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Supplementary Figure 2: Gating strategy for flow cytometry. Live CD45+ cells were gated after live/dead discrimination and removal of doublets as shown.

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen–specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.

Detailed list of used antibodies

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen–specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.

Patients with muscle-invasive urothelial carcinoma achieving pathological complete response (pCR) upon neoadjuvant chemotherapy (NAC) have improved prognosis. Molecular subtypes of bladder cancer differ markedly regarding sensitivity to cisplatin-based chemotherapy and harbor FGFR treatment targets to various content. The objective of the present study was to evaluate whether preoperative assessment of molecular subtype as well as FGFR target gene expression is predictive for therapeutic outcome—rate of ypT0 status—to justify subsequent prospective validation within the “BladderBRIDGister”. Formalin-fixed paraffin-embedded (FFPE) tissue specimens from transurethral bladder tumor resections (TUR) prior to neoadjuvant chemotherapy and corresponding radical cystectomy samples after chemotherapy of 36 patients were retrospectively collected. RNA from FFPE tissues were extracted by commercial kits, Relative gene expression of subtyping markers (e.g., KRT5, KRT20) and target genes (FGFR1, FGFR3) was analyzed by standardized RT-qPCR systems (STRATIFYER Molecular Pathology GmbH, Cologne). Spearman correlation, Kruskal–Wallis, Mann–Whitney and sensitivity/specificity tests were performed by JMP 9.0.0 (SAS software). The neoadjuvant cohort consisted of 36 patients (median age: 69, male 83% vs. female 17%) with 92% of patients being node-negative during radical cystectomy after 1 to 4 cycles of NAC. When comparing pretreatment with post-treatment samples, the median expression of KRT20 dropped most significantly from DCT 37.38 to 30.65, which compares with a 128-fold decrease. The reduction in gene expression was modest for other luminal marker genes (GATA3 6.8-fold, ERBB2 6.3-fold). In contrast, FGFR1 mRNA expression increased from 33.28 to 35.88 (~6.8-fold increase). Spearman correlation revealed positive association of pretreatment KRT20 mRNA levels with achieving pCR (r = 0.3072: p = 0.0684), whereas pretreatment FGFR1 mRNA was associated with resistance to chemotherapy (r = −0.6418: p < 0.0001). Hierarchical clustering identified luminal tumors of high KRT20 mRNA expression being associated with high pCR rate (10/16; 63%), while the double-negative subgroup with high FGFR1 expression did not respond with pCR (0/9; 0%). Molecular subtyping distinguishes patients with high probability of response from tumors as resistant to neoadjuvant chemotherapy. Targeting FGFR1 in less-differentiated bladder cancer subgroups may sensitize tumors for adopted treatments or subsequent chemotherapy.

543 Background: Patients with muscle invasive urothelial carcinoma achieving pathological complete response (pCR) upon neoadjuvant chemotherapy (NACT) have improved prognosis. Molecular subtypes of bladder cancer differ markedly with regard to sensitivity to cisplatinum based chemotherapy and harbour FGFR treatment targets to various content. Previously we did show that lumina tumors respond better to NACT, while FGFR1 expression is associated with chemo resistance (Ecke et al. 2022). The objective of this study was to determine wether radioligand therapy may be an appropriate option in chemoresistent tumors to justify subsequent prospective validation within the "Bladder BRIDGister". Methods: Formalin fixed paraffin embedded (FFPE) tissues from transurethral resections (TUR) before chemotherapy and cystectomy samples after NACT of 36 patients were retrospectively collected. RNA from FFPE tissues were extracted by commercial kits, relative gene expression of subtyping markers (KRT5, KRT20, FGFR1) and radioligand target genes (CXCR4, FAP) were analyzed by standardized RT-qPCR systems (STRATIFYER Molecular Pathology GmbH, Cologne). Spearman correlation, hierarchical clustering, Kruskal-Wallis, chi square and contingency tests were done by JMP 9.0.0 (SAS software). Results: The neoadjuvant cohort consisted of 36 patients (median age: 69, male 83% vs. female 17%) with 92% of patients being histopathologically node negative. When comparing pretreatment with post treatment samples the median expression of KRT20 dropped 128fold, while FGFR1, CXCR4 and FAP mRNA expression increased 6,8fold, 1,9 fold and 2,9 fold, respectively. FAP was positively associated with KRT5, FGFR1 and CXCR4 in treatment naïve TUR biopsies (r=0.4051 p=0.0141, r=0.6458 p2 4.314 p=0,0378). Combining elevated FAP and CXCR4 mRNA expression did identify 28% of the patients to be at high risk of NACT resistance (90%). Conclusions: Expression of the radioligand targets CXCR4 and FAP are associated with basal/stromal enriched tumors and resistance to NACT. Theranostic targeting of CXCR4 and FAP before NACT might increase response towards NACT in this poor prognosis group.

545 Background: Patients with muscle invasive urothelial carcinoma achieving pathological complete response (pCR) upon neoadjuvant chemotherapy (NACT) have improved prognosis. Molecular subtypes of bladder cancer differ markedly with regard to sensitivity to cisplatinum based chemotherapy. Previously we did show that luminal tumors respond better to NACT, while FGFR1 expression is associated with chemo resistance (Ecke et al. 2022). The objective of this study was to determine which patients may benefit from Antibody Drug Conjugate (ADC) treatment in addition to NACT to justify subsequent prospective analysis within the "Bladder BRIDGister". Methods: Formalin fixed paraffin embedded (FFPE) tissues from transurethral resections (TUR) before chemotherapy and cystectomy samples after NACT of 36 patients were retrospectively collected. RNA from FFPE tissues were extracted by commercial kits, relative gene expression of subtyping markers (KRT5, KRT20, FGFR1) and radioligand target genes (NECTIN4, TROP2) were analyzed by standardized RT-qPCR systems (STRATIFYER Molecular Pathology GmbH, Cologne). Spearman correlation, hierarchical clustering, Kruskal-Wallis, chi square and contingency tests were done by JMP 9.0.0 (SAS software). Results: The neoadjuvant cohort consisted of 36 patients (median age: 69, male 83% vs. female 17%) with 92% of patients being pathohistologically node negative. When comparing pretreatment with post treatment samples the median expression of KRT20 dropped 128fold, while FGFR1 expression increased 6.8 fold. Interestingly, TROP2 and NECTIN4 mRNA expression also dropped significantly upon NACT by 5.7 fold and 7.1 fold, respectively. TROP2 and NECTIN4 were positively associated with the response marker KRT20 in therapy naïve TUR biopsies (r=0.5562 p=0.0004; r=0.5833 p=0.0002), but negatively associated with the resistance marker FGFR1 (r=-0.2903 p=0,0858; r=-0.3396 p=0,0427). However, TROP2 and NECTIN4 were not associated with pCR in spearman analysis with minor trend for TROP2 (r=0,2139 p=0,2103). Cluster analysis revealed a subgroup of KRT20 positive and FGFR1 negative tumors expression TROP2 and NECTIN4, which achieved 80% pCR. In addition elevated TROP2 and NECTIN4 expression was found in KRT20 positive tumors coexpressing FGFR1 and being resistant to NACT. Conclusions: Expression of the ADC targets TROP2 and NECTIN4 is associated with KRT20 positive, luminal tumors being highly sensitive to neoadjuvant chemotherapy alone. KRT5 positive, basal tumors do exhibit only very low expression of TROP2 and NECTIN4 mRNA. In view of toxicities the addition of TROP2 and NECTIN4 treatment to NACT might be considered only in luminal tumors exhibiting elevated FGFR1 expression as resistance mechanism and therefore do not respond to NACT.

BACKGROUND: The aim of this study was to determine whether mRNA expressions and dynamic changes of immune-related genes before and after starting first-line treatment with the PD-1 inhibitor pembrolizumab in patients with NSCLC were of predictive value. METHODS: CD3, CD8, PD-1, PD-L1 and CTLA-4 mRNA expression levels were measured from peripheral blood before and after three weeks of treatment with the PD-1 inhibitor. Univariate and multivariate analyses were performed retrospectively. Response, progression-free survival (PFS) and overall survival (OS) were determined. RESULTS: In univariate analysis an increase of CD3 and CD8 mRNA expression after the first cycle of pembrolizumab were each associated with improved PFS and OS. In contrast, patients with no change or with a decrease in CD3 and CD8 mRNA expression showed significantly worse outcome. CD8 mRNA increase remained an independent predictive factor for PFS and OS in the multivariate analysis with p values of 0.011 and 0.006, respectively. CONCLUSIONS: An increase of CD8 mRNA expression predicts favorable outcome after first line monotherapy with pembrolizumab, while no change or decrease might serve as an indicator of poor outcome and might give cause for early treatment escalation for instance by addition of chemotherapy or additional ICI treatment, e.g., against CTLA-4.

Patients with muscle-invasive bladder cancer (MIBC) that underwent neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) show improved overall survival, especially those with pathological complete response (pCR). The response to NAC according to molecular subtypes has been discussed. Molecular targets such as estrogen receptor (ESR1) and human epidermal growth factor receptor 2 (ERBB2) play an important role in breast cancer management and have also been associated with urothelial bladder cancer. Hence, the association of Keratin 20 (KRT20) Keratin 5 (KRT5), ESR1, and ERBB2 mRNA expression in MIBC at transurethral resection (TUR-BT) with pCR after NAC was analyzed retrospectively. Formalin-fixed paraffin-embedded tumour tissue samples from TUR-BT of 54 patients (42 males, 12 females, median age of 64) with MIBC were analyzed for KRT20, KRT5, ESR1, and ERBB2 mRNA expression. After NAC, RC was performed, and the specimens were evaluated for pCR. Statistical analyses comprised nonparametric and chi2 testing, partition models, and Spearman correlation analyses. After NAC, 22 out of 54 patients (40.7%) had pCR. Tumours with an elevated expression of markers associated with luminal differentiation (KRT20, ERBB2, ESR1) were associated with a higher chance of pCR (55% vs. 15.8%, p = 0.009). Elevated ERBB2 expression was positively correlated with luminal expression features such as KRT20, and negatively with basal characteristics such as KRT5. Patients with MIBC showing a high expression of ERBB2, ESR1, or KRT20 have a significantly higher chance of pCR following NAC. These findings might improve patient selection for NAC in MIBC., publishedVersion

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen-specific B cells and induced responses of autologous T cells

469 Background: The objective of the present study was to assess FGFR mutattions and fusions from matched urine and tissue samples from patients suspicious of bladder cancer and undergoing first TURB within BRIDGister RealWorld Experience trial. Methods: FFPE samples from the first TURB of 39 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits and analyzed by Therascreen FGFR IVD kit (Qiagen GmbH, Hilden). In addition extracellular vesicles were centrally isolated for subsequent RNA extraction (exoRNA.Exosome Diagnostics GmbH, Martinsried) and centrall analysis by QIAcuity digital PCR (Qiagen, Hilden). In addition mRNA based profiling of urine was done by dPCR. Concordance, Spearman, Kruskal-Wallis and MannWhitney were analyzed by JMP 9.0.0 (SAS software). Results: The pilot cohort of the BRIDGister trial consisted of 39 patients (median age: 75, male 69% vs female 31%) of diverse clinical stages (Benign lesions/no tumor 23%, pTa 31%, pT1 26%, pT2 21%) and WHO 1973 grade (G1 8%, G2 39%, G3 34%). Based on FFPE tissue testing using Therascreen FGFR IVD kit and exosomal RNA extraction follwoed by dPCR 12 out of 39 patients exhibited FGFR alterations (31%). Comparison with tissue testing as probable gold standard revealed 67% sensitivity, 85% specificity, 67% PPV and 85%NPV. There were 4 patients being FGFR positive for exoRNA from urine with no mutation found in the corresponding TUR biopsy. Determining ERBB2 mRNA by dPCR from urine revealed that high ERBB2 mRNA correlated with higher WHO1973grade, while high FGFR3 mRNA correlated with lower grade tumors (Spearman r=0.4386 p=0.0075 and r=-0.4663 p=0.0042). Similiarly, trends were seen for association of ERBB2 and FGFR3 mRNA with clinical stage tumors in this pilote cohort (Spearman r=0.2359 p=0.0923 and r=-0.2249 p=0.1089). Conclusions: Extraction of exosomal RNA from urine followed by highly sensitive dPCR mutation testing is feasible with good concordance to matched tissue testing. Urine testing might evolve as alternative approach for FGFR3 screening in a non invasive fashion without the need of transurethral biopsy. Interestingly, mRNA assessment of exosomal RNA from urine before TURB correlated with clinical parameters such as WHO Grade 1973 with ERBB2 mRNA being associated with high grade tumors and FGFR3 mRNA being associated with low grade tumors, which is in line with previous tissue tsting results. This indicates the potential to clinically characterize tumor grade and stage before TUR biopsy or surgery which might be helpful for future risk stratification and planning of surgical intervention. Further exploration is warranted and includes the potential of monitoring patients with regard to urine based mutation detection and risk stratification.

560 Background: The objective of the present study was to evaluate whether preoperative standard urine parameters such as leucocyte count in urine samples dominate in particular molecular subtypes of bladder cancer and reflect the immune infiltration status in the tissue in matched urine and TUR biopsy samples from patients being suspicious of bladder cancer and undergoing first TURB within the prospective Real World Experience registry trial "BRIDGister". Methods: For this pilot study paraffin fixed pretreatment tissue samples from the first TURB of 48 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits- Relative gene expression of subtyping markers (KRT5, KRT20) by standardized RT-qPCR systems for PD-1, PD-L1, CTLA4 (STRATIFYER Molecular Pathology GmbH, Cologne). In addition urine samples were analyzed for leucocyte and erythrocyte count. Spearman correlation, Kruskal-Wallis, MannWhitney and Sensitivity/Specificity tests were done by JMP 9.0.0 (SAS software). Results: The pilot cohort of the BRIDGister trial consisted of 48 patients (median age: 77, male 65% vs. female 35%) of diverse clinical stages (Benign lesions/no tumor 38%, pTa 23%, pT1 20%, pT2 19%) and WHO 1973 grade (G1 11%, G2 43%, G3 23%). Presence of leucocytes but not erythrocytes was negatively associated with KRT20 (r=-0.4249, p=0.0216) but positive with KRT5 (r=0.3704, p=0.0479). Moreover high levels of leucocytes in urine were positively associated with tumor expression of PD-L1 (r=0.5081, p=0.0041), PD-1 (r=0.5342, p=0.00024) and CTLA4 (r=0.4244, p=0.0194). In contrast there was no significant association of tumor PDL-1, PD-1 and CTLA4 expression with erythrocyte count in urine. Conclusions: Presence of leucotytes in urine is strongly associated with basal subtype and immune cell infiltration into early bladder cancer. As the presence of erythrcytes did not reveal these significant associations, the presence of leucocytes is not due to simple bleeding or tissue vulnerability. Moreover, the strong tumor subtype specificity further demonstrate the basal tumor specificity of urine leucocytes and therefore may be helpful for detecting and monitoring basal bladder cancer in a non invasive fashion. Given the prognostic value of tissue determination of PD-L1, PD-1 and CTLA4 quantitation on mRNA level, these results warrant further investigation to conclude on its impact on outcome prediction (BCG responsiveness, recurrence, progression, etc.), which will be prospectively analyzed in the framework of the ongoing multicenter BRIDGister Real World Experience trial.

471 Background: The objective of the present study was to prospectively evaluate FGFR mutation detetion in matched urine and tissue samples from patients suspicious of bladder cancer and undergoing first TURB within the framework of the BRIDGister RealWorld Experience trial. Methods: For this pilot study paraffin fixed pretreatment tissue samples from the first TURB of 48 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits and analyzed by Therascreen FGFR IVD kit (Qiagen GmbH, Hilden). In addition urine samples were filtered at local urology and filters were shipped for central extraction of cellular DNA (Uromonitor, Porto). Concordance, Kruskal-Wallis, MannWhitney and Sensitivity/Specificity tests were analyzed by JMP 9.0.0 (SAS software). Results: The pilote cohort of the BRIDGister trial consisted of 47 patients (median age: 77, male 65% vs female 35%) of diverse clinical stages (benign lesions/no tumor 38%, pTa 23%, pT1 20%, pT2 19%) and WHO 1973 grade (G1 11%, G2 43%, G3 23%). Based on FFPE tissue testing using Therascreen FGFR IVD kit 10 out of 47 patients exhibited FGFR alterations (25%), while urine filtering for cellular components and subsequent PCR testing revealed 13 out of 40 matched urine sampels were FGFR positive (33%). Comparison with tissue testing as probable gold standard revealed 100% sensitivity, 90% specificity, 77% PPV, 100% NPV as well as high concordance (kappa 0,82, p < 0,0001). There were 3 patients being FGFR positive for Uromonitor from urine with no mutation found in the corresponding TUR biopsy. Conclusions: Filtering urine for cells and subsequent DNA extraction followed by PCR detection results in highly sensitive mutation testing being feasible with good concordance to matched tissue testing. Prospective testing validated the diagnostic accuracy of the Uromonitor FGFR test in a real world setting.

// Markus Eckstein 1, * , Ralph M. Wirtz 2, 3, * , Carolin Pfannstil 1, * , Sven Wach 4, * , Robert Stoehr 1, * , Johannes Breyer 6, * , Franziska Erlmeier 7, * , Cagatay Gunes 8, * , Katja Nitschke 5, * , Wilko Weichert 7, * , Wolfgang Otto 6, * , Bastian Keck 4, * , Sebastian Eidt 3, * , Maximilian Burger 6, * , Helge Taubert 4, * , Bernd Wullich 4, * , Christian Bolenz 8, * , Arndt Hartmann 1, * and Philipp Erben 5, * 1 Institute of Pathology, University of Erlangen-Nuremberg, Erlangen, Germany 2 STRATIFYER Molecular Pathology GmbH, Cologne, Germany 3 Institute of Pathology at The St. Elisabeth Hospital Koln-Hohenlind, Cologne, Germany 4 Department of Urology, University of Erlangen-Nuremberg, Erlangen, Germany 5 Department of Urology Mannheim, University of Heidelberg, Mannheim, Germany 6 Department of Urology, University of Regensburg, Regensburg, Germany 7 Institute of Pathology, Technical University Munich, Munich, Germany 8 Department of Urology, University of Ulm, Ulm, Germany * On behalf of the BRIDGE-Consortium Germany Correspondence to: Markus Eckstein, email: markus.eckstein@uk-erlangen.de Keywords: bladder cancer; PD-L1; checkpoint inhibitors; molecular therapy stratification; immunohistochemistry Received: October 26, 2017 Accepted: February 10, 2018 Epub: February 19, 2018 Published: March 13, 2018 ABSTRACT Background: Immunohistochemical PD-L1 assessment is currently used to identify responders towards checkpoint inhibitors although it is limited by inter-observer effects. Here, we conducted a multi-center round robin test to prove the possibility of assessing the PD-L1 status by gene expression to avoid inter-observer effects. Patients and methods: Gene expression of PD-L1 was analyzed in a total of 294 samples (14 cases non-muscle invasive and muscle-invasive bladder cancer; MIBC) in seven centers by a RT-qPCR kit and compared with immunohistochemical scoring of three pathologists (DAKO, 22c3). Both assays were compared towards prognosis prediction in a cohort of 88 patients with MIBC. Results: PD-L1 gene expression revealed very high inter center correlation (centrally extracted RNA: r = 0.68–0.98, p ≤ 0.0076; locally extracted RNA: r = 0.81–0.98, p ≤ 0.0014). IHC Inter-observer concordance was moderate to substantial for immune cells (IC), fair for combined IC/ tumor cell (TC) (IC: κ = 0.50–0.61; IC + TC: κ = 0.50), and fair for TC scoring (κ = 0.26–0.35). Gene expression assessment resulted in more positive cases (9/14 cases positive vs. 6/14 cases [IHC]) which could be validated in the independent cohort. Positive mRNA status was associated with significantly better overall and disease-specific survival (5-year OS: 50% vs. 26%, p = 0.0042, HR = 0.48; 5 year DSS: 65% vs. 40%, p = 0.012, HR = 0.49). The 1% IHC IC cut-off also revealed significant better OS (5 year OS: 58% vs. 31%, p = 0.036, HR = 0.62). Conclusion: Gene expression showed very high inter-center agreement. Gene expression assessment also resulted in more positive cases and revealed better prognosis prediction. PD-L1 mRNA expression seems to be a reproducible and robust tool for PD-L1 assessment.

e16532 Background: The objective of the present study was to assess FGFR mutattions and fusions from matched urine and tissue samples from patients suspicious of bladder cancer and undergoing first TURB at the pilot center of the multicentric BRIDGister RealWorld Experience trial Methods: For this pilot study paraffin fixed pretreatment tissue samples from the first TURB of 28 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits and analyzed by Therascreen FGFR IVD kit (Qiagen GmbH, Hilden). In addition urine samples were shipped for central isolation of extracellular vesicles and extraction of RNA (exoRNA.Exosome Diagnostics GmbH, Martinsried) and subsequently centrally analyzed by QIAcuity digital PCR (Qiagen, Hilden). Additional urine testing was performed by further technologies including central cytology. Concordance, Kruskal-Wallis, MannWhitney and Sensitivity/Specificity tests were analyzed by JMP 9.0.0 (SAS software). Results: The pilot cohort of the BRIDGister trial consisted of 28 patients (median age: 73, male 71% vs female 29%) of diverse clinical stages (Benign lesions/no tumor 21%, pTa 32%, pT1 21%, pT2 21%) and WHO 1973 grade (G1 7%, G2 43%, G3 21%). Based on FFPE tissue testing using Therascreen FGFR IVD kit 9 out of 28 patients exhibited FGFR alterations (32%). Based on exosomal RNA (exoRNA) and subsequent dPCR testing 8 out of 21 matched urine sampels were FGFR positive (38%). Comparison with tissue testing as probable gold standard revealed 71% sensitivity, 78% specificity, 63% PPV and 85%NPV. There were 3 patients being FGFR positive for exoRNA from urine with no mutation found in the corresponding TUR biopsy. One of these mutations could be validated by independent urine test. Furthermore one tumor harbored two tissue mutations (R248C, Y373C) but three urine mutations (R248C, Y373C, G370C) indicating substantial tumor heterogeneity. One FGFR3-TACC3 fusion was detected from a benign lesion, which was not found by the exosomal urine test. Conclusions: Extraction of exosomal RNA from urine followed by highly sensitive dPCR mutation testing is feasible with good concordance to matched tissue testing. Urine testing bears the potential of detecting additional mutations in a real world setting and might evolve as alternative approach for FGFR3 screening in a non invasive fashion without the need of transurethral biopsy. Discordant cases are further followed up and might reveal validation of mutation status in upcoming recurrences.Further exploration is warranted and includes the potential of monitoring patients with FGFR before and after therapeutic intervention. By the time of the congress an update of the data with approximately 50 matched pairs will be presented.

e16533 Background: The objective of the present study was to identify molecular charactersitics associated with FGFR positive tumors in matched urine and TUR biopsy samples from patients being suspicious of bladder cancer and undergoing first TURB at the pilot center of the multicentric BRIDGister Real World Experience trial that are helpful for patient management and prognosis. Methods: For this pilot study paraffin fixed pretreatment tissue samples from the first TURB of 28 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits and analyzed by Therascreen FGFR IVD kit (Qiagen GmbH, Hilden). Relative gene expression of subtyping markers (KRT5, KRT0) as well as FGFR1, FGFR2, FGFR3, FGFR4, ERBB2 was centrally tested by standardized test systems (STRATIFYER Molecular Pathology GmbH, Cologne). In addition urine samples were analyzed by commercially available urine tests (UBC Rapid, BTA stat, NMP22). Spearman correlation, Kruskal-Wallis, MannWhitney and Sensitivity/Specificity tests were done by JMP 9.0.0 (SAS software). Results: The pilot cohort of the BRIDGister trial consisted of 28 patients (median age: 73, male 71% vs female 29%) of diverse clinical stages (Benign lesions/no tumor 21%, pTa 32%, pT1 21%, pT2 21%) and WHO 1973 grade (G1 7%, G2 43%, G3 21%). Based on FFPE tissue testing using Therascreen FGFR IVD kit 9 out of 28 patients exhibited FGFR alterations (32%). FGFR positive tumors were associated with high expression of FGFR3 mRNA (r = 5.951, p = 0.0011) but low FGFR1 mRNA as well as FGFR4 mRNA (r = -0.3882, p = 0.0412 and r = -0.6305, p = 0.0004, respectively). Interestingly, FGFR alteration was positively associated with the basal marker KRT5 (r = 0.3929, p = 0.0386), but not with luminal KRT20 mRNA expression (r = -0.0208, p = 0.9179). Moreover FGFR3 altered bladder cancer was associated with elevated NMP22 levels in pretreatment urine (r = 0.3978, p = 0.0361). Conclusions: In early bladder cancer FGFR3 alterations are tightly associated with a characteristic FGFR mRNA signature. Mutation/Fusion of FGFR3 results in high FGFR3 but low FGFR1 and FGFR4 mRNA expression, which might be i.a. relevant for the response to FGFR inhibition and important to predict outcome of FGFR inhibitors. Morever FGFR alteration was associated with elevated NMP22 urine levels, which might be helpful for detecting and monitoring FGFR altered bladder cancer in a non invasive fashion. These results warrant further investigation and its impact on outcome prediction (BCG responsiveness, recurrence, proegression, etc) will be prospectively analyzed in the framework of the ongoing multicenter BRIDGister Real World Experience trial.

Background Quantitative determination of estrogen receptor mRNA expression in luminal breast tumors is predictive for benefit from adjuvant tamoxifen compared to placebo treatment as has been shown in the large randomized NSABP B-14 trial, while protein determination by IHC or LBA is not (Kim et al JCO 2011). Interestingly, the ultrahigh expression of ESR1 mRNA (above ER score 10 by Oncotype test) has been indicative for tamoxifen benefit. This predictive cut-off value of mRNA expression is significantly higher than the diagnostic cut-off (at ER score 6.5). Here we tested wether the ultrahigh expression of ESR1 mRNA determined by commercial MammaTyper® testing is predictive for survival after neoadjuvant chemotherapy treatment of advanced breast cancer. Materials and Methods Pretreatment core cut biopsies from n=54 patients with PBC treated within a randomized phase II trial (2) of anthracyline/taxane based NAC with available clinical follow-up information were examined. RNA was extracted from the FFPE sections and ESR1 mRNA from each section was measured by commercial assays. For technical comparison of ESR1 mRNA values by Oncotype DX versus MammaTyper® from n=113 surgical samples were analyzed by both commercial assays in a blinded fashion. Statistical analysis was performed using the SAS JMP® 9.0.0 software. Results Quantification of ESR1 mRNA expression after RNA extraction from separate slices of 113 primary breast tumors and determination by different commercial RT-qPCR assays resulted in high correlation of continuous expression results (Spearman r=0,85; p Conclusion Previous data suggest that ultrahigh expression of ESR1 mRNA is predictive for improved overall survival and tamoxifen benefit (1). Here we show that ultrahigh expression of ESR1 mRNA is also prognostic in more advanced breast tumors after neoadjuvant chemotherapy. These findings validate the importance of quantitative determination of estrogen receptor expression and substantiate the understanding of receptor expression being a continuous determinant with indication specific cut-off values. Ultrahigh expression of ESR1 seems to identify a distinct subset of luminal breast tumors with superior prognosis and benefit from tamoxifen treatment. These findings warrant further investigation, which are currently being done in independent large breast cancer cohorts. Citation Format: Wirtz RM, Scheffen I, Marme F, Laible M, Sclombs K, Schumacher C, Schneeweiss A, Eidt S, Sinn H-P. Predictive value of ultra-high ESR1 mRNA expression in early breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-25.

Differential expression of cytokeratins (CK) is a characteristic feature of chemoresistant luminal (KRT20) and chemosensitive intrinsic aggressive basal (KRT5) subtypes in muscle-invasive bladder cancer (MIBC). We investigated mRNA expression of KRT5 and KRT20 and its predictive value in stage pT1 bladder cancer. In retrospective analysis of clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with stage pT1 NMIBC who underwent transurethral resection of the bladder, a single-step RT-qPCR was used to measure mRNA expression. Furthermore, immunohistochemical (IHC) staining of CK20, panCK, and MIB1 was performed. Valid measurements were obtained from 231 samples out of a series of 284 patients. Spearman correlation revealed significant associations between mRNA and protein expression of KRT20/CK20 (ρ 0.6096, p

Pathological staging and grading are crucial for risk assessment in non-muscle-invasive bladder cancer (NMIBC). Molecular grading might support pathological evaluation and minimize interobserver variability. In this study, the well-established breast cancer markers ESR1, PGR, ERBB2, and MKI67 were evaluated as potential molecular markers to support grading and staging in NMIBC. We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with NMIBC. Messenger RNA (mRNA) expression of the aforementioned markers was measured by single-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) using RNA-specific TaqMan assays. Relative gene expression was determined by normalization to two reference genes (CALM2 and B2M) using the 40-ΔΔCT method and correlated to histopathological stage and grade. Pathological assessment was performed by an experienced uropathologist. Statistical analysis was performed using the SAS software JMP 9.0.0 version and GraphPad Prism 5.04. Of 381 cases of NMIBC, samples of 100 pTa and 255 pT1 cases were included in the final study. Spearman rank correlation revealed significant correlations between grade and expression of MKI67 (r = 0.52, p

Recently, muscle-invasive bladder cancer (MIBC) has been subclassified by gene expression profiling, with a substantial impact on therapy response and patient outcome. We tested whether these complex molecular subtypes of MIBC can be determined by mRNA detection of keratin 5 (KRT5) and keratin 20 (KRT20). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was applied to quantify gene expression of KRT5 and KRT20 using TaqMan®-based assays in 122 curatively treated MIBC patients (median age 68.0 years). Furthermore, in silico analysis of the MD Anderson Cancer Center (MDACC) cohort (GSE48277 + GSE47993) was performed. High expression of KRT5 and low expression of KRT20 were associated with significantly improved recurrence-free survival (RFS) and disease-specific survival disease specific survival (DSS: 5-year DSS for KRT5 high: 58%; 5-year DSS for KRT20 high: 29%). KRT5 and KRT20 were associated with rates of lymphovascular invasion and lymphonodal metastasis. The combination of KRT5 and KRT20 allowed identification of patients with a very poor prognosis (KRT20+/KRT5−, 5-year DSS 0%, p < 0.0001). In silico analysis of the independent MDACC cohorts revealed congruent results (5-year DSS for KRT20 low vs. high: 84% vs. 40%, p = 0.042). High KRT20-expressing tumors as well as KRT20+/KRT− tumors were significantly enriched with aggressive urothelial carcinoma variants (micropapillary, plasmacytoid, nested).

Background Immune cell infiltrates have been shown to play a prominent role with regard to prognosis of node negative breast cancer (Schmidt et al 2008). Moreover, it has been shown that B-cells and T-Cells are of particular importance for the response to chemotherapy (Schmidt et al. 2012). However, the role of macrophages remains unclear. It has been assumed, that tumor-associated macrophages play a role in promoting tumorigenesis. Still their role with regard to therapy prediction is unknown. Here we tested wether the infiltration of macrophages is associated with the response to neoadjuvant chemotherapy. Materials and Methods Pretreatment core cut biopsies from n = 100 patients with PBC treated within a randomized phase II trial (1) of anthracyline/taxane based NAC were examined. RNA from formalin-fixed, paraffin embedded (FFPE) routine biopsies were extracted using a bead-based extraction method (STRATIFYER XTRAKT kits). CD68 and ESR1, PGR, HER2, Ki67 as well as CALM2 as a house keeping gene were measured via a multiplex quantitative RT-PCR (RT-qPCR). Correlation analyses and partitioning tests were performed using the SAS JMP® 9.0.0 software. Results CD68 mRNA exhibited a normal data distribution (Median expression 40-DCT 34,66) in the core needle biopsies of advanced breast tumors. CD68 mRNA correlated strongly with Ki67 mRNA levels (Spearman r = 0,46; p Conclusion The presence of macrophages indicates tumors being sensitive to chemotherapy beyond the prospectively validated ESR1 and HER2 mRNA determinations (2), both in luminal and triple negative tumors. The mRNA expression levels of CD68 provide additional information beyond conventional subtyping and might therefore be useful for early assessment of non-response to chemotherapy und subsequent treatment planning. 1) Schneeweiss et al, Ann Oncol 2011 2) Denkert et al, Ann Oncol 2013. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-07.

Background: Tumor immune cell infiltrates influence prognosis of node negative breast cancer (BC), and intratumoral B-cells and T-cells are of importance for achieving response to chemotherapy in triple negative BC. The role of tumor infiltrating macrophages remains unclear, but they might promote tumorigenesis. We investigated the prognostic value of CD68 mRNA levels within the luminal, HER2-positive and triple negative subtypes in the FinHer trial patient population, and evaluated their predictive value on survival outcomes achieved with adjuvant trastuzumab and chemotherapy in early breast cancer. Methods: RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue of 917 (90.8%) patients out of the 1010 patients who participated in the FinHer trial. Intratumoral macrophage infiltration was assessed by measuring breast tumor CD68 mRNA content using RT-qPCR from representative FFPE tissue samples. Breast cancer molecular subtypes (luminal, HER2 positive and triple-negative) were approximated using immunohistochemistry (IHC) and central CISH testing data obtained from the FinHer trial datafile. Prognostic significance of CD68 on distant disease-free survival (DDFS) was assessed using Kaplan-Meyer analysis and log-rank test. Results: Tumor CD68 mRNA expression was normally distributed with median expression of 33.56 (dCt). CD68 mRNA levels correlated weakly with estrogen receptor (ER) mRNA expression and ER protein levels (r = 0.11 and r = 0.15, respectively; p Conclusions: The study validates tumor CD68 concentration as a prognostic biomarker in HER2-positive early breast cancer. Patients with HER2-positive cancer and few tumor macrophages (low tumor CD68 mRNA content) benefitted from addition of trastuzumab to chemotherapy, whereas patients with HER2-positive BC with high tumor macrophage content derived no benefit from adjuvant trastuzumab. Trastuzumab may be effective only for macrophage-poor HER2-positive cancers that are prone to antibody-dependent cellular cytotoxicity (ADCC), whereas it may have little efficacy for cancers that progress despite or due to high intratumoral macrophage content that interferes with ADCC. Other agents, such as T-DM1, might work better than trastuzumab in the subset of women who have HER2-positive BC with a high tumor macrophage content. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-06.

There is some controversy regarding the precise role and need for adjuvant therapy in patients with pT1a/pT1bN0 breast cancer, although studies have indicated that a HER2-positive status is one of the most powerful poor prognostic factors.We retrospectively evaluated disease-free survival (DFS), distant disease-free survival (DDFS), and overall survival (OS) among 960 patients diagnosed between 2000 and 2008 with T1N0 primary breast cancer treated at 3 German centers, and determined prognostic risk factors. Univariate analysis was used to determine associations with potential risk factors.With a median follow-up of 23 months, DFS was 94.8%, DDFS 96.3%, and OS 97.5%. Risk factors for decreased 1-year DFS were: peritumoral lymphatic invasion (L1) (p = 0.031), negative hormone receptor status (p = 0.003), non-use of hormonal therapy (p = 0.001), and a positive HER2 status (p = 0.003). Amongst the HER2-positive patients only 2.7% (n = 1/37) of those treated with trastuzumab had a DFS event compared with 20% (n = 10/50) without trastuzumab.Patients with HER2-positive T1 breast cancer should be considered for inclusion in prospective trials of trastuzumab in combination with chemotherapy to determine the risk-to-benefit ratio and association with other prognostic factors.Hintergrund: Die Bedeutung und der Nutzen einer adjuvanten Therapie bei Patientinnen mit einem pT1a/pT1bN0-Mammakarzinom wird kontrovers diskutiert. In einigen Studien konnte ein positiver HER2-Status als einer der stärksten prognostischen Faktoren nachgewiesen werden. Patientinnen und Methoden: In dieser Studie wurde retrospektiv an 3 deutschen Brustzentren das krankheitsfreie Überleben (DFS), das distant krankheitsfreie Überleben (DDFS) und das Gesamtüberleben (OS) von 960 Patientinnen, die zwischen 2000 und 2008 an einem primären Mammakarzinom2 cm (T1N0) erkrankten, ausgewertet. Zusätzlich wurden die prognostischen Faktoren untersucht. Der Zusammenhang mit potentiellen Risikofaktoren wurde durch univariate Analysen untersucht. Ergebnisse: Die mediane Nachbeobachtungszeit betrug 23 Monate, DFS war 94,8%, DDFS 96,3% und OS 97,5%. Risikofaktoren für ein kürzeres 1-Jahres-DFS waren: peritumorale lymphatische Invasion (L1) (p = 0,031), negativer Hormonrezeptorstatus (p = 0,003), keine adjuvante hormonelle Therapie (p = 0,001) und ein positiver HER2-Status (p = 0,003). Bei Patientinnen, die mit Trastuzumab behandelt wurden, trat nur bei 2,7% (n = 1/37) ein DFS-Ereignis auf, bei Patienten ohne Trastuzumab-Therapie war dies bei 20% (n = 10/50) der Fall. Schlussfolgerung: Patienten mit einem HER2-positiven Mammakarzinom2 cm sollten in prospektive Studien eingeschlossen werden, um die Bedeutung der Trastuzumab-Therapie in Kombination mit Chemotherapie, das Nutzen-Risiko-Verhältnis und die Bedeutung anderer Riskiofaktoren untersuchen zu können.

Background/Aims/Objectives: It is difficult to identify patients with a non-muscle-invasive bladder cancer (NMIBC) at stage pT1 with concomitant carcinoma in situ (Cis) who will benefit from an early cystectomy. Methods: We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues of patients with NMIBC. Messenger ribonucleic acid (mRNA) expression of progesterone receptor (PGR), estrogen receptor (ESR1), ERBB2, and marker of proliferation Ki-67 (MKI67) was measured by single-step reverse transcription quantitative real-time polymerase chain reaction using RNA-specific TaqMan assays. Relative gene expression was determined by the normalization of 2 reference genes (CALM2, B2M) using the 40 ΔΔCT method and relative gene expression was correlated to the histopathological stage and oncological outcome. Results: Of 302 patients with pT1 NMIBC in the initial transurethral resection of the bladder, 65 had a concomitant Cis. Elevated ERBB2 expression (>40.1) significantly correlated with progress in patients with and without concomitant Cis (p = 0.020 and p = 0.049, respectively). For the subgroup of pT1 with concomitant Cis, elevated ERBB2 expression significantly discriminated between a high-risk group of 55% progression-free survival (PFS) and a low-risk group of 90% PFS after a 5-year follow-up (p = 0.020). Cox-regression analysis revealed ERBB2 expression as the only independent prognostic factor for PFS (p = 0.0037). Conclusions: High mRNA expression of ERBB2 can identify patients with pT1 NMIBC with concomitant Cis, who have a high risk of progression and might benefit from an early cystectomy.

Background: Stratification of molecular subtypes is of outmost importance for clinical decision making according to the 12th St Gallen Consensus conference (1). The molecular subtypes were originally identified by genome-wide mRNA analysis of fresh tissue specimen (2). Conventional immunehistochemistry assessment using ER, PR, HER2 and Ki67 approximate these subtypes with ∼ 80% concordance (3). Here we have tested a predefined Single-Sample-Predictor for molecular subtyping based on mRNA assessment of ESR1, PGR, HER2, Ki67 and RACGAP1 in a prospective-retrospective study design. Materials and Methods: Pretreatment core cut biopsies from n=100 patients with PBC treated within a randomized phase II trial (1) of anthracylin/taxane based NAC were examined. Immunohistochemistry was performed for the Ki67 antigen on an automated IHC platform (Dako Techmate 500). Ki-67 were assessed either by visual scoring (vIHC) or by quantitative image analysis (qIHC). RNA from formalin-fixed, paraffin embedded (FFPE) routine biopsies was extracted using a bead-based extraction method (STRATIFYER XTRAKT kits). Fresh tissue samokes were prepared by using Qiagen kits. ESR1, PGR, HER2, Ki-67 and RACGAP1 as well as CALM2 as a house keeping gene were measured via a multiplex quantitative RT-PCR (qPCR). Stratification into molecular subtypes was done by stepwise analysis of singular genes using predefined cut-off values. Kaplan-Meier survival estimates, patitioning test and correlation analyses were performed using the SAS JMP® 9.0.0 software. Results: Concordance between RNA based and IHC-based subtyping into Luminal A, Luminal B, HER2 positive and Triple Negative breast cancer by using ESR1, PGR, HER2, Ki-67 and RACGAP1 was at 85%. mRNA based analysis of fixed tumor specimen performed equally well as fresh tissue analysis. The rate of pathological complete Remission for the predefined molecular subtypes has been for Luminal A 0%, Luminal B 19%, HER2 positive 21% (without Trastuzumab) und Triple Negative Tumors 22%. qPCR from fresh tissue specimen resulted in almost identical classifications. The 5-year overall survival rate was significantly different between subtypes as expected: Luminal A 100%, Luminal B 85%, HER2 positiv 80% und triple negativ 60%. Luminal tumors predominantly metastasized into the bones. Cancer related deaths within the first three years were restricted tot he Triple negative Tumortype. Conclusion: The Single-Sample-Predictor based on assessment of only five mRNA Markers (ESR1, PGR, HER2, Ki-67 and RACGAP1) reliably stratified into molecular subtypes by using either fixed or fresh pretreatment core needle biopsies. The mRNA based assessment enables objective and highly reproducible subtyping for clinical routine diagnostics. 1) Goldhirsch et al., Annals of Oncology 2011 2) Perou et al., Nature 2000 3) Sotiriou&Pusztai, NEJM 2009 4) Schneeweiss et al, Ann Oncol 2011 Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-19.

Background: Presence of immune cell infiltrates in tumor may influence outcome of patients with node negative breast cancer (BC) (Schmidt et al 2008). High expression of CXCL13 (B lymphocyte chemoattractant chemokine) was strongly associated with favorable survival in one recent series consisting of BC patients treated with adjuvant chemotherapy (Razis et al. 2012). Here we examined the clinical significance of breast tumor CXCL13 mRNA levels within the context of the FinHer trial, where patients were randomly assigned to receive 3 cycles of either docetaxel or vinorelbine followed by 3 cycles of FEC. Patients with HER2-positive BC had a second randomization for trastuzumab vs. control. Since intratumoral B cells are of particular importance for obtaining response to chemotherapy in triple negative BC (TNBC) and of limited value in luminal BC (Schmidt et al. 2012), and trastuzumab treatment is a confounding factor in HER2+ BC, we focused on TNBC. Methods: RNA was extracted from FFPE tumor tissue of 917 (90.8%) out of the 1010 patients who participated in the FinHer trial. CXCL13 mRNA expression was measured using RT-qPCR. The molecular subtypes (luminal, HER2-enriched and triple-negative) were determined using IHC and central chromogenic in situ hybridization (CISH) testing. Prognostic significance of factors was assessed using univariate and multivariate analyses. Distant disease-free survival (DDFS) between groups was compared using the log-rank test. Results: Tumor CXCL13 mRNA expression showed a bimodal distribution and correlated negatively with tumor ESR1 mRNA levels (r = −0,31; p < 0.0001). 146 (15.9%) out of the 917cancers were classified as TNBC. High tumor CXCL13 mRNA levels above the median were strongly associated with favorable 5-yr DDFS in TNBC compared to low levels (85% vs. 60%; p < 0.0001). The prognostic value of tumor CXCL13 was evident in the subset of women with TNBC treated with vinorelbine (5-year DDFS 90% vs. 40%; p < 0.0001), whereas no significant association was found in the docetaxel arm (5-year DDFS 80% vs. 80%; p = 0.76). When only patients with TNBC and low CXCL13 mRNA levels were assessed, patients treated with docetaxel had better DDFS compared to those treated with vinorelbine (5-year DDFS 80% versus 40%; p < 0.0064). There was no DDFS difference between treatment arms in patients exhibiting high CXCL13 expression. Conclusions: Tumor CXCL13 mRNA expression is a favorable prognostic factor for women with TNBC treated with adjuvant chemotherapy providing further evidence that the host immune response may influence outcome of patients with early BC. The clinical significance of the host B cell immune response may vary substantially pending on the type of chemotherapy administered. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-06.

Background: Pathologic complete response (pCR, ypT0/is) is one of the strongest prognostic factors for primary breast cancer (PBC) in the setting of neoadjuvant chemotherapy (NAC). Factors predictive for response to NAC can help to stratify for an individualized therapy. As chemotherapeutic agents are most active in highly proliferative tumors, it was the aim of the present study to evaluate the potential of Ki-67 as a single proliferation marker, quantitatively measured on the mRNA or protein level, to predict response of PBC to NAC. Material and methods: Pretreatment core cut biopsies from n=72 patients with PBC treated within a randomized phase II trial (1) of anthracylin/taxane based NAC were examined. Immunohistochemistry was performed for the Ki67 antigen on an automated IHC platform (Dako Techmate 500). Ki-67 were assessed either by visual scoring (vIHC) or by quantitative image analysis (qIHC). For quantitative IHC analysis tumor proliferation was analyzed after tumor stroma segmentation using the Aperio Image Analysis toolbox. RNA was extracted from formalin-fixed, paraffin embedded (FFPE) routine biopsies using a bead-based extraction method (STRATIFYER XTRAKT kits). Ki-67, TOP2A and RACGAP1 as well as CALM2 as a house keeping gene were measured via a multiplex quantitative RT-PCR (qPCR). Kaplan-Meier survival estimates, patitioning test and correlation analyses were performed using the SAS JMP® 9.0.0 software. Results: There was only a moderate correlation between Ki-67 mRNA or Ki-67 measured by immunohistochemistry (IHC) and histologic grade (Spearman r = 0.52 p < 0.0001; r = 0.23 p = 0.033, respectively). Conventional visual scoring and qIHC correlated well (r = 0,78 p < 0,0001), while qPCR and qIHC correlated moderately (r = 0,50 p < 0,0001). For Ki-67 determined by visual scoring of standard IHC there was an optimal discrimination at a cut-off of 20% stained nuclei with regard to chemotherapy response. Yet, the difference between pCR-rates for high and low Ki-67 on IHC was non-significant, with 16.3% and 12.5% pCRs in the IHC Ki-67 high and low group, respectively. For Ki-67 IHC analysed by quantitative image analysis there was optimal cut-off at 35% stained nuclei resulting in 37,5% versus 8,7% pCR rate and 54% of pCRs classified correctly. For qPCR determination, high Ki-67 mRNA expression was associated with a high pCR-rate of 36.4% as opposed to 5.8% in tumors with low Ki-67 mRNA levels and 82% classified correctly. Conclusion: High Ki-67 mRNA expression measured by RT-qPCR was predictive for the achievement of pCR to NAC and in this respect was superior to Ki-67 determined by vIHC or qIHC. This is consistent with data on Ki-67 IHC as a predictor of pCR on larger neoadjuvant cohorts (1). RT-qPCR based measurements of Ki-67 mRNA ensures an objective and highly reproducible quantification of proliferative activity from FFPE tissue from routine core cut biopsies. As such it seems to be more robust and meaningful compared to protein-based determination of Ki-67 by IHC, both by visual scoring or quantitative image analysis. 1) Schneeweiss et al, Ann Oncol 2011 2) Fasching et al., BMC Cancer 2011 Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-08.

Background: Molecular subtyping of breast cancer has become an integral part of standard evaluation of breast cancer patients. Their assessment requires combining data from analyses on ER, PR, HER2 and cell proliferation markers. However, their immunohistochemical (IHC) testing carries an up to 20% risk of erroneous results. Similarly, assessment of cell proliferation by Ki-67 staining is hampered by lack of standardization of laboratory methods and agreement on cut-offs. Here we tested the prognostic value of objective quantitation of ESR1, PgR, HER2 and the proliferation markers RACGAP1 using RT-qPCR and compared the results with local and central IHC assessments. Methods: RNA was extracted from FFPE tumor tissue of 917 patients who participated in the FinHer trial. ESR1, PgR, HER2 and RACGAP1 mRNA expression were measured using RT-qPCR. The molecular subtypes (luminal, HER2−enriched and triple-negative) were determined. Prognostic significance of proliferation markers was assessed using univariate and multivariate analyses. The RT-qPCR results were compared with local and central IHC results. Results: HER2 mRNA showed a bimodal distribution with 197 (21.4%) out of the 917 tumors being above the predefined cut-off. HER2 mRNA expression increased in parallel with HER protein expression. Overall concordance of HER2 mRNA testing with central IHC and CISH was good, while local IHC testing suffered higher false positive rates. RACGAP1 mRNA expression was the greater the higher the histological grade. ESR1 and PgR mRNA correlated negatively with the histological grade (r=-0.38 and r=-0.33; p Conclusions: Molecular subtyping of breast cancer by RT-qPCR using RNA isolated from FFPE tissue proved successfully in this large patient cohort. RACGAP1 mRNA expression distinguished high and low risk luminal breast cancers. In a multivariate analysis mRNA-based molecular markers outperformed the immunohistochemical markers ER, PgR and Ki-67. Of note, quantitative assessment of the proliferation marker RACGAP1 was superior to semiquantitative assessment of Ki-67 from routine FFPE tissues using IHC. We conclude that quantitative assessment of ESR1, PgR, HER2 and RACGAP1 mRNA by RT-qPCR is a robust and reproducible method to assess these key tumor biological factors from archival FFPE tumor tissue. RACGAP1 is novel cell proliferation marker in breast cancer that warrants further validation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-04.

Die Autoren berichten uber eine 83-jahrige Frau, die 6 Wochen vor stationarer Aufnahme uber plotzlich einsetzende Ruckenschmerzen klagte. Die Rontgenubersichtsaufnahme der Lendenwirbelsaule zeigte eine Wirbelkorperdeformitat. Die Magnetresonanztomographie erbrachte den Befund eines diffusen Odems des Knochenmarks des betroffenen Wirbelkorpers. In der weiteren Labordiagnostik ergab sich kein pathologischer Befund. Bei fortgesetzter Schmerzsymptomatik wurde die Indikation zu einer Vertebroplastie gestellt. Im Verlauf dieser Prozedur wurde eine Knochenbiopsie entnommen, und das Biopsat zeigte eine diffuse Infiltration des Markraums durch ein myeloisches Sarkom (Chlorom), eine Entitat, die als Erstmanifestation einer akuten myeloischen Leukamie vorausgehen kann. Dieser Fall unterstreicht die Bedeutung einer sorgfaltigen Abklarung bei Patienten mit fortgesetzter Schmerzsymptomatik bei einer vermeintlich osteoporotischen Wirbelkorperdeformitat. Das myeloische Sarkom stellt dabei eine sehr seltene Differentialdiagnose einer Wirbelkorperdeformitat beim alteren Menschen dar.

Background. No predictive or prognostic biomarker is available for patients with locally advanced rectal cancer (LARC) undergoing perioperative chemoradiotherapy (CRT). Members of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases EGFR (HER1,ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) are therapeutic targets in several cancers. The analysis was performed to assess expression levels and study the potential prognostic impact for disease-free and overall survival in patients with LARC.Patients and Methods.ERBB1–4mRNA expression and tumor proliferation using Ki-67 (MKI67) mRNA were evaluated using RT-quantitative PCR in paraffin-embedded tumor samples from 86 patients (median age: 63) treated with capecitabine or 5-fluorouracil-based CRT within a phase 3 clinical trial.Results. A positive correlation of HER4 and HER2, HER3 and HER2, and HER4 and HER3 with each other was observed. Patients with high mRNA expression ofERBB1(EGFR, HER1) had significantly increased risk for recurrence and death. Patients with high mRNA expression ofMKI67had reduced risk for relapse.Conclusion. This analysis suggests a prognostic impact of bothERBB1and MKi67 mRNA expression in LARC patients treated with capecitabine or fluorouracil-based chemoradiotherapy.

Background MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. Methods Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. Results Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and 0.20 Cqs accompanied by >94 % concordant single marker assignments for all four markers. In platform 2, the inter-site SD estimates were between 0.40 and 0.66 Cqs while the concordance for single marker assignments was >94 % for all four markers. The agreement reached between the two qPCR systems located in one site was 100 % for ERBB2, 96.9 % for ESR1, 97.2 % for PGR and 98.6 % for MKI67. RT-qPCR for individual markers was stable up to a 64-fold dilution for a typical clinical sample. There was no change in assay performance detected at the level of individual markers or subtypes after using different RNA isolation methods. The presence of up to 80 % of surrounding non-tumor tissue including in situ carcinoma did not affect the assay output. Sixteen out of 20 RNXtract eluates yielded more than 50 ng/μl of RNA (average RNA output: 233 ng/μl), whereas DNA contamination per sample was restricted to less than 15 ng/μl. Median recovery rate of RNA extraction was 91.0 %. Conclusions In this study the performance characteristics of MammaTyper were successfully validated. The various sources of analytical perturbations resulted in negligible variations in individual marker assessments. Therefore, MammaTyper may serve as a technical improvement to current standards for decentralized FFPE-based routine assessment of the commonly used breast cancer biomarkers and for molecular subtyping of breast cancer specimens. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2476-x) contains supplementary material, which is available to authorized users.

Hintergrund: Aufgrund ihrer Seltenheit sind bei primaren malignen Lymphomen der Leber bislang keine standardisierten therapeutischen Konzepte entwickelt worden. Fallbericht: Wir berichten uber den Fall eines diffusen groszelligen B-Zell-Lymphoms der Leber bei einer 48-jahrigen Frau. Es handelt sich um eine solitaren Tumor, dessen maximaler Durchmesser 12,5 cm betrug. Nach neoadjuvanter Chemotherapie konnte das Lymphom vollstandig reseziert werden. Histologisch zeigte sich eine hochgradige therapieinduzierte Tumorregression. Eine Infiltration regionarer Lymphknoten oder anderer Organe war im Rahmen des Stagings nicht erkennbar (Stadium I EA). Nach sechs Zyklen einer adjuvanten Chemotherapie ist die Patientin seit mehr als 5 Jahren beschwerdefrei, ohne dass sich Hinweise auf ein Rezidiv des Leberlymphoms ergeben haben. Schlussfolgerung: Der vorliegende Fall dokumentiert den Effekt einer systemischen Chemotherapie. Daher wird sie in vielen Zentren als alleinige Therapie eingesetzt. Die neoadjuvante Chemotherapie stellt jedoch moglicherweise eine wichtige Erganzung der therapeutischen Strategien bei unilokularem primarem Leberlymphom dar.

$('.header-date').hide();$('#titleAuthors').hide(); $('#abstractHeader').hide(); Alexander Shimabukuro-Vornhagen 1 , 2 , * , Hans A. Schloser 1 , 3 , * , Luise Gryschok 1 , Joke Malcher 1 , Kerstin Wennhold 1 , Maria Garcia-Marquez 1 , Till Herbold 1 , 3 , Laura S. Neuhaus 1 , Hans J. Becker 1 , Anne Fiedler 1 , Pascal Scherwitz 4 , Thomas Koslowsky 5 , Roland Hake 6 , Dirk L. Stippel 3 , Arnulf H. Holscher 3 , Sebastian Eidt 6 , Michael Hallek 2 , Sebastian Theurich 1 , 2 , Michael S. von Bergwelt-Baildon 1 , 2 1 Cologne Interventional Immunology, University of Cologne, Germany. 2 Department I of Internal Medicine, University of Cologne, Cologne, Germany. 3 Department of General, Visceral and Cancer Surgery, University of Cologne, Germany. 4 Department of Surgery, Marien Hospital, Bruhl, Germany. 5 Department of Surgery, St. Elisabeth Hospital, Cologne, Germany. 6 Institute of Pathology, St. Elisabeth Hospital, Cologne, Germany. * This authors contributed equally to this work. Correspondence to: Michael S. von Bergwelt-Baildon, e-mail: michael.von-bergwelt-baildon@uk-koeln.de Received: December 14, 2013 Accepted: April 28, 2014 Published: May 09, 2014 ABSTRACT Purpose: A precise understanding of the mechanisms by which human immune cell subsets affect tumor biology will be critical for successful treatment of cancer using immunotherapeutic approaches. Recent evidence suggests that B cells can both promote and inhibit the development and progression of tumors. The aim of this study was to characterize the composition of the B-cell infiltrates in colorectal cancers (CRC) in order to gain further insight into the role of B cells in CRC. Experimental Design: In this study we characterized B-cell subsets in primary tumors (n=38), metastases (n=6) and blood (n=46) of 51 patients with a diagnosis of CRC and blood of 10 healthy controls. B-cell subsets were analyzed by flow cytometry or immunohistochemistry. Results: Peripheral blood of CRC patients contained a higher percentage of memory B cells than that of age-matched healthy controls. Furthermore, the percentage of B cells within tumors was higher than that in the peripheral blood of CRC patients while metastases were typically devoid of tumor-infiltrating B cells. Tumor-associated B cells were enriched for activated and terminally differentiated B cells. Relevant proportions of regulatory B cells could only be detected in advanced cancer and metastases. Conclusion: B cells constitute a significant proportion of the immune infiltrate in CRC. The B-cell infiltrate of primary CRC is characterized by an accumulation of terminally differentiated memory B cells or plasma cells suggestive of a specific immune response against the tumor. However advanced tumors and metastases are also infiltrated by a considerable number of regulatory B cells.

For evaluation of the prognostic relevance of p53 expression in gastric cancer, the immunohistochemical tissue status of 133 primary gastric cancer patients was investigated for p53 expression and the association between p53 tissue status and clinicopathological parameters was analyzed. P53 immunoreactivity was detected in the nuclei of cancer cells in 35 cases (26.3%). The nuclear p53 immunoreaction was closely associated with tumor location, lymph node metastasis, and curability. Tumors with positive p53 stain reactions frequently metastasized to lymph nodes (metastatic rate: 91.4%) in contrast to tumors with negative p53 stain reactivity (71.4%, P = 0.021). Immunohistochemical analysis of primary gastric cancer appears to be an accurate and simple method of screening for p53 expression. In combination with common prognostic parameters, determination of p53 tissue status might help to detect prognostically unfavorable subgroups of gastric cancer patients.

Low-grade gastric lymphomas of mucosa-associated lymphoid tissue (MALT) may be extremely difficult to differentiate from atypical lymphoid infiltrations of the gastric mucosa caused by Helicobacter pylori (H. pylori) infection, especially if lympho-epithelial destructions are not detected. In order to clarify this diagnostic problem and to evaluate the potential benefit of clearing H. pylori-induced lymphoid infiltration a prospective study was performed. Our study included biopsy specimens from 61 patients, that showed excessive lymphoid infiltrations. Using histology and immunohistochemistry no definite diagnosis could be delivered. In 46 (76.4%) cases histological regression occurred 4 weeks after the end of therapy suggesting a reactive infiltrate. In 7 patients (11.5%) the histological picture remained unchanged, and in 8 (13.1%) the follow-up biopsies revealed lympho-epithelial destructions allowing the diagnosis of low-grade MALT lymphoma. On the basis of our data we conclude that eradication of H. pylori could provide an additional tool in the differential diagnosis between atypical lymphoid infiltrations of the gastric mucosa caused by H. pylori and an early primary gastric MALT lymphoma.

e12045Background: Ultrahigh ESR1 mRNA expression in luminal breast tumors is predictive for benefit from adjuvant tamoxifen compared to placebo treatment in NSABP B14, while protein determination b...

Zielsetzung: Die pathologisch komplette Remission nach NACT ist von groser Bedeutung fur das Uberleben von Mammakarzinompatientinnen mit hormonrezeptornegativen Tumoren. Da Chemotherapeutika insbesondere in zellteilungsaktiven Tumoren wirksam sind, sollte untersucht werden inwieweit die quantitative Einzelgenmessung des Proliferationsmarker Ki67 auf mRNA Basis das Ansprechen von invasiven Mammakarzinomen vorhersagen kann. Material und Methoden: Pratherapeutische Stanzbiopsien von neoadjuvant behandelten Tumoren aus einer Phase II Studie wurden untersucht (n=100). Ki67 mRNA wurde nach Extraktion aus formalin fixierten Gewebeproben mittels RT-qPCR gemessen. Ki67 Immunhistochemie (IHC) wurde sowohl konventionell semiquantitativ als auch quantitativ mittels automatisierter Bildanalyse (Aperio Technologies) ausgewertet. Korrelationsanalyen, Partitionstests, Kaplan-Meier Uberlebensanalysen wurden mittels der JMP®9.0.0 Software von SAS durchgefuhrt. Ergebnisse: Die Ki67 mRNA korrelierte moderat mit der immunhistochemischen Bestimmung von Ki67 und dem histologischen Grade (Spearman r=0,52p

The prevalence of antral intestinal metaplasia (IM) in underlying Helicobacter pylori (HP) gastritis was studied in biopsy specimens of the antral mucosa obtained from 2,692 patients with histologically proven HP gastritis. The prevalence of IM varied in the groups investigated: 47.4% in gastric ulcers, 30.5% in pyloric ulcers, 16.8% in duodenal ulcers, 21.8% in gastritis with no lesions and 9.8% in chronic antral erosions (p

The female breast rarely constitutes the primary localization for non-Hodgkin’s lymphoma (NHL). The incidence of primary breast lymphoma (PBL) lies between 0.04 and 1.1% for all breast tumors and 1.7–2.2% for all extranodal NHL. Mostly it occurs during the child-bearing period, during pregnancy or lactation. In general, children between the ages of 8 and 10 years are most frequently affected by Burkitt’s lymphoma. Methods for classification, detection and especially treatment of this condition continue being a subject of discussions and research. We present and evaluate the rare case of a 12-year-old girl with bilateral gigantic breast tumors treated during a surgical mission with “Doctors without borders” (MSF, “medecins sans frontiers”) in the zone of civil war in the Democratic Republic of Congo. The monstrous bilateral breast hypertrophy, symmetrical, rapidly growing, consequently ulcerating and severely bleeding had to be treated by bilateral mastectomy as a salvage procedure. Examinations through histopathology was not feasible and other facilities like ultrasound, CT, MRI and further laboratory examinations were not available.. The patient died 27 days after the surgical procedure. The histological result received by later examination in Germany showed a bilateral high malignant B cell lymphoma (Burkitt lymphoma) of the breast.

The authors report on an 83-year-old woman who presented with back pain with sudden onset 6 weeks prior to admission. A radiograph of the lumbar spine revealed a vertebral deformity. An MRI (magnetic resonance imaging) scan showed bone marrow edema of the affected vertebra. Blood counts on laboratory tests were normal at the time of admission. Due to continuing pain vertebroplasty was performed, and bone biopsies were taken during the procedure. The biopsies showed infiltration of the marrow space with granulocytic sarcoma (chloroma), a tumor, which may precede the onset of acute myelogenous leukemia.This case underlines the need for a thorough diagnostic work-up in patients with ongoing complaints of an assumed osteoporotic vertebral fracture. Granulocytic sarcoma represents a rare differential diagnosis of vertebral deformity.

Background: Subtyping of breast cancer has become an integral part of standard evaluation of breast cancer patients1. However, assaying of ER, PgR and Her-2/neu by immunohistochemistry (IHC) carries an up to 20% risk of erroneous results2,3. Moreover, reliable assessment of Ki-67 by IHC in grade 2 breast cancer is challenging due to high intra- and interobserver variations4,5. Interobserver concordance for ESR1, PGR, HER2, and KI67 determination on mRNA level using MammaTyper® reagents were 96.8%, 97.2%, 100%, 97.6%, respectively, based on predefined cutoffs (Laible et al., abstract submitted). Here we tested the clinical concordance and prognostic value of MammaTyper® determinations in the FinHer trial patient population. Methods: RNA was extracted from formalin-fixed paraffin-embedded (FFPE) breast tumor tissue, and candidate gene expression was analyzed using the RNXtract® IVD and MammaTyper® IVD kits (BioNTech Diagnostics GmbH, Mainz) from 791 patients who participated in the FinHer trial. RNA levels of ESR1, PGR, HER2, and KI67 mRNA expression were measured using RT-qPCR,normalized according to the 40-DDCT method and compared with IHC or CISH results. Associations with distant disease-free survival (DDFS) and overall survival (OS) were assessed using the log-rank test. Results: ESR1, PGR, HER2, and KI67 mRNA levels exhibited strong correlations with the respective clinical assays (each p-value 95%). Conclusions: Determination of ESR1, PGR, HER2, and KI67 without macrodissection of routine whole tissue FFPE specimens results in highly concordant results when compared to clinical assays. A significant minority of HER2 negative breast cancers expressed PGR mRNA despite low ESR1 mRNA levels and had superior outcome. HER2 mRNA levels differed substantially between ER-positive and ER negative tumors. This may explain why HER2 determination using a single cut-off for HER2 mRNA with the Oncotype DX assay frequently results in a false negative finding6. The MammaTyper-defined ESR1, PGR, HER2, and KI67 expression showed strong correlations with the corresponding clinical assays and survival. 1) Goldhirsch et al., Annals of Oncology 2013 2) Hammond et al., JCO 2010 3) Wolff et al., JCO 2007 4) Varga et al., PloS 2012 5) Polley et al., JNCI 2013. Citation Format: Ralph M Wirtz, Pirkko-Liisa Kellokumpu-Lehtinen, Jorma Isola, Vesa Kataja, Petri Bono, Taina Turpeenniemi-Hujanen, Sirkuu Jyrkkiö, Harri Sitho, Sebastian Eidt, Ugur Sahin, Heikki Joensuu. Comparison of ESR1, PGR, HER2 and KI67 expression by central IHC and MammaTyper® RT-qPCR kit in the FinHer trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-02-01.

Since primary malignant lymphomas of the liver represent a rare entity, no commonly accepted therapeutic strategy has been developed so far.We report the case of a diffuse large-cell B-cell lymphoma of the liver in a 48-year-old female patient. The tumor presented as a solitary mass measuring 12.5 cm in maximum diameter. After neoadjuvant chemotherapy, the lymphoma was completely resected (R0). A massive therapy-induced tumor regression was found histologically. No involvement of regional lymph nodes or other organs was detected during staging procedures (stage I EA). After six courses of adjuvant chemotherapy, the patient has been alive and well for more than 5 years and shows no evidence of tumor relapse.This case documents the effect of systemic chemotherapy on lymphoma cells. In many centers systemic chemotherapy is used as the only therapeutic regimen. Neoadjuvant chemotherapy might, however, represent an important addition to the therapeutic strategies concerning unilocular primary hepatic lymphomas.

Summary Infection with Helicobacter pylori causes chronic active gastritis and has been associated with gastric and duodenal ulcer disease. In biopsy samples of 110 patients with clinical symptoms of active gastritis, H. pylori was detected by means of the polymerase chain reaction (PCR), using species-specific primers defining a 858 by DNA fragment of H. pylori urease βsubunit. Sensitivity and specificity of the PCR was compared with culture, histology and Warthin-Starry stain (WSs), detection of H. pylori urease antibodies in serum and urease testing with the Campylobacter-like organism (CLO) test. PCR yielded specific amplification products in 53 cases, whereas culture of the organisms was positive in a subset of 50 cases. Only direct detection in histological sections of biopsy specimens had a higher sensitivity, with 65 positive samples. In contrast, the CLO test was negative in eleven culture-positive and PCR-positive cases. Significant urease antibody titres were found in 39 patients with histologically confirmed diagnosis. These results placed the sensitivity of PCR between that of the Warthin-Starry stain (WSs) and that of culture. Therefore, PCR can be proposed as a useful rapid and time-saving technique for the detection of H. pylori in gastritis. For epidemiological purposes, fingerprinting with arbitrarily chosen primers by AP-PCR was evaluated. Strain-specific patterns with up to 13 fragments were achieved with 10nucleotide or longer primers (21-nt) with a G+C content ≥ 55%. Thirty-five of 40 strains investigated by this method were distinguishable with a single primer. These results suggest a high level of DNA sequence diversity within this species with the possibility of confirming the clonality in consecutive isolates from a single individual. Alternatively, an increased in-vivo mutation rate could be responsible for DNA divergence, resulting in specific strains for each individual patient.

Aims: Hormone receptors are the prototype predictive marker in breast and prostate cancer. Hormone receptor positive cancers have a better prognosis, increased tropism to metastasize into the bones and respond to endocrine treatment options. The prognostic value of hormone receptor in urothelial carcinoma of the bladder (UCB) is less established. This may in part result from technical limitations of immunhistochemical detection methods. Interestingly, female gender has recently been identified as strong adverse factor in advanced UCB (May et al., 2011). By analyzing whole genome expression data from non-muscle invasive bladder cancer patients, we have evaluated the potential of top candidate genes (ESR1, PGR, AR, CYP19, HER2, RACGAP1) commonly used to stratify breast cancer patients to predict bladder cancer progression. In view of the gender specific effects, we have focused on the prognostic role of androgen receptor expression on tumor invasion, disease progression and survival. Methods: Affymetrix microarray data from 41 non-metastatic bladder cancer patients undergoing curative surgery were analyzed. Prognostic value of androgen receptor mRNA expression was analyzed by unsupervised Cluster analysis, partitioning tests, Mann Whitney tests and Kaplan Meier estimates of cancer specific survival. Results: Cluster analysis in the microarray date of the superficial UCB cohort identified a hormone receptor positive subtype and a proliferation dominated subtype of equal size. Androgen receptor expression was negatively associated with cancer specific death (r=−0,42; p=0,005), while proliferation correlated with increased risk of cancer specific death (r=0,46; p=0,003). In addition, low androgen receptor expression was associated with higher tumor stage (pTa vs pT1-4; p=0,017). In Kaplan Meier analysis, the cancer specific survival was significantly better in tumors exhibiting high androgen receptor levels (80% vs. 20 %; p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3654. doi:1538-7445.AM2012-3654