a set of patients diagnosed with pancreatic cancer and compared with age- and sex-matched normal subjects. To account for patient-to-patient variability, we used a rank-based non-parametric statistical testing approach in which proteins eliciting significant differences in the humoral response in cancer compared with control samples were identified. The prediction analysis for microarrays classification algorithm was used to explore the classification power of the proteins found to be differentially expressed in cancer and control sera. The generalization error of the classification analysis was estimated using leave-one-out cross-validation. A serum diagnosis of pancreatic cancer in this set was predicted with 86.7% accuracy, with a sensitivity and specificity of 93.3 and 80%, respectively. Candidate autoantibody biomarkers identified using this approach were studied for their classification power by performing a humoral response experiment on recombinant proteins using an independent sample set of 238 serum samples. Phosphoglycerate kinase-1 and histone H4 were noted to elicit a significant differential humoral response in cancer sera compared with age- and sex-matched sera from normal patients and patients with chronic pancreatitis and diabetes. This work demonstrates the use of natural protein arrays to study the humoral response as a means to search for the potential markers of cancer in serum.

Analysis of protein glycosylation and phosphorylation using liquid phase separation, protein microarray technology, and mass spectrometry.

Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis.

Nano level detection of Cd(II) using poly(vinyl chloride) based membranes of Schiff bases.

The construction and performance characteristics of polymeric membrane electrodes based on two neutral ionophores, 2,2'-(1Z,1'Z)-(1E,1'E)-(1,2-phenylenebis(methan-1-yl-1-ylidene))bis(azaan-1-yl-1-ylidene)bis(methylene)bis(azan-1-yl-1-ylidene)bis(methan-1-yl-ylidene)diphenol (L(1)) and 4,4'-(1E,1'E)-(butane-1,4-diylbis(azan-1-yl-1-ylidene))bis(methan-1-yl-1-ylidene)dinaphthalen-1-ol (L(2)) for quantification of cadmium ions, are described. The influences of membrane compositions on the potentiometric response of the electrodes have been found to substantially improve the performance characteristics. The best performance was obtained with the electrode having a membrane composition (w/w) of (L(1)) (2.6%):PVC (31.6%):DOP (63.2%):NaTPB (2.6%). The proposed electrode exhibits Nernstian response in the concentration range 5.0 x 10(-9) to 1.0 x 10(-1)M Cd(2+) with limit of detection 3.1 x 10(-9), performs satisfactorily over wide pH range (2.0-8.5) with a fast response time (11s). The electrode has been found to work satisfactorily in partially non-aqueous media up to 40% (v/v) content of methanol, ethanol and acetonitrile and could be used for a period of 2.5 months. The analytical usefulness of the proposed electrode has been evaluated by its application in the determination of cadmium in cigarette samples. The practical utility of the membrane electrode has also been observed in the presence of surfactants.

Humoral response profiling reveals pathways to prostate cancer progression.

There is considerable evidence for an association between prostate cancer development and inflammation, which results in autoantibody generation against tumor proteins. This immune system-driven amplification of the autoantibody response to intracellular antigens can serve as a sensitive tool to detect low abundance serum proteomic tumor markers for prostate cancer as well as provide insight into biological processes perturbed during cancer development. Here we examine serum humoral responses in a cohort of 34 patients with either benign prostatic hyperplasia or clinically localized prostate cancer (PCa). The experimental strategy couples multidimensional liquid-phase protein fractionation of localized and metastatic prostate cancer tissue lysates to protein microarrays and subsequent mass spectrometry. A supervised learning analysis of the humoral response arrays generated a parsimonious predictor having 78% sensitivity and 75% specificity in distinguishing PCa from benign prostatic hyperplasia in a cohort of American males with elevated prostate-specific antigen. Enrichment analysis of the PCa-specific humoral signature revealed large scale immune reprogramming mediated by STAT transcription factors and the generation of autoantibodies to enzymes involved in nitrogen metabolism. Meta-analysis of independent prostate cancer gene expression data validated the presence of STAT-induced immunomodulation. Concomitant validation of elevated levels of the nitrogen metabolism pathway was obtained by direct measurement of metabolic levels of glutamate and aspartate in prostate cancer tissues. Thus, in addition to functioning as markers in prostate cancer detection, humoral response profiles can serve as powerful tools revealing pathway dysregulation that might otherwise be suppressed by the complexity of the cancer proteome.

Automated integration of monolith-based protein separation with on-plate digestion for mass spectrometric analysis of esophageal adenocarcinoma human epithelial samples.

A unique approach of automating the integration of monolithic capillary HPLC-based protein separation and on-plate digestion for subsequent MALDI-MS analysis has been developed. All liquid-handling procedures were performed using a robotic module. This automated high-throughput method minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and analysis. Also, precise positioning of the droplet from the capillary HPLC separation onto the MALDI plate allows for preconcentration effects of analytes for improved sensitivity. Proteins from primary esophageal Barrett's adenocarcinoma tissue were prefractionated by chromatofocusing and analyzed successfully by this automated configuration, obtaining rapid protein identifications through PMF and sequencing analyses with high sequence coverage. Additionally, intact protein molecular weight values were obtained as a means to further confirm protein identification and also to identify potential sequence modifications of proteins. This simple and rapid method is a highly versatile and robust approach for the analysis of complex proteomes.

Toward high sequence coverage of proteins in human breast cancer cells using on-line monolith-based HPLC-ESI-TOF MS compared to CE MS.

A method is developed toward high sequence coverage of proteins isolated from human breast cancer MCF10 cell lines using a 2-D liquid separations. Monolithic-capillary columns prepared by copolymerizing styrene with divinylbenzene are used to achieve high-resolution separation of peptides from protein digests. This separation is performed with minimal sample preparation directly from the 2-D liquid fractionation of the cell lysate. The monolithic column separation is directly interfaced to ESI-TOF MS to obtain a peptide map. The protein digests were also analyzed by MALDI-TOF MS and an accurate M(r) of the intact protein was obtained using an HPLC-ESI-TOF MS. The result is that these techniques provide complementary information where nearly complete sequence coverage of the protein is obtained and can be compared to the experimental M(r) value. The high sequence coverage provides information on isoforms and other post-translational modifications that would not be available from methods that result in low sequence coverage. The results from the use of monolithic columns are compared to that obtained by CE-MS. The monolithic column separations provide a rugged and highly reproducible method for separating protein digests prior to MS analysis and is suited to confidently identify biomarkers associated with cancer progression.