Christine Böger

Her2/neu testing in gastric cancer: evaluating the risk of sampling errors

Background We evaluated the risk of sampling errors in specimens of biopsy size, which may be caused by heterogeneous overexpression of Her2/neu in gastric cancer (GC). Patients and methods The study cohort comprised 454 gastrectomy patients with adenocarcinoma of the stomach or esophago-gastric junction. Tissue micro-arrays (TMAs) served as ‘biopsy procedure’ and were generated from formalin-fixed and paraffin-embedded tissue: five tissue cylinders were collected randomly from each tumor, rendering 2230 core cylinders. These were compared with 454 whole tissue sections obtained from the same paraffin blocks. Her2/neu expression and gene amplification were analyzed by immunohistochemistry and in situ hybridization. The Her2/neu status was determined according to GC scoring system by two independent observers. Results In whole tissue sections, 37 (8.1%; observer 1) and 38 (8.4%; observer 2) of the GCs, and in the corresponding TMAs, 28 (6.3%; observer 1) and 28 (6.3%; observer 2) of the GCs were classified as Her2/neu-positive (kappa value 98.5% and 96.2%; P < 0001). Comparison of whole tissue sections with corresponding TMAs showed a false-negative rate of 24% and a false-positive rate of 3% for TMAs. Conclusion Assessment of the Her2/neu status in tissue biopsies carries a significant risk of sampling errors, thereby rendering patients unsuitable for treatment with trastuzumab.

The Spatial Distribution of LGR5+ Cells Correlates With Gastric Cancer Progression

In this study we tested the prevalence, histoanatomical distribution and tumour biological significance of the Wnt target protein and cancer stem cell marker LGR5 in tumours of the human gastrointestinal tract. Differential expression of LGR5 was studied on transcriptional (real-time polymerase chain reaction) and translational level (immunohistochemistry) in malignant and corresponding non-malignant tissues of 127 patients comprising six different primary tumour sites, i.e. oesophagus, stomach, liver, pancreas, colon and rectum. The clinico-pathological significance of LGR5 expression was studied in 100 patients with gastric carcinoma (GC). Non-neoplastic tissue usually harboured only very few scattered LGR5+ cells. The corresponding carcinomas of the oesophagus, stomach, liver, pancreas, colon and rectum showed significantly more LGR5+ cells as well as significantly higher levels of LGR5-mRNA compared with the corresponding non-neoplastic tissue. Double staining experiments revealed a coexpression of LGR5 with the putative stem cell markers CD44, Musashi-1 and ADAM17. Next we tested the hypothesis that the sequential changes of gastric carcinogenesis, i.e. chronic atrophic gastritis, intestinal metaplasia and invasive carcinoma, are associated with a reallocation of the LGR5+ cells. Interestingly, the spatial distribution of LGR5 changed: in non-neoplastic stomach mucosa, LGR5+ cells were found predominantly in the mucous neck region; in intestinal metaplasia LGR5+ cells were localized at the crypt base, and in GC LGR5+ cells were present at the luminal surface, the tumour centre and the invasion front. The expression of LGR5 in the tumour centre and invasion front of GC correlated significantly with the local tumour growth (T-category) and the nodal spread (N-category). Furthermore, patients with LGR5+ GCs had a shorter median survival (28.0±8.6 months) than patients with LGR5− GCs (54.5±6.3 months). Our results show that LGR5 is differentially expressed in gastrointestinal cancers and that the spatial histoanatomical distribution of LGR5+ cells has to be considered when their tumour biological significance is sought.

PD-L1 is an independent prognostic predictor in gastric cancer of Western patients

Targeting the PD-1/PD-L1 immune checkpoint signaling is a novel promising treatment strategy in several tumor entities, and it is suggested that PD-L1/PD-1 expression is predictive for a PD-1/PD-L1 checkpoint inhibitor treatment response. We investigated the expression of PD-L1 and PD-1 by immunohistochemistry in a large and well characterized gastric cancer (GC) cohort of Caucasian patients, consisting of 465 GC samples and 15 corresponding liver metastases. Staining results were correlated with clinico-pathological characteristics and survival. PD-L1 expression was found in tumor cells of 140 GCs (30.1%) and 9 liver metastases (60%) respectively in immune cells of 411 GCs (88.4%) and 11 liver metastases (73.3%). PD-1 was expressed in tumor infiltrating lymphocytes in 250 GCs (53.8%) and in 11 liver metastases (73.3%). PD-L1 expression was significantly more prevalent in men, GCs of the proximal stomach, unclassified, papillary, Her2/neu-positive, Epstein-Barr-virus-positive, microsatellite instable, and PIK3CA-mutated GCs. A high PD-L1/PD-1 expression was associated with a significantly better patient outcome, and PD-L1 turned out to be an independent survival prognosticator. The correlation of PD-L1/PD-1 expression with distinct clinico-pathological patient characteristics may serve as a surrogate marker of PD-L1-positive GCs and may direct the use of immune checkpoint treatment strategies.

Nuclear Death Receptor TRAIL-R2 Inhibits Maturation of Let-7 and Promotes Proliferation of Pancreatic and Other Tumor Cells

BACKGROUND & AIMS Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

Prognostic and putative predictive biomarkers of gastric cancer for personalized medicine.

We investigated various phenotypic and genotypic biomarkers of gastric cancer (GC) testing the following hypotheses: are these biomarkers suitable for the identification of GC subtypes, are they of prognostic significance, and should any of these biomarkers be considered to tailor patient treatment in the future. The study cohort consisted of 482 patients. pTNM-stage was based on surgical pathologic examination. The Laurén and mucin phenotype was assessed. Helicobacter pylori and Epstein-Barr virus infections were documented. The following biomarkers were determined: BRAF, KRAS, NRAS, and PIK3CA genotype, microsatellite instability, mucin 1, mucin 2, mucin 5, and mucin 6, CD10, E-cadherin, &bgr;-catenin, and lysozyme. The histologic phenotype correlated with 10/13 (77%) clinicopathologic patient characteristics and 6/13 (46%) immunohistochemical/molecular biological biomarkers. Inversely, immunohistochemical biomarkers (mucin phenotype, E-cadherin, &bgr;-catenin, and lysozyme) were unsuitable for subclassification of GC. It showed too much overlap between the different subtypes. Among the genotypes, only microsatellite instability correlated with tumor type being more prevalent in intestinal and unclassified GCs. Patient survival correlated significantly with 8 (62%) clinicopathologic and 5 (36%) immunohistochemical/molecular biomarkers. Interestingly, in proximal GCs, KRAS mutation was associated with worse prognosis, as was persistent H. pylori infection in unclassified GCs. Mucin 2 (all patients, proximal GCs) and PIK3CA (exon 20; intestinal type GC) prognosticated independently patient survival. The biomarkers examined herein are unsuitable to aid histologic classification of GC. However, several of them show a correlation with either phenotype and/or prognosis and may be considered to tailor patient treatment in the future, such as KRAS, PIK3CA, MSI, and H. pylori status.

MET in gastric cancer – discarding a 10% cutoff rule

We aimed to develop a putative predictive biomarker score for future hepatocyte growth factor receptor (MET)‐targeted therapy of gastric cancer (GC).

The novel negative checkpoint regulator VISTA is expressed in gastric carcinoma and associated with PD-L1/PD-1: A future perspective for a combined gastric cancer therapy?

ABSTRACT A combined blockade of V-domain Ig suppressor of T-cell activation (VISTA) and PD-1 is a promising new cancer treatment option, which was efficient in murine tumor models and is currently tested in first phase I studies. Here, we analyzed the VISTA expression in a large and well-characterized gastric cancer (GC) cohort on 464 therapy-naive GC samples and 14 corresponding liver metastases using immunohistochemistry. Staining results were correlated with clinico-pathological characteristics, genetic alterations and survival. VISTA expression in tumor cells was detected in 41 GCs (8.8%) and 2 corresponding liver metastases (14.3%). Moreover, VISTA expression in immune cells was observed in 388 GCs (83.6%) and 6 liver metastases (42.9%). VISTA expression was associated with the Laurén phenotype, tumor localization, Epstein–Barr virus infection, KRAS- and PIK3CA-mutational status and PD-L1 expression. There was no significant correlation with patient outcome. Moreover, a change of VISTA expression in immune cells during tumor progression was observed. The co-incidence of VISTA and PD-L1 expression indicates a dual immune evasion mechanism of GC tumor cells and makes GC an interesting target for novel combined immune checkpoint inhibitor treatments.

Reproducibility of Her2/neu scoring in gastric cancer and assessment of the 10% cut-off rule

The application of Trastuzumab on gastric cancer patients is based on Her2/neu immunostaining. The testing method relies on visual estimation of both membranous staining intensity, and positive tumor ratio with respect to a 10% cutoff. We evaluated the effect of inter‐ and intraobserver variations of both factors on therapeutic decision, especially if the positive tumor ratio hovers around the 10% cutoff. Ten pathologists scored 12 Her2/neu immunohistologically stained whole sections of gastric cancer. Applying the common rules for Her2/neu testing for gastric cancer, they separately noted the strongest identifiable staining intensity and the corresponding positive tumor ratio. Scoring was done repeatedly using the microscope, plain virtual microscopy, and virtual microscopy with a manual outline drawing function. Agreements on the strongest identified staining intensities were moderate. Overall concordance correlation coefficients of positive tumor ratios ranged from 0.55 to 0.81. Reproducibility was not improved by virtual microscopy. Pathologists have a good ability to estimate ratios of clearly demarcated areas, but gradients in staining intensities hinder reproducible visual demarcation of positive tumor areas. When hovering around the 10% positive tumor ratio cutoff there is a risk of misinterpretation of the staining results. This could lead to a denial of Trastuzumab therapy. Assessment of Her2/neu expression should be carried out by experienced pathologists because they can more reproducibly rate membranous staining intensities. The low reproducibility of positive tumor ratio is inherent in the testing method and cannot be improved by virtual microscopy. Therefore, we propose to reconsider the 10% cut‐off limit.

Clinicopathologic Characteristics of Microsatellite Instable Gastric Carcinomas Revisited: Urgent Need for Standardization

Microsatellite instable gastric cancer (MSI-GC) is a specific molecular subtype of GC. We studied the phenotypes, genotypes, and clinicopathologic characteristics of MSI-GC in a white GC cohort and compared our findings with an extended literature review. The study cohort consisted of 482 patients. Specimens were available from 452 cases and were used for immunostaining (MLH1, PMS2, MSH2, MSH6) and molecular biological analyses (BAT-25, BAT-26, NR-21, NR-24, NR-27; Epstein-Barr virus in situ hybridization). Thirty-four (7.5%) GCs were MSI. Loss of MLH1 and/or PMS2 was found in 30 (88%) MSI-GC, 3 (9%) showed loss of MSH2 and/or MSH6. One (3%) MSI-GC was identified only by molecular biological testing. A single case was heterogeneous and contained microsatellite-stable and instable tumor areas. Twenty-one (62%) MSI-GCs showed unusual histologic features. MSI-GC was not found in diffuse-type or Epstein-Barr virus-positive GC. MSI-GC was significantly more prevalent in elderly patients, distal stomach, and was associated with a significantly lower number of lymph node metastases and a significantly better overall and tumor-specific survival. MSI-GC constitutes a small but relevant subgroup of GC with distinct clinicopathologic characteristics. Our literature review illustrates the shortcomings of missing standardized testing algorithms with prevalences of MSI-GC ranging from 0% to 44.5%. Future studies should test the hypothesis that patients with MSI-GCs may not need adjuvant/perioperative chemotherapy. However, this will require a standardized, quality-controlled diagnostic algorithm of MSI for GC.

Ki67—An unsuitable marker of gastric cancer prognosis unmasks intratumoral heterogeneity

Due to contradictious findings of previous studies regarding Ki67s value in gastric cancer (GC), we reevaluated the expression of Ki67 in whole tissue sections (WTS) and tissue microarrays (TMAs) of GC testing the following hypotheses: does Ki67 show intratumoral heterogeneity; are TMAs representative in the determination of the Ki67 proliferation index (PI); is the Ki67 PI subject to an intralaboratory variability; and is the Ki67 PI related to clinico‐pathological patient characteristics and/or prognostically relevant in GC.