Interpreting whole genome and exome sequencing data of individual gastric cancer samples.

BACKGROUND: Gastric cancer is the fourth most common cancer and the second leading cause of cancer death worldwide. In order to understand the genetic background, we sequenced the whole exome and the whole genome of one microsatellite stable as well as one microsatellite unstable tumor and the matched healthy tissue on two different NGS platforms. We here aimed to provide a comparative approach for individual clinical tumor sequencing and annotation using different sequencing technologies and mutation calling algorithms. RESULTS: We applied a population-based whole genome resource as a novel pathway-based filter for interpretation of genomic alterations from single nucleotide variations (SNV), indels, and large structural variations. In addition to a comparison with tumor genome database resources and a filtering approach using data from the 1000 Genomes Project, we performed pyrosequencing analysis and immunohistochemistry in a large cohort of 428 independent gastric cancer cases. CONCLUSION: We here provide an example comparing the usefulness and potential pitfalls of different technologies for a clinical interpretation of genomic sequence data of individual gastric cancer samples. Using different filtering approaches, we identified a multitude of novel potentially damaging mutations and could show a validated association between a mutation in GNAS and gastric cancer.

Progressive histoplasmosis with hemophagocytic lymphohistiocytosis and epithelioid cell granulomatosis: A case report and review of the literature.

Histoplasmosis in central Europe is a rare fungal disease with diverse clinical presentations. Apart from acute pulmonary histoplasmosis and involvement of the central nervous system, the most serious clinical presentation is progressive disseminated histoplasmosis which is generally associated with severe immunodeficiency and, in particular, advanced human immunodeficiency virus infection. Here, we report on an immunocompetent female residing in a non-endemic area, presenting with progressive disseminated histoplasmosis after a remote travel history to Thailand and Costa Rica. Diagnosis was delayed by several months due to misinterpretation of epithelioid cell granulomatosis of the intestine as Crohn's disease and of similar lung lesions as acute sarcoidosis. Prompted by clinical deterioration with signs and symptoms consistent with hemophagocytic lymphohistiocytosis, a bone marrow aspiration was performed that documented hemophagocytosis and intracellular organisms interpreted as Leishmania sp., but later identified by molecular methods as Histoplasma capsulatum. Treatment with liposomal amphotericin B followed by posaconazole led to prompt clinical improvement and ultimately cure.

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.

Integrins αvß3 and αvß5 as prognostic, diagnostic, and therapeutic targets in gastric cancer.

BACKGROUND: We investigated the expression of two αv integrins, αvß3 and αvß5, in gastric cancer (GC) by testing the following hypotheses: that these molecules are expressed in GC; that they are implicated in GC biology; that they help to distinguish between the two major histological subtypes of GC, according to Laurén; and that they are prognostically relevant. METHODS: Formalin-fixed and paraffin-embedded tissue samples from 482 GC samples were stained immunohistochemically using rabbit monoclonal antibodies directed against αvß3 (EM22703) and αvß5 (EM09902). Immunostaining of tumor, stroma, and endothelial cells was evaluated separately by the quantity and intensity, generating an immunoreactivity score. The immunoreactivity score of both antibodies was correlated with clinicopathology data and patient survival. RESULTS: Each integrin was expressed in at least one tumor component in all GCs. Both were expressed significantly more often in the intestinal phenotype according to Laurén. Moreover, patients who grouped as "positive" for expression of αvß3 on endothelial cells, and patients with an intestinal type GC, grouped as "negative" for expression of αvß5 on stroma cells, had significantly longer survival. The expression of αvß5 on stroma cells was confirmed to be an independent prognostic factor of intestinal-type GC. CONCLUSION: The expression of αvß3 and αvß5 in at least one tumor component in all GC samples is an interesting new result that should form a basis for further investigations; for example, regarding selective integrin antagonists and the value of αvß3 and αvß5 as putative prognostic biomarkers. Moreover, both markers might be helpful in the routine classification of GC subtypes.

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.

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, ß-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, ß-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.

Evaluation of two different vacuum-assisted breast biopsy systems: Mammotome(R) system 11G/8G vs. ATEC(R) system 12G/9G.

BACKGROUND: Breast cancer screening programs have been established worldwide and early detection of breast cancer has increased steadily. The most common way to confirm dignity of non-palpable and sonographically-occult suspicious findings on mammography is the stereotactically-guided vacuum-assisted breast biopsy PURPOSE: To compare two stereotactically guided vacuum-assisted breast biopsy systems measuring time effectiveness and quality of harvested material in clinical practice. MATERIAL AND METHODS: One hundred and forty-six patients presenting with suspicious microcalcifications on mammography were included in the study. Biopsies were carried out with either the Mammotome(®) system (11-gauge and 8-gauge) or the ATEC(®) system (12-gauge and 9-gauge). Lesions with a diameter <15 mm on mammography were biopsied with 11-gauge or 12-gauge devices whereas lesions >15 mm were targeted with 8-gauge and 9-gauge. Mammotome(®) system 8-gauge device was used in 34 patients, the 11-gauge system in 37 patients. The ATEC(®) system 9-gauge system was used in 37 patients and 12-gauge in 38 patients. Time was taken, focusing on preparing the system, time of collecting the samples, preparing the samples, and cleaning the site. During the biopsies 24 samples were taken. The histologic quality of the tissue samples was judged by a pathologist in a blinded fashion according to a specimen grading classification concerning tissue fragmentation, artefacts, and the adequacy of the tissue for diagnosis. RESULTS: The median overall time for the Mammotome(®) system was 879 s (11-gauge) and 934 s (8-gauge) and for the ATEC(®) system 671 s (12-gauge) and 673 s (9-gauge). The ATEC(®) system displays a significantly shorter overall time for small and large biopsy devices (U-test, P < 0.001). Concerning the mean time difference of the overall time comparing small and large systems the ATEC(®) system was 267.6 s faster using the small and 244.8 s faster using the large system. Comparing the histologic quality of tissue samples the Mammotome(®) system shows significantly higher values for the large and the small system (Chi-square test, P < 0.001). CONCLUSION: Both biopsy systems meet all requirements for daily practice and confirm the diagnosis of suspicious microcalcifications. The ATEC(®) system was observed to be faster but this difference of about 250 s might not be relevant in daily practice. The Mammotome(®) system provides a better histologic quality of tissue samples.

LGR4 and LGR6 are differentially expressed and of putative tumor biological significance in gastric carcinoma.

Gastric cancer (GC) is one of the most common causes of cancer-related deaths worldwide. We investigated the differential expression and putative tumor biological significance of five G-protein-coupled receptors (GPCRs) in GC, i.e., LGR4, LGR6, GPR34, GPR160, and GPR171. Based on our previous microarray analyses, we identified five candidate genes in human GC samples. Real-time RT-PCR was carried out to validate their expression in malignant and non-malignant tissues on an independent collective comprising 32 GC patients with and without lymph node metastases. Selected protein targets LGR4 and LGR6 were further validated on paraffin-embedded sections of ten intestinal and ten poorly cohesive (diffuse)-type GCs and their corresponding non-malignant tissue using immunohistochemistry. Additionally, the putative tumor biological significance of LGR4 and LGR6 was studied using tissue microarrays obtained from a cohort of 481 GC patients. On transcriptional level, GPR34, GPR160, and GPR171 were not differentially expressed in GC compared with non-neoplastic mucosa. LGR4 and LGR6 were up-regulated on transcriptional (real-time RT-PCR) and translational (immunohistochemistry) levels in GC. Furthermore, in tissue microarray analysis, LGR6 expression was significantly associated with local tumor growth (T-category; p = 0.04) and correlated with patient survival. LGR4 expression was significantly correlated with nodal spread (N-category; p = 0.025). Our systematic analysis indicates that LGR4 and LGR6 may play a role in GC biology. Future studies will have to demonstrate whether these are also putative diagnostic, prognostic, and/or therapeutic targets for GC.

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.

Cohort study based on the seventh edition of the TNM classification for gastric cancer: proposal of a new staging system.

PURPOSE: We investigated the effect of the new TNM classification on gastric cancer staging. PATIENTS AND METHODS: From hospital records, information from patients with gastric cancer, who had undergone either total or partial gastrectomy for adenocarcinomas of the stomach or esophagogastric junction, was retrieved. The pathologic TNM stage was determined according to the sixth and seventh editions of the International Union Against Cancer guidelines and was based on surgical pathologic examination. RESULTS: Five hundred fifty-four patients (338 men and 216 women; median age, 68 years) had undergone partial or complete gastrectomy for intestinal (n = 209) or diffuse (n = 249) adenocarcinoma of the esophagogastric junction and stomach. Survival data and date of death were available for all patients. Patient death correlated significantly with age at diagnosis, tumor type, histologic grade, local tumor growth (T category), number of metastatic lymph nodes, lymph node ratio, lymph node status (N category), and tumor stage. No major difference was noted between the sixth and seventh editions of the TNM classification. On the basis of survival data, we revised the stage grouping system; stage I and II tumors were confined to nonmetastatic tumors, and stage III and IV tumors were confined to metastatic tumors. The Kaplan-Meier plots of this modified stage grouping showed statistically significant differences between individual stage subgroups without crossing curves and demonstrated improved survival of patients with stage II disease. CONCLUSION: The seventh edition of the TNM classification is associated with a stage migration in 60% of patients with esophagogastric and stomach cancer. This change did not improve the assessment of patient prognosis, and therefore, a revised staging system is proposed.

Genotypic and phenotypic characterization of side population of gastric cancer cell lines.

The side population (SP) of tumor cell lines shares characteristics with tumor stem cells. The objective of this study was to phenotypically and genotypically characterize the SP of gastric cancer cell lines. SP cells were obtained from AGS and MKN45 gastric cancer cells using Hoechst 33342 staining and fluorescence-activated cell sorting. The cells were subsequently studied morphologically at cytology and immunocytochemistry, on the transcriptional level via gene array, and in cell culture using recultivation assays. Genes differentially expressed in SP cells were evaluated at immunohistochemistry in tissue samples from 486 patients with gastric cancer. The SP cells were smaller and rounder then non-SP cells. SP cells self-renewed in recultivation experiments and differentiated into SP and non-SP cells. Recultivated SP and non-SP cells exhibited distinct phenotypes in culture insofar as cell shape and colony formation. SP cells demonstrated increased levels of the stem cell markers CD133 and Musashi-1. Transcriptional analyses demonstrated that SP cells express genes that encode for stem cell properties including FZD7, HEY1, SMO, and ADAM17. It was observed that ADAM17 and FZD7 are differentially expressed in human gastric cancer, and FZD7-positive cancers are associated with significantly shorter patient survival. In conclusion, human gastric cancer cell lines enclose a phenotypically and genotypically distinct cell population with tumor stem cell features. Phenotypic characteristics of this distinct cell population are also present in gastric cancer tissue, and correlate with patient survival.

Precarious preoperative diagnostics and hints for the laparoscopic excision of uterine adenomatoid tumors: two exemplary cases and literature review.

OBJECTIVE: To present two exemplary cases of adenomatoid tumors. DESIGN: Case report. SETTING: Two laparoscopic centers in Germany. PATIENT(S): Two women of reproductive age with uterine adenomatoid tumors. INTERVENTION(S): Laparoscopic tumor excision. MAIN OUTCOME MEASURE(S): Differential diagnostics of uterine masses. RESULT(S): Intraoperatively, tumor excision is difficult because of the missing capsule that allows simple enucleation of fibroids. Adenomatoid tumors need to be cut out of the myometrium because they are densely adherent, with no clear plane of cleavage. Definite exclusion of a malignant tumor is only possible by histologic workup. CONCLUSION(S): The above might lead to an intraoperative dilemma in the case of young women because a malignant tumor would implicate a more radical operative strategy. Nevertheless, owing to the benign nature of adenomatoid tumors and the lack of recurrence, simple resection with conservation of the surrounding tissue is the treatment of choice. The proper laparoscopic handling of such cases is illustrated and placed in context within the existing literature.