Publications by authors named "Robert Kubiak"

44 Publications

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation ( - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

Bioanalysis 2021 Mar 3;13(6):415-463. Epub 2021 Feb 3.

Intellia Therapeutics, Cambridge, MA, USA.

The 14 edition of the Workshop on Recent Issues in Bioanalysis (14 WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.
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http://dx.doi.org/10.4155/bio-2021-0007DOI Listing
March 2021

A new method for identification of outliers in immunogenicity assay cut point data.

J Immunol Methods 2020 Sep - Oct;484-485:112817. Epub 2020 Jun 29.

AstraZeneca PLC.

The cut point is an important parameter for immunogenicity assay validation and critical to immunogenicity assessment in clinical trials. FDA (2019) recommends using a statistical approach to derive cut point, with an appropriate outlier removal procedure. In general, the industry follows the methods described in Shankar et al. (2008) and Zhang et al. (2013) among others to determine cut point. Outlier removal is a necessary step during the cut point determination exercise to reduce potential false negative classifications. However, the widely used statistical outlier removal method, namely, Tukey's box-plot method (1.5 times inter-quartile range, IQR), is often found to be overly conservative in the sense that it removes too many "outliers". Tukey's box-plot method can be used to flag potential outliers for further investigation, however, it is not a hypothesis testing based statistical method. Removing these suspected "outliers" will lead to lower cut point which might confound immunogenicity assessment due to the presence of many low false positives. Besides, the very nature of assay analytical variability has a non-negligible adverse impact on the reliability of ADA classification in terms of false positive and false negative, demanding as large as possible contribution from biological variability relative to analytical variability. A new outlier removal procedure, which takes into account the relative magnitude between biological variability and analytical variability within the sample population, is proposed and statistically justified. After sequential removal of analytical and biological outliers, a 5% false positive rate and 1% false positive rate in screening and confirmatory assays, respectively, are still targeted without increasing potential false negatives. Internal data shows that this practice has minimal impact on assay sensitivity and has the advantage of selecting true positive samples. It is shown that the new procedure is more appropriate for cut point determination.
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http://dx.doi.org/10.1016/j.jim.2020.112817DOI Listing
March 2021

Confirmatory cut point has limited ability to make accurate classifications in immunogenicity assays.

Bioanalysis 2020 Feb 24;12(4):245-256. Epub 2020 Feb 24.

Clinical Pharmacology and Safety Sciences, AstraZeneca PLC 1, One MedImmune Way, Gaithersburg, MD 20878, USA.

Competitive inhibition with excess unlabeled drug is used to confirm the presence of antidrug antibodies (ADA) in study samples. We evaluated specific and nonspecific responses from both drug-naive and drug-treated subjects to identify conditions required by the confirmatory assay to make accurate ADA classifications. Nonspecific signal measured in drug-naive samples used to determine assay cut points was uniformly low and close to the screening cut point. Confirmatory assays performed on incurred study samples with nonspecific responses significantly above the level observed during cut point determination resulted in incorrect ADA classifications. Intensity of confirmatory response should be proportional to the screening response and therefore, to ensure accurate ADA classifications, the confirmatory responses cannot be considered as independent but need to be evaluated in relation to the screening responses.
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http://dx.doi.org/10.4155/bio-2019-0283DOI Listing
February 2020

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

Bioanalysis 2019 Dec 10;11(24):2207-2244. Epub 2019 Dec 10.

Janssen R&D, Spring House, PA, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of , issues 22 and 23 (2019), respectively.
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http://dx.doi.org/10.4155/bio-2019-0271DOI Listing
December 2019

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

Bioanalysis 2018 Dec 29;10(24):1973-2001. Epub 2018 Nov 29.

Amador Bioscience, Pleasanton, CA, USA (formerly of OncoMed, Redwood City, CA, USA).

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of , issues 22 and 23 (2018), respectively.
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http://dx.doi.org/10.4155/bio-2018-0287DOI Listing
December 2018

Excessive outlier removal may result in cut points that are not suitable for immunogenicity assessments.

J Immunol Methods 2018 12 9;463:105-111. Epub 2018 Oct 9.

MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, United States.

Cut point determination is an important aspect of immunogenicity assay development. The cut point can be influenced by a myriad of factors. Key among those is the analytical variability of the assay itself and biological variation due to test samples. Since a smaller cut point value may result in improved sensitivity, the existing procedures often employ statistical techniques such as outlier removal to produce a conservative cut point. Although such practices are intended to yield acceptable assay sensitivity, they may fail to fully account for biological variability in the data, thus generating higher than expected number of false positive results. In this paper, we introduce the concept of minimum cut point. It is defined as the cut point that is determined in the absence of biological variability. Under the log-normal assumption of the data used for cut point analysis, closed-form formulas are derived for the minimum cut point. This minimum cut point can be used to benchmark whether a cut point derived from a procedure can compromise assay specificity by being too low.
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http://dx.doi.org/10.1016/j.jim.2018.10.001DOI Listing
December 2018

Rhodium-Catalyzed Intermolecular C-H Functionalization as a Key Step in the Synthesis of Complex Stereodefined β-Arylpyrrolidines.

Org Lett 2018 07 21;20(13):3771-3775. Epub 2018 Jun 21.

Department of Chemistry , Emory University , 1515 Dickey Drive , Atlanta , Georgia 30322 , United States.

The synthesis of β-arylpyrrolidines via a catalytic enantioselective intermolecular allylic C(sp)-H functionalization of trans-alkenes followed by immediate reduction, ozonolysis, and then in situ diversification of the resulting cyclic hemiaminal to furnish highly substituted, stereoenriched β-arylpyrrolidines is reported. This methodology utilizes 4-aryl-1-sulfonyl-1,2,3-triazoles as carbene precursors and the dirhodium tetracarboxylate catalyst Rh( S-NTTL). A variety of β-arylpyrrolidines were prepared in good yields with high levels of diastereo- and enantioselectivity over four linear steps, requiring only a single purification procedure.
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http://dx.doi.org/10.1021/acs.orglett.8b01362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232104PMC
July 2018

Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays.

J Immunol Res 2016 10;2016:1485615. Epub 2016 Jul 10.

Drug Metabolism, Pharmacokinetics and Biological Safety Assessment, MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, USA.

Consistent performance of anti-drug antibody (ADA) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB) had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC) serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.
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http://dx.doi.org/10.1155/2016/1485615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958433PMC
March 2017

Enantioselective Intermolecular C-H Functionalization of Allylic and Benzylic sp(3) C-H Bonds Using N-Sulfonyl-1,2,3-triazoles.

Org Lett 2016 07 22;18(13):3118-21. Epub 2016 Jun 22.

Department of Chemistry, Emory University , 1515 Dickey Drive, Atlanta, Georgia 30322, United States.

The enantioselective intermolecular sp(3) C-H functionalization at the allylic and benzylic positions was achieved using rhodium-catalyzed reactions with 4-phenyl-N-(methanesulfonyl)-1,2,3-triazole. The optimum dirhodium tetracarboxylate catalyst for these reactions was Rh2(S-NTTL)4. The rhodium-bound α-imino carbene intermediates preferentially reacted with tertiary over primary C-H bonds in good yields and moderate levels of enantioselectivity (66-82% ee). This work demonstrates that N-sulfonyltriazoles can be applied to the effective C-H functionalization at sp(3) C-H bonds of substrates containing additional functionality.
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http://dx.doi.org/10.1021/acs.orglett.6b01298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5521008PMC
July 2016

Ectopic expression of follicle-stimulating hormone receptors in thyroid tumors.

Arch Med Sci 2015 Dec 11;11(6):1314-7. Epub 2015 Dec 11.

Department of Neuroendocrinology, Chair of Laboratory Medicine, Medical University of Lodz, Lodz, Poland.

Introduction: In normal conditions follicle-stimulating hormone receptors (FSHR) are expressed in the ovary and the testis. They can also be expressed in gonadal tumors. However, recently we have found FSHR immunostaining in pituitary adenomas, adrenal tumors and neuroendocrine tumors (carcinoids). The aim of this study was to determine whether the same occurs in thyroid tumors.

Material And Methods: Thirty-six samples of surgically excised thyroids were examined. Follicle-stimulating hormone receptors immunostaining was performed on paraffin sections using the rabbit anti-human FSHR polyclonal antibody raised against a 1-190 amino acid sequence from the human FSHR (sc-13935, Santa Cruz).

Results: Normal thyroid follicles do not show immunopositivity for FSHR. The same concerns the majority of benign lesions, diagnosed as hyperplasia nodularis or thyroid adenomas. However, positive FSHR immunostaining in some follicles was observed. In all but one thyroid cancer (15 papillary, 10 follicular cancers and one case of anaplastic thyroid cancer) 10-100% of tumor cells exhibit positive FSHR immunostaining. In about 40% of samples FSHR immunoreactivity can be observed also in the endothelia of intrathyroidal blood vessels. This immunopositivity was more frequent in the samples of thyroid cancers (13/27) than in benign lesions (2/9).

Conclusions: Ectopic positive FSHR immunostaining is also present in thyroid cancers, and, to a lesser degree, in benign lesions but not in the normal thyroid epithelium.
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http://dx.doi.org/10.5114/aoms.2015.56357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697062PMC
December 2015

Lack of CD151/integrin α3β1 complex is predictive of poor outcome in node-negative lobular breast carcinoma: opposing roles of CD151 in invasive lobular and ductal breast cancers.

Br J Cancer 2015 Nov 29;113(9):1350-7. Epub 2015 Sep 29.

School of Cancer Sciences of the University of Birmingham, Birmingham B15 2TT, UK.

Background: The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3β1 in patients with ILC in context of the data of our recent IDC study.

Methods: Expression of CD151 and/or integrin α3β1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3β1 complex in patients with HER2-negative tumours.

Results: Unlike in the IDCs, neither CD151 nor CD151/α3β1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3β1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(-) cases. The CD151(-)/α3β1(-) patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(-) patients.

Conclusions: Biological role of CD151/α3β1 varies between ILC and IDC. Assessment of CD151/α3β1 might help to identify ILC N(-) patients with increased risk of distant metastases.
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http://dx.doi.org/10.1038/bjc.2015.344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815791PMC
November 2015

Expression of follicle stimulating hormone receptors (FSHR) in thyroid tumours - a marker of malignancy?

Thyroid Res 2015 5;8(1). Epub 2015 Feb 5.

Department of Neuroendocrinology, Chair of Laboratory Medicine, Medical University of Lodz, Lodz, Poland.

Background: In normal conditions FSHR are expressed in granulosa cells of the ovary and Sertoli cells of the testis. They can be expressed also in gonadal tumours. However, recently the expression of FSHR was found in tumoral cells and intra-tumoral blood vessels of many other tumours, including thyroid tumours. Aim of this study was to see whether the expression of FSHR can be useful in the differentiation of benign and malignant thyroid lesions.

Methods: 44 samples of surgically excised thyroids were immunostained with anti- FSHR antibody raised against 1-190 amino acid sequence from the human FSHR.

Results: Non-neoplastic thyroid follicles (i.e. the follicles situated outside the tumour) do not show the immunostaining for FSHR. The same concerns the majority of follicular adenomas. In contrast, 87.5% of follicular cancers, the same percentage of papillary cancers and all the examined undifferentiated cancers showed the FSHR immunopositivity of tumoral cells. A tendency towards the higher frequency of FSHR - positive blood vessels also concerns malignant thyroid tumours.

Conclusions: The ectopic FSHR immunostaining seems to be useful to differentiate malignant from benign lesions, especially follicular cancers from follicular adenomas. However, the further studies on larger material are needed.
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http://dx.doi.org/10.1186/s13044-015-0014-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4328168PMC
February 2015

Association of microRNA-93, 190, 200b and receptor status in core biopsies from stage III breast cancer patients.

DNA Cell Biol 2014 Sep 27;33(9):624-9. Epub 2014 May 27.

1 Department of Surgical Oncology, Cancer Center , Lodz, Poland .

Oncologists now favor more personalized treatment strategies in breast cancer patients. Gene expression analysis has been widely used, but less is known about epigenetic factors, for example, microRNAs (miRNAs). The aim of this study was to determine the relationship between selected miRNAs and receptor status in core biopsies sampled before preoperative chemotherapy in stage III locally advanced breast cancer (LABC) patients. In 37 LABC core biopsies, three miRNAs per sample were analyzed: hsa-miR-93-5p, hsa-miR-190a, and hsa-miR-200b-3p, and hsa-miR-103a-3p as an endogenous control (TaqMan(®) RT-PCR; Applied Biosystems). Receptor status was determined by a dedicated pathologist. The Mann-Whitney U, Shapiro-Wilk, and Levene's tests were used to compare related samples. Levels of miRNA-93 differed significantly in core biopsies of LABC patients with different expressions of ER (estrogen receptor) and PR (progesterone receptor). Higher levels of miRNA-93 were found in ER-negative (p=0.0027) and PR-negative patients (p=0.0185). Levels of miRNA-190 and 200b did not differ significantly in core biopsies of LABC patients who expressed ER and PR differently (p=0.7727, p=0.9434, p=0.6213, and p=0.1717). Levels of miRNA-93, 190, and 200b were not significantly different in core biopsies of LABC patients with different HER2 (human epidermal growth factor 2) expressions (p=0.8013, p=0.2609, and p=0.3222). The assessment of core biopsy miRNA profiles and receptor-based subtypes may identify new signaling pathways for improved breast cancer classification.
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http://dx.doi.org/10.1089/dna.2014.2419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144366PMC
September 2014

Association of microRNAs and pathologic response to preoperative chemotherapy in triple negative breast cancer: preliminary report.

Mol Biol Rep 2014 May 29;41(5):2851-7. Epub 2014 Jan 29.

Department of Surgical Oncology, Copernicus Memorial Hospital, Cancer Center, Paderewskiego 4, 93-509, Lodz, Poland,

Triple negative breast cancer (TNBC) has caught the attention of oncologists worldwide because of poor prognosis and paucity of targeted therapies. Gene pathways have been widely studied, but less is known about epigenetic factors such as microRNAs (miRNAs) and their role in tailoring an individual systemic and surgical approach for breast cancer patients. The aim of the study was to examine selected miRNAs in TNBC core biopsies sampled before preoperative chemotherapy and the subsequent pathologic response in mastectomy or breast conservation specimens. Prior to treatment, core needle biopsies were collected from 11 female patients with inoperable locally advanced TNBC or large resectable tumors suitable for down-staging. In all 11 TNBC core biopsies we analyzed 19 miRNAs per sample: 512, 190, 200, 346, 148, 449, 203, 577, 93, 126, 423, 129, 193, 182, 136, 135, 191, 122 and 222 (miRCURY LNA™ Universal RT microRNA polymerase chain reaction Custom Pick & Mixpanels). The Wilcoxon signed-rank test was used to compare related samples. Ingenuity pathway analysis was used to evaluate potential functional significance of differentially expressed miRNAs. Statistical analysis showed that 3 of 19 miRNAs differed in relation to pathologic response i.e. good versus poor. These differences failed to reach statistical significance, although a trend was observed (p=0.06). Among these miRNAs, we identified-miR-200b-3p, miR-190a and miR-512-5p. In summary, our results indicate that higher miR-200b-3p, higher miR-190a and lower miR-512-5p expression levels in core biopsies sampled from TNBC patients may be associated with better pathologic response to chemotherapy and the increased feasibility of breast conserving surgery in these patients. Although these results were from a small cohort, they provide an important basis for larger, prospective, multicenter studies to investigate the potential role of miRNAs in neoadjuvant setting.
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http://dx.doi.org/10.1007/s11033-014-3140-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013446PMC
May 2014

[The value of Ki-67 antigen expression in tissue microarray method in prediction prognosis of patients with endometrioid endometrial cancer].

Ginekol Pol 2013 Jun;84(6):444-9

Zaklad Radioterapii, Katedra Onkologii, Uniwersytet Medyczny w Lodzi, Polska.

Objectives: To assess the prognostic significance of Ki-67 expression in the tissue microarray method (TMA) for disease free survival (DFS) and overall survival (OS) in endometrioid endometrial cancer (EEC).

Material And Methods: The study examined 159 consecutive patients aged 37-86 (62.82 +/- 9.95) with EEC stages I-III according to FIGO, treated surgically at the Pirogow Memorial Hospital of Lodz between 2000 and 2007. Afterwards they were subsequently treated and examined at the Regional Cancer Center Copernicus Memorial Hospital of Lodz. Tissue cores 2 mm in size, in duplicate, were taken from the formalin-fixed and paraffin-embedded tissue donor blocks from surgery and constructed into the TMA recipient blocks. Using TMA method, the relationship between Ki-67 expression, DFS and OS was examined. DFS was defined as a period from primary surgery until relapse. OS was defined as a period from primary surgery until the end of the follow-up (60 months) or until the death of the patient. The study was approved by the Ethics Committee of the Medical University of Lodz (RNN/82/11/KE; KE/1673/12).

Results: The follow-up time varied between 3-60 months (51.42 +/- 15.87). In 31 patients (19.50%) the relapse of was diagnosed 1-59 months (24.97 +/- 16.08) after commencement of the treatment. During follow-up 32 patients (20.12%) died. DFS and OS were 80.50% and 79.88%, respectively The lack of Ki-67 expression was found in 37 cases (23.27%) while in 122 patients (76.73%) the expression was present (p < 0.001). The expression of Ki-67 in 1-10%, 11-20% and > 20% was present in 76 cases, 26 cases and 20 cases, respectively Positive correlation between the expression of Ki-67 and staging was present (r = 0.353; p < 0.001). In EEC patients with no relapse diagnosed during follow-up the expression of Ki-67 was present in 7.63 +/- 7.57% of EEC cells, when compared to 23.06 +/- 22.93% in EEC patients in relapsed disease (p < 0.001). The relationship between increased Ki-67 expression and increased grading was not statistically significant (r = 0.149; p = 0.061). The expression of Ki-67 did not depend on patient age (r = 0.040; p = 0.617). In univariate analysis negative correlation was found between the expression of Ki-67 and DFS (p < 0.001) and OS (p = 0.01). In multivariate analysis worse DFS was related to higher staging of EEC (p < 0.0 01) and increased expression of Ki-67 (p < 0.001). Worse OS was related to higher staging in multivariate analysis (p < 0.001). Ki-67 expression was not related to OS in multivariate analysis. Age of patients and grading of the EEC were not related to DFS and OS.

Conclusions: The expression of the Ki-67 can significantly affect therapeutic decisions in selected EEC patients. The high Ki-67 expression in EEC patients is related to increased risk of relapse. The TMA technique is a good method for the assessment of the Ki-67 in studies conducted in EEC patients and makes it easier to carry out immunohistochemistry in large populations of patients.
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http://dx.doi.org/10.17772/gp/1602DOI Listing
June 2013

[The value of progesterone and estrogen receptors expression in tissue microarray method in prognosis of patients with endometrioid endometrial cancer].

Ginekol Pol 2013 Feb;84(2):95-101

Zakład Radioterapii, Katedra Onkologii, Uniwersytet Medyczny w Łodzi, Polska.

Objectives: To assess prognostic significance of progesterone receptors (PR) and estrogen receptors (ER) expression in the tissue microarray (TMA) technique for disease free survival (DFS) and overall survival (OS) in endometrioid endometrial cancer (EEC).

Material And Methods: The study included 151 consecutive patients, aged 37-86 years (62.80 +/- 9.99), with the EEC in stages I-III (FIGO), treated surgically at the Pirogow Memorial Hospital of Lodz between 2000 and 2007. Afterwards, they were subsequently treated and examined at the Regional Cancer Center, Copernicus Memorial Hospital of Lodz. Tissue cores 2 mm in size, in duplicate, were taken from the formalin-fixed and paraffin-embedded tissue donor blocks from surgery and constructed into the TMA recipient blocks. Using TMAs, the expression of PR and ER was examined and presented as Total Score (TS). The TS was determined by adding the intensity and marker distribution scores in a given case. The relationship between PR and ER expression, DFS and OS was examined. DFS was defined as the period from primary surgery until relapse. OS was defined as the period from primary surgery until the end of the follow-up (60 months) or until the death of the patient. The study was approved by the Ethics Committee of the Medical University of Lodz (RNN/82/11/KE).

Results: Lack of the PR and ER expression was found in 46 cases (30.46%) and 67 cases (44.37%), respectively. The expression of the PR and ER was weak in 24 cases (15.89%) and 22 cases (14.57%), respectively. Strong PR and ER expression was found in 81 patients (53.65%) and 62 patients (41.06%), respectively. Follow-up after surgery varied from 3 to 60 months (50.95 +/- 16.36). In 30 patients (19.87%) relapse was diagnosed 1-54 months (22.17 +/- 15.59) after surgery. During follow-ups, 29 patients (19.21%) died. In univariate analysis better DFS was related to the presence of PR (p = 0.010), higher TS of PR (HR = 0.81; 95% CI 0.71-0.94), the presence of ER (p = 0.001) and higher TS of ER (HR = 0.88; 95% CI 0.78-0.99). DFS differed significantly between the groups: without PR and ER expression (A), with presence of the PR but not ER expression (B), with the ER but not PR expression (C) and with the PR and ER expression (D) (p = 0.004). In univariate analysis OS was not related to PR expression (p = 0.110), TS of PR (HR = 0.89; 95% CI 0.80-1.02) and ER expression (p = 0.070). TS of ER was connected to better OS (HR = 0.83; 95% CI 0.72-0.96). The OS differed between groups A, B, C and D (p = 0.006). In multivariate analysis variants of PR/ER expression influenced the DFS (p = 0.039) and OS (p = 0.016).

Conclusions: The expression of the PR and ER can significantly affect therapeutic decisions in selected patients with EEC. In EEC, common assessment of PR and ER expression is of higher prognostic value, than compared to single evaluation of PR and ER receptors.
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http://dx.doi.org/10.17772/gp/1547DOI Listing
February 2013

Membrane expression of TRAIL receptors DR4, DR5, DcR1 and DcR2 in the normal endometrium, atypical endometrial hyperplasia and endometrioid adenocarcinoma: a tissue microarray study.

Arch Gynecol Obstet 2013 Oct 13;288(4):889-99. Epub 2013 Apr 13.

Department of Radiotherapy, Medical University of Lodz, ul Paderewskiego 4, 93-509, Lodz, Poland,

Purpose: To evaluate the membrane expression of DR4, DR5, DcR1 and DcR2 in the normal endometrium (NE), atypical endometrial hyperplasia (AEH) and endometrioid adenocarcinoma (EAC).

Methods: The study comprised 197 patients: 20 NE, 18 AEH and 159 EAC. Tissue microarrays were constructed. Membrane expression of DR4, DR5, DcR1 and DcR2 was examined and presented as total score (TS).

Results: In EAC, the membrane expression of DR4, DR5 and DcR2 was less common compared to NE (p < 0.001; p < 0.001; p = 0.018) and AEH (p < 0.001; p < 0.001; p = 0.004). In EAC the membrane expression of DcR1 did not differ when compared to NE (p = 0.055) and AEH (p = 0.173). A strong correlation was found between the type of endometrial tissue (NE/AEH/EAC) and the TS of DR4 (p < 0.001), DR5 (p < 0.001), DcR1 (p = 0.033) and DcR2 (p < 0.001). In EAC, the TS of DR4, DR5, DcR1 and DcR2 was not related to grading and staging. In EAC, the membrane expression of DR5, but not DR4, DcR1 and DcR2, was related to better disease-free survival (DFS). The overall survival (OS) was not related to membrane TRAIL receptors expression.

Conclusions: The membrane expression of the receptors for TRAIL DR4, DR5, DcR1 and DcR2 is greater in NE than EAC. The level of membrane staining of the receptors in EAC is not dependent on grading and staging. In EAC patients, membrane expression of DR4, DR5, DcR1 and DcR2 are not independent predictors of survival.
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http://dx.doi.org/10.1007/s00404-013-2840-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778234PMC
October 2013

Statistical methods and tool for cut point analysis in immunogenicity assays.

J Immunol Methods 2013 Mar 7;389(1-2):79-87. Epub 2013 Jan 7.

Abbvie Inc, One North Waukegan Road, North Chicago, IL 60064, United States.

Administration of biopharmaceutical products can generate immune response that may severely impact the safety or efficacy of the products. Immunogenicity evaluation, required by regulatory agencies, relies on well developed and validated assays. Key to such assay development is the determination of a cut point during assay validation. Although many methods have been suggested in literature, they are either too complicated to be of practical use by scientists without ready assistance of statisticians or lacking statistical justification. In this paper, we discuss statistical considerations on cut point analysis in immunogenicity assay validation, with a focus on data normalization, outlier detection, and cut point calculation. Also provided is a statistical procedure for cut point determination which maintains a good balance between statistical rigor and implementation simplicity. To facilitate the implementation of the procedure, a software tool is developed, using R language, to automate the proposed process. Taken together the proposed approach renders scientists a practical guide for cut point determination.
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http://dx.doi.org/10.1016/j.jim.2012.12.008DOI Listing
March 2013

Correlation of screening and confirmatory results in tiered immunogenicity testing by solution-phase bridging assays.

J Pharm Biomed Anal 2013 Feb 2;74:235-45. Epub 2012 Nov 2.

Clinical Testing Laboratory, MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, United States.

Biotherapeutic proteins induce undesired immune responses that can affect drug efficacy and safety. For this reason, immunogenicity assessment is an integral part of drug development and is mandated by the regulatory authorities. Immunogenicity is typically evaluated by a tiered approach consisting of a screening assay followed by a competitive inhibition with unlabeled drug serving as confirmatory assay and additional characterization of the immune response. The confirmatory assay is intended to reduce the number of false positive responses generated in the screening tier and ensure that all samples are correctly classified as positive or negative. The positive-negative sample decisions are based on screening and confirmatory assay cut points that are statistically derived through evaluation of drug-naive samples. In this paper, we describe the analysis of cut point data for the presence of statistical correlation between the screening and confirmatory results. Data were obtained from validations of solution-phase bridging assays for detection of anti-drug antibodies against monoclonal antibody therapeutics. All data sets showed moderate to strong positive correlation, indicating that the screening and confirmatory assays were not independent and were likely to generate similar information. We present theoretical evidence that correlated results may be a general feature of the tiered approach when the same test platform is used for both screening and confirmatory assays. The competitive inhibition test, therefore, may be of limited value beyond reduction of the overall false positive rate. Our results indicate that similar sample results could be obtained by using just the screening assay with the false positive rate set to 1%.
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http://dx.doi.org/10.1016/j.jpba.2012.10.027DOI Listing
February 2013

Immunohistochemical detection of FSH receptors in pituitary adenomas and adrenal tumors.

Folia Histochem Cytobiol 2012 Oct 8;50(3):325-30. Epub 2012 Oct 8.

Department of Immunoendocrinology, Medical University of Lodz, Poland.

Objectives: Follicle stimulating hormone (FSH) receptors (FSHR) are physiologically expressed in the ovary and testis. It is well known that FSHR are also expressed in gonadal cancers, but the data on their incidence in extra-gonadal tumors are scarce. Recently, the expression of FSHR in the vascular endothelium within different human cancers was found, but nothing is known on FSHR appearance in non-gonadal endocrine tumors. The present paper reports on the immunohistochemical detection of FSHR in human pituitary adenomas and adrenal tumors.

Materials And Methods: The study included samples of 28 pituitary adenomas and 36 adrenal tumors. Moreover, 2 samples of non-tumoral adrenal glands were also studied. FSH receptor immunostaining was performed on paraffin sections using the rabbit anti-human FSH-R polyclonal antibody raised against 1-190 amino acid sequence from the human FSH-R (sc-13935). The pituitary adenomas were immunostained to reveal the pituitary hormones and the proliferation marker Ki-67.

Results: In the pituitary adenomas, positive immunostaining with anti-FSHR antibody occurred in the adenoma cells cytoplasm and endothelia of the intra- and peritumoral blood vessels. The cytoplasmic immunostaining was found in the majority of investigated tumors but the intensity of staining was weak to moderate. There is some tendency towards the higher cytoplasmic FSHR score in tumors with higher Ki-67 index (atypical adenomas). In contrast to the cytoplasm, the FSHR immunostaining in blood vessels is strong and concerns all the investigated samples. Strong FSHR immunostaining is present in the endothelium of intra- and/or peritumoral blood vessels in the majority of pheochromocytomas, approximatively one half of the adrenocortical adenomas and both cases of the adrenal cancers. The immunostaining is detectable also in the tumoral cell cytoplasm in all but one examined pheochromocytomas. All the investigated adrenocortical adenomas presented strong immunostaining of cell membranes. No immunostained cell membranes were found. in adrenal cancers. The positive immunostaining was found in glandular cells, but not in blood vessels, of non-tumoral adrenal cortex and medulla.

Conclusions: Immunostaining of FSHR often occurs in the endothelium of intra- and/or peritumoral blood vessels of pituitary adenomas and benign and malignant adrenal tumors. The immunostaining may be also present in tumoral cells. A role of FSHR expression in these tumors (stimulation of angiogenesis? stimulation of cell growth?) needs further studies to be clarified.
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http://dx.doi.org/10.5603/17850DOI Listing
October 2012

[Effectiveness of tissue microarray technique for the assessment of estrogen and progesterone receptors expression in endometrioid endometrial cancer--preliminary report].

Ginekol Pol 2012 May;83(5):342-6

Oddział Medycyny Paliatywnej, Wojewódzki Specjalistyczny Szpital im. M. Kopernika w Łodzi, Polska.

Objectives: To assess the effectiveness of the donor-block biopsies with a 2 mm-size needle in endometrioid endometrial cancer (EEC) in the tissue microarray (TMA) technique and the application of the TMA for estrogen receptors (ER) and progesterone receptors (PR) expression in EEC.

Material And Methods: The study examined EEC tissues from 60 patients. Tissue cores, 2 mm in size, in duplicate, were taken from the formalin-fixed and paraffin-embedded tissue donor blocks and constructed into the TMA recipient block. The presence of EEC tissue in the TMAs was analyzed, and the ER and PR expressions were examined.

Results: EEC tissue in TMAs was confirmed in 56 cases (93.33%). In 49 of them (81.67%), both cores presented with cancer tissues. In 4 cases (6.67%) EEC tissue was absent. All cases with ECC present on the TMA slides were appropriate for the ER and PR analysis. In 29 EEC cases (51.98%) both ER and PR were expressed. In 3 cases (5.36%) only ER was expressed, in 8 cases (14.29%) only PR was expressed, and in 16 cases (28.57%) ER and PR were assessed as negative.

Conclusions: Two 2 mm-sized tissue cores from donor-block biopsies constructed into the TMA recipient block were sufficient to diagnose EEC and enabled the assessment of ER and PR expression in 93.3% of the cases. The use of the described TMA technique makes the immunohistochemical study of EEC easier and more time-efficient.
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May 2012

Comparison of prognosis in patients with endometrioid endometrial cancer staged IB in FIGO 1988 and 2009 classifications.

Arch Gynecol Obstet 2012 Oct 25;286(4):995-1000. Epub 2012 May 25.

Department of Palliative Radiotherapy and Palliative Medicine, Regional Cancer Center, Copernicus Memorial Hospital, Ciolkowskiego Str. 2, 93-509 Lodz, Poland.

Purpose: Since 2009 the new FIGO Staging System of endometrial cancer, which changed the previous FIGO 1988 Staging System, has been in use. The aim of the study was to compare prognosis in patients with endometrioid endometrial cancer at stage IB of the 2009 FIGO Staging System and of the 1988 FIGO Staging System.

Methods: We analyzed 173 patients: 108 patients (group A) at stage IB in FIGO 1988 Staging System, and 68 patients (group B) at stage IB in FIGO 2009 Staging System from 262 consecutive endometrioid endometrial cancer patients. The disease-free survival (DFS) and overall survival (OS) were compared between these groups.

Results: The DFS rate was 96.3 % in group A and it was 87.7 % in group B (p = 0.029). Relapses were observed in 12 patients (6.4 %) from 6 to 57 months (mean 28.1; SD = 14.6) after initial surgery, and occurred in four patients from group A (3.7 %) and eight patients from group B (12.3 %) (p = 0.032). The OS rate was 94.4 % in group A and it was 83.1 % in group B (p = 0.018). During follow-up, 17 patients (9.8 %) died: six patients from group A (5.6 %), and 11 patients from group B (16.9 %).

Conclusions: Stage IB in FIGO 2009 Staging System is associated with worse prognosis compared to stage IB according to FIGO 1988 classification. There seems to be a need to use exclusively the new FIGO 2009 classification worldwide to avoid therapeutic mistakes, which can be caused by diverse nomenclature.
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http://dx.doi.org/10.1007/s00404-012-2378-3DOI Listing
October 2012

[Endometrioid endometrial cancer--the prognostic value of selected clinical and pathological parameters].

Ginekol Pol 2011 Oct;82(10):743-8

Pracownia Medycyny Paliatywnej Katedry Onkologii, Uniwersytet Medyczny w Łodzi, Polska.

Objectives: To assess the relationship between selected clinical and pathological factors and disease free survival (DFS) and overall survival (OS) in endometrioid endometrial cancer patients.

Material And Methods: A retrospective review of 262 patients aged 37-86 (6.0 +/- 9.0) was performed. Selected clinical and pathological data were correlated with DFS and OS.

Results: Follow-up was 8-123 months (64.9 +/- 27.1). In 4 patients (1.5%) clinical progression was diagnosed during the treatment. In 43 patients (16.4%) relapse was diagnosed 2-61 months (23.9 +/- 15.7) after commencing treatment. DFS and OS were 82.1% and 81.3% respectively. In univariate analysis worse DFS was related to older patients (p = 0.007) and non-radical surgery (p < 0.001). In multivariate analysis worse DFS was related to older patients (HR = 1.058; 95% CI = 1.024-1.093; p < 0.001), younger at menopause (HR = 0.910; 95% CI = 0.851-0.973; p = 0.006), with higher staging (HR = 2.639; 95% CI = 1.968-3.539; p < 0.001) operated non-radically (HR = 0.220; 95% CI = 0.096-0.504; p < 0.001). In univariate analysis worse OS was connected with older patients (p = 0.018), diabetes type II (p = 0.019) and non-radical surgery (p < 0.001). In multivariate analysis worse OS was related to younger age at menopause (HR = 0.932; 95% CI = 0.873-0.996; p = 0.039), diabetes type II (HR = 2.372; 95% CI = 1.260-4.466; p = 0.008), higher staging (HR = 2.053; 95% CI = 1.482-2.845; p < 0.001), and non-radical surgery (HR = 0.240; 95% CI = 0.091-0.636; p = 0.004).

Conclusions: Relapsed endometrial cancer developed in 90.7% during four years after commencing treatment. In 79.1% of these patients distant metastases were present. Most significant prognostic factors were radicality of surgery age of patients and staging. The presence of diabetes type II and early menopause were connected with worse prognosis.
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October 2011

Gene expression and pathologic response to neoadjuvant chemotherapy in breast cancer.

Mol Biol Rep 2012 Jul 9;39(7):7435-41. Epub 2012 Feb 9.

Department of Surgical Oncology, Cancer Center, Copernicus Memorial Hospital, Paderewskiego 4, 93-509 Lodz, Poland.

Pathologic complete response after neoadjuvant systemic treatment appears to be a valid surrogate for better overall survival in breast cancer patients. Currently, together with standard clinicopathologic assessment, novel molecular biomarkers are being exhaustively tested in order to look into the heterogeneity of breast cancer. The aim of our study was to examine an association between 23-gene real-time-PCR expression assay including ABCB1, ABCC1, BAX, BBC3, BCL2, CASP3, CYP2D6, ERCC1, FOXC1, GAPDH, IGF1R, IRF1, MAP2, MAPK 8, MAPK9, MKI67, MMP9, NCOA3, PARP1, PIK3CA, TGFB3, TOP2A, and YWHAZ receptor status of breast cancer core biopsies sampled before neoadjuvant chemotherapy (anthracycline and taxanes) and pathologic response. Core-needle biopsies were collected from 42 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan low density arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to multiple hypothesis testing. Statistical analysis showed that seven genes out of a 23-gene real-time-PCR expression assay differed significantly in relation to pathologic response regardless of breast cancer subtypes. Among these genes, we identified: BAX (p = 0.0146), CYP2D6 (p = 0.0063), ERCC1 (p = 0.0231), FOXC1 (p = 0.0048), IRF1 (p = 0.0022), MAP2 (p = 0.0011), and MKI67 (p = 0.0332). The assessment of core biopsy gene profiles and receptor-based subtypes, before neoadjuvant therapy seems to predict response or resistance and to define new signaling pathways to provide more powerful classifiers in breast cancer, hence the need for further research.
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http://dx.doi.org/10.1007/s11033-012-1576-1DOI Listing
July 2012

Differential expression of somatostatin receptor subtype-related genes and proteins in non-functioning and functioning adrenal cortex adenomas.

Mol Med Rep 2011 Sep-Oct;4(5):963-9. Epub 2011 Jun 29.

Department of Neuroendocrinology, Medical University of Łódź, Łódź 91-425, Poland.

Adrenocortical adenomas display highly variable expressions of somatostatin receptor (SSTR) subtypes, whose expression is mandatory (although not always sufficient) to achieve the positive effects of somatostatin (SST) analog therapy. Immunohistochemistry (IHC) is the main method used to investigate receptor protein expression. The molecular biology method - polymerase chain reaction (PCR) - is also often used to investigate receptor expression. Nevertheless, the expression of receptor mRNA and the respective receptor protein is not always synchronized. The aim of this study was to investigate SSTR expression by IHC in adrenal adenomas, to compare the results to data obtained by real-time PCR and to determine whether hormonally functioning and non-functioning adenomas differ in this respect. Adrenocortical adenomas were removed surgically from 13 females and 2 males. The tissues were obtained from 9 non-functioning and 6 functioning adenomas. The intensity of IHC reaction was scored semiquantitatively by two independent observers. Real-time PCR was performed using pairs of primers in a reaction amplified along a gradient of temperatures. Amplified DNA was measured by monitoring SYBR-Green fluorescence. In non-functioning tumors, compatibility between IHC and PCR results was observed for SSTR 1 and 2 in 62.5% of the samples. Fifty percent of patients demonstrated compatibility for SSTR 4 and 5 and 37.5% for SSTR 3. In hormonally active adenomas, total compatibility of both methods was noted for SSTR 2 (100%). The compatibility obtained for SSTR 5 was 66.6%. We conclude that receptor gene and respective receptor protein expression are not always synchronized. Messenger RNA detection alone is not sufficient to predict the presence of the receptor protein acting as a target for SST and its analogs.
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http://dx.doi.org/10.3892/mmr.2011.519DOI Listing
November 2011

TRAIL protein expression in breast cancer cells correlates with nuclear grade.

Arch Med Sci 2010 Aug 7;6(4):545-51. Epub 2010 Sep 7.

Department of Surgical Oncology, Chair of Oncology, Medical University of Lodz, Poland.

Introduction: TRAIL protein may serve as an escape mechanism for cancer cells from the immune response. The aim of the study was to assess whether the presence of TRAIL protein correlates with unfavourable prognostic factors in breast carcinoma.

Material And Methods: The study group was composed of breast cancer patients treated surgically in the Department of Surgical Oncology, Medical University of Lodz, Poland, from January to December 2003. Inclusion criteria for the study were fulfilled by 117 women. The immunohistochemical study of TRAIL protein expression was performed in 118 breast carcinomas diagnosed in the study group. TRAIL protein expression was correlated with other variables: tumour size, lymph node status, grade, histological type of carcinoma, oestrogen and progesterone receptor status, HER2 expression, presence of lymphovascular invasion and age of the patient.

Results: Expression of TRAIL protein was present in 73% of breast carcinomas. The percentage of TRAIL-expressing breast carcinoma cells correlated with the nuclear grade (τ = 0.26, p < 0.05; Tau Kendall test). The intensity of TRAIL expression (intensity of staining) in breast carcinoma cells correlated with the nuclear grade (τ = 0.15, p < 0.05; Tau Kendall test). TRAIL expression in breast carcinoma did not correlate with other studied variables.

Conclusions: Our analysis revealed that expression of TRAIL protein in breast carcinoma cells correlates with nuclear grade of carcinoma.
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http://dx.doi.org/10.5114/aoms.2010.14466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3284069PMC
August 2010

SSTR1 and SSTR5 subtypes are the dominant forms of somatostatin receptor in neuroendocrine tumors.

Folia Histochem Cytobiol 2010 Jan;48(1):142-7

Department of Neuroendocrinology, Medical University of Łódź, Poland.

The effectiveness of the long acting somatostatin analogues like octreotide and lanreotide depends on the expression of specific somatostatin receptors on the target cells. The immunohistochemical method performed on surgically removed tumors searches the expression of receptors at the level of receptor protein and gives us insight into receptor's cellular localization. The aim of study was to assess the presence of all the 5 subtypes of SSTR 1-5 (including 2A and 2B SSTR isoforms) in surgically treated human neuroendocrine tumors (NETs) to establish which receptor subtype is the dominant form of somatostatin receptor in particular tumor and thus to be able to predict which somatostatin analog will be effective in NETs treatment. 18 samples of neuroendocrine tumors (surgically excised tumors or biopsies) were immunostained with specific antibodies. Expression of SSTR was scored semiquantitatively. Only strong or moderate immunostaining was considered as positive reaction. The summarized expression pattern of SSTR in the investigated neuroendocrine tumors in our material was: SSTR 1> SSTR 5> SSTR 3> SSTR 2A> SSTR 2B. The receptors were distributed mainly in the area of cells cytoplasm with a few specimens showing only membranous or mixed: membranous--cytoplasmic localization. The observed pattern suggests that apart from octreotide and lanreotide, newly synthesized multiligand analogs such as SOM 230, KE 108 or SSTR 1 and SSTR 5 selective analogs could be effective in NETs treatment.
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http://dx.doi.org/10.2478/v10042-008-0103-7DOI Listing
January 2010

[The thymidine phosphorylase as the platelet-derived endothelial cell growth factor of endometrial cancer].

Ginekol Pol 2009 Aug;80(8):596-601

Zakład Patologii Nowotworów, Katedra Onkologii UM w Lodzi.

Objectives: The aim of the study was to assess the correlation between the activity of thymidine phosphorylase (TP) and the platelet derived-endothelial cell growth factor (PD-ECGF) expression in endometrial carcinoma.

Methods: The study group consisted of 40 tissue samples taken from patients with endometrial carcinoma, who underwent surgery in First Clinic of Gynecology and Oncologic Gynecology of Medical University in Lodz. The control tissue samples were taken from patients who were operated on for non-oncologic reason. The activity of TP was measured by the spectrophotometric method in the cytosol of tumor cells, and the immunohistochemical staining of PD-ECGF was performed in the same tumors. The results of TP activity were compared with the microvessel density (MD) assessed by immunohistochemical analysis and with clinico-pathological features like tumor grade and FIGO stage.

Result: A positive correlation between the enzyme activity and expression of TP/PD-ECGF protein was found. Moreover a significantly higher TP activity was confirmed in malignant tumors from endometrial cancer patients when compared to the controls. A positive correlation between the enzyme activity and MD was also stated, but there was no connection to the grade of tumors and FIGO stage. Since the TP activity proved to be related to PD-ECGF expression and angiogenesis, we can state that TP seems to be an active form of PD-ECGF growth factor in endometrial carcinoma. This is in agreement with the results of many publications on other malignancies. The proper modulation of this activity may be useful in adjuvant therapies.
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August 2009

[Influence of the selected pyrimidine compounds on the activity of thymidine phosphorylase from normal and tumor endometrial cells].

Ginekol Pol 2009 Aug;80(8):590-5

Zakład Biochemii Medycznej, Katedra Biochemii Medycznej w todzi.

Objectives: The aim of this study was to evaluate the influence of the selected pyrimidine compounds on the activity of thymidine phosphorylase (TP) of normal and tumor endometrial cells.

Materials And Methods: Influence of 28 chemical compounds on the TP activity in the cytosol of the endometrial cells was studied by the spectrophotometric method. The studied group comprised postmenopausal women with endometrial cancer: adenocarcinoma endometrialis (Adeno Ca E). The second group included women with normal endometrium after surgery due to non-oncologic reasons.

Results: The most potent inhibitor of TP activity from cancer and endometrium was synthesized 5-bromo-6-acetyloaminouracil, which at the 0.2 mM concentration, by 0.2 mM concentration thymidine reduced the cytosol TP activity by about 80%. 5-bromo-6-aminouracil, 5-nitrouracil and 5-bromouracil reduced this TP activity in statistically significant manner. From among synthesized 1-N-allyloxymethylpyrimidine derivatives 1-N-allyloxymethylthymine was the strongest inhibitor of the TP activity in endometrium, and 1-N-allyloxymethyl-4-hydrokxy-5-nitro-6-oxopyrimidine in endometrial cancer respectively. The most potent activators of TP in endometrial cancer was 5-bromodeoxyuridine and 1-N-allyloxymethyl-5-nitrouracil, which increased the TP activity about 100%. 5-fluorodeoxyuridine, 5-jododeoxyuridine and 2'-deoxyuridine activated the TP in statistically significant manner too, but stronger in case of endometrial cancer than in normal endometrium. The synthesized 5-bromo-6-acetyloaminouracil strongly inhibited the TP activity of endometrial cells and might be useful in reducing endometrial cancer angiogenesis. On the other hand 5-bromodeoxyuridine and the synthesized 1-N-allyloxymethyl-5-nitrouracil might increase the effect of antitumor therapy with the cytostatics. These conclusions ought to be confirmed by analyzing more tumor cases.
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August 2009

Expression of somatostatin receptor subtypes in human thyroid tumors: the immunohistochemical and molecular biology (RT-PCR) investigation.

Thyroid Res 2009 Jan 27;2(1). Epub 2009 Jan 27.

Department of Neuroendocrinology, Medical University, Łódź, Poland.

Human endocrine tumors often express the somatostatin receptors SSTR 1-5 with different intensity. It has been widely investigated their distribution in pituitary adenomas, brain tumors, adrenal tumors and neuroendocrine tumors in gastrointestinal tract (NET). Some of studies also concern the expression of SSTRs in thyroid tumors but they are mainly limited to parafollicular C cells - derived medullary thyroid carcinomas (MTC). Results of SSTR 1-5 detection in other thyroid pathologies like follicular adenomas and papillary cancers are still scarce and often controversial, depending of investigation method used. The aim of this study was to report the presence of all the 5 subtypes of SSTR (including 2A and 2B SSTR isoforms) in some surgically treated human thyroid tumors by means of immunohistochemistry and real-time PCR method and to correlate the results obtained with both techniques. SSTR 1 protein was expressed in 88.8% of investigated cases, SSTR 2A and 2B both in 44.4%, SSTR 3 in 55.5%, SSTR 4 in 11.2% and SSTR 5 in 33.3%. SSTR 1 is the dominant form in the thyroid gland tumor and hyperplasia. We found positive confirmation of both methods in 88.8% for SSTR 1, 2A, 3 subtypes, in 22.2% for SSTR 4 and in 100% for SSTR 5. It suggests that somatostatin multiligand analogs or selective SSTR 1 agonists may be used in thyroid tumors treatment.
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http://dx.doi.org/10.1186/1756-6614-2-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646698PMC
January 2009