A 'waterfall' transfer-based workflow for improved quality of tissue microarray construction and processing in breast cancer research.

A major focus in cancer research is the identification of biomarkers for early diagnosis, therapy prediction and prognosis. Hereby, validation of target proteins on clinical samples is of high importance. Tissue microarrays (TMAs) represent an essential advancement for high-throughput analysis by assembling large numbers of tissue cores with high efficacy and comparability. However, limitations along TMA construction and processing exist. In our presented study, we had to overcome several obstacles in the construction and processing of high-density breast cancer TMAs to ensure good quality sections for further research. Exemplarily, 406 breast tissue cores from formalin-fixed and paraffin embedded samples of 245 patients were placed onto three recipient paraffin blocks. Sectioning was performed using a rotary microtome with a "waterfall" automated transfer system. Sections were stained by immunohistochemistry and immunofluorescence for nine proteins. The number and quality of cores after sectioning and staining was counted manually for each marker. In total, 97.1 % of all cores were available after sectioning, while further 96 % of the remaining cores were evaluable after staining. Thereby, normal tissue cores were more often lost compared to tumor tissue cores. Our workflow provides a robust method for manufacturing high-density breast cancer TMAs for subsequent IHC or IF staining without significant sample loss.

[Diagnostics and therapy for neuroendocrine neoplasia of an unknown primary - a plea for open exploration]. / Diagnostik und Therapie bei neuroendokrinen Neoplasien mit unklarem Primarius - ein Plädoyer für die offene Exploration.

BACKGROUND: Neuroendocrine neoplasia (NEN) are a rare and heterogenous tumour entity. The subgroup with unknown primary tumour (N-CUP) seems to have a worse prognosis as resection of the primary is necessary for cure. The diagnostics and therapeutic algorithms for N-CUP in a German single centre are presented. PATIENTS/METHODS: Analysis of the surgical databank showed 35 cases of N-CUP in 261 cases with NEN from gastroenteropancreatic and lung origin over 2 decades (03/1990-03/2011). Three groups were built: K1 - primary detection after operative exploration (n = 10), K2 - unknown primary after operative exploration (n = 10) and K3 - no operative exploration for various reasons (n = 13). RESULTS: Initially 13.4 % (35/261) of patients presented as N-CUP, after intensified diagnostics 12.7 % (33/261) and after operative exploration 8.8 % (23/261) remained with unknown primary tumour. The sex ratio was 1 : 1, the median age is significantly higher in N-CUP [63.8 years (y) vs. 55.9 y, p = 0.004), the 5-year-survival is lower (58 vs. 72 %, n. s.). compared to NEN with known primary. Operative exploration was performed in 60.6 % (20/33), 30 % (6/20) of them were found to have inoperable situations, in 20 % (4/20) single site metastases were removed completely and in 50 % (10/20) a primary tumour was detected (8 × midgut, 2 × pancreas) intraoperatively. In these cases 70 % (7/10) got complete tumour resection (R0) and in 30 % (3/10) primary tumour resection with debulking of liver metastasis was done. In K3 (39.4 %, 13/33) most patients [69.2 % (9/13)] were treated with chemotherapy. The median age in K1 was significantly lower than in K3 (54.9 y vs. 68.3 y, p = 0.028), male dominance was seen in K3 (3,3 : 1, n. s.). The average Ki-67 index was 4.3, 23.8 and 53 % in K1, K2 and K3 (p < 0.0001 for K1 and K3 and p = 0.035 for K2 and K3), respectively. The death rate was 20, 30 and 76.9 % in K1, K2 and K3, respectively. CONCLUSION: Primary tumours of the midgut and pancreas are often found in the subset of well differentiated neuroendocrine CUP syndrome after open surgical exploration. A high rate of complete tumour resection and cure can be achieved in these cases. After common diagnostic tools (CT, MRI and somatostatin receptor scintigraphy), immunhistochemistry can give important hints (CDX-2 for midgut, TTF-1 for lung and thyroid) for a primary lesion. Also in single site metastasis without primary tumour detection a good clinical outcome is seen after complete resection.

Impact of the HPV-positivity definition on the prognostic value of HPV status in patients with locally advanced squamous cell carcinoma of the head and neck.

BACKGROUND AND PURPOSE: This study re-evaluated the prognostic value of HPV status for loco-regional control (LRC), metastases-free survival (MFS), and survival (OS) in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN). A modified definition of HPV positivity was used in the current study compared to the authors' previous study. PATIENTS AND METHODS: In the previous study of the same 170 patients, a tumor was defined as HPV-positive if it showed a positive in situ hybridization result in ≥10% of tumor cells and/or positive p16 immunostaining. In the current analysis, tumors were considered HPV-positive only if they showed positive results for both in situ hybridization and p16 immunostaining. In addition to HPV status, the same 11 potential prognostic factors were investigated for treatment outcomes as in the preceding study. RESULTS: In the multivariate analysis of the current study, HPV positivity was significantly associated with improved LRC [risk ratio (RR) 9.78; p<0.001], MFS (RR 7.17; p=0.008), and OS (RR 6.61; p<0.001). In the previous study, HPV positivity was associated with LRC (RR 2.34; p=0.014) and OS (RR 2.19; p=0.019), but not with MFS (RR 2.04; p=0.11). CONCLUSIONS: Applying the new definition of HPV positivity, the impact of HPV status on the prognosis of patients irradiated for locally advanced SCCHN was more prominent than in our previous study and associated with all three investigated endpoints.

[Round robin test for detection of genomic aberrations in non-Hodgkin lymphoma by in situ hybridization]. / Ringversuch zum Nachweis genomischer Veränderungen bei Non-Hodgkin-Lymphomen mittels In-situ-Hybridisierung.

BACKGROUND: The detection of characteristic genomic aberrations by fluorescence in situ hybridization (FISH) has a high diagnostic impact on lymphomas according to the World Health Organization (WHO). To investigate the reproducibility of non-isotopic ISH results a multicenter trial was carried out involving eight institutes for hematopathology. MATERIAL AND METHODS: Analyses were performed on two diffuse large B-cell lymphomas (DLBCL) without known aberrations, on one follicular lymphoma with a IGH/BCL2 translocation and BCL6 split and on two B-cell lymphomas intermediate between DLBCL and Burkitt's lymphoma with c-MYC and BCL2 rearrangements, one with an additional BCL6 split. Break-apart probes for BCL6 and c-MYC, as well as fusion probes for the c-MYC/IGH and the IGH/BCL2 translocations were used. RESULTS: All aberrations were correctly detected by all centres and no false positive or false negative results were obtained. The numbers of positive cells varied from 25% to 94%. Pearson's correlation coefficient between the centres was always > 0.8. CONCLUSIONS: The ISH analysis of recurrent genomic aberrations in formalin-fixed paraffin-embedded (FFPE) tissue is a highly reproducible technique which yields substantial additive help for lymphoma diagnostics.

Pancreatic carcinoma cell lines reflect frequency and variability of cancer stem cell markers in clinical tissue.

BACKGROUND: Pancreatic cancer is one of the most deadly malignancies with insufficient therapeutic options and poor outcome. Cancer stem cells (CSCs) are thought to be responsible for progression and therapy resistance. We investigated the potential of pancreatic cell lines for CSC research by analyzing to what extent they contain CSC populations and how representative these are compared to clinical tissue. METHODS: Six pancreatic cancer cell lines were analyzed by flow cytometry for CD326, CD133, CD44, CD24, CXCR4 and ABCG2. Subsequently, 70 primary pancreatic tissues were evaluated for CD326, CD133 and CD44 by immunohistochemistry. RESULTS: All the cell lines but one showed a stable expression pattern throughout biological replicates. Marker expression in clinical tissue of CD44 distinguished normal patients from pancreatic carcinoma patients with a sensitivity of 50% at 80% specificity and metastasized from nonmetastasized carcinomas with 69% sensitivity at 100% specificity. CONCLUSIONS: Our results indicate a link between elevated CD44 expression, malignancy and metastasis of pancreatic tissue. Furthermore, individual pancreatic cell lines show a substantial amount of cells with CSC properties which is comparable with interpatient variability detected in primary tissue. These pancreatic cancer cell lines could thus serve for urgently needed pharmacological CSC in vitro research.

The detection of mupirocin resistance and the distribution of methicillin-resistant Staphylococcus aureus at the University Hospital of the West Indies, Jamaica.

OBJECTIVES: The objectives of this study were to determine the susceptibility of Methicillin Resistant Staphylococcus aureus (MRSA) isolates to Mupirocin and other antimicrobial agents and to record the prevalence and distribution of this organism at the University Hospital of the West Indies (UHWI). METHODS: MRSA isolates collected between January 1, 2008 and December 31, 2008, were tested for low and high level resistance to Mupirocin. Susceptibility testing to other antibiotics including cotrimoxazole, minocycline, tetracycline, clindamycin, erythromycin, gentamicin and vancomycin was also done. Laboratory records for all patients from whom MRSA was recovered were reviewed and data on type and source of isolates, clinical diagnosis, history of previous hospitalization and use of mupirocin were extracted. In addition, the laboratory records for 2004 and 2005 were also reviewed to determine prevalence during these periods. RESULTS: Seven per cent of Staphylococcus aureus isolates were resistant to methicillin (MRSA) and of these, 30% and 24% showed low level and high level resistance to mupirocin, respectively. Ninety-four per cent of MRSA strains were resistant to erythromycin while 52% showed resistance to clindamycin. Resistance to tetracycline, co-trimoxazole and minocycline was 27%, 12% and 6%, respectively, while about one-third of the isolates were resistant to gentamicin. There was no resistance to vancomycin. More than half (58%) of the isolates were from skin and soft tissue specimens while isolates from respiratory and urinary tracts and the bloodstream accounted for 19%, 13% and 4%, respectively. There has been a steady increase in prevalence from 4% in 2004 to 5% in 2007 and 7% in 2008. CONCLUSION: Resistance of MRSA to mupirocin appears to be an emerging problem at the UHWI and must be monitored carefully. There is also significant resistance to commonly used antimicrobial agents and strict adherence to antibiotic policy is required to preserve the usefulness of these agents.

Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications (a study from the Lunenburg Lymphoma Biomarker Consortium).

BACKGROUND AND AIMS: The results of class prediction and the determination of prognostic markers in diffuse large B-cell lymphoma (DLBCL) have been variably reported. Apart from biological variations, this may be caused by differences in laboratory techniques, scoring definitions and inter- and intra-observer variation. In this study, an international collaboration of clinical lymphoma research groups has concentrated on validation and standardisation of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL. METHODS: Sections of a tissue microarray with 36 cases of DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM-1 and Ki-67 according to local methods. The study was performed in two rounds, firstly focused on the evaluation of laboratory staining variation, and secondly on the scoring variation. RESULTS: Different techniques resulted in highly variable results and poor reproducibility for almost all markers. Reproducibility of the nuclear markers was highly sensitive to technical variations, including immunological enhancement techniques (agreements 34%). With elimination of variation due to staining and uniformly agreed on scoring criteria, significant improvement was seen; however less so for bcl-6 and Ki-67 (agreement 53-58%). Absence of internal controls that preclude scoring, significantly influenced the results for CD10 and bcl-6. CONCLUSION: Semi-quantitative immunohistochemistry for subclassification of DLBCL is feasible, but with varying rates of concordance for different markers and only using optimised techniques and strict scoring criteria. These findings may explain the wide variation in prognostic impact reported in the literature. Harmonisation of techniques and centralised consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

Hibernoma-like brown fat in the bone marrow: report of a unique case.

We report on a bone-marrow biopsy of a 61-year-old female patient that was performed because of the clinical suspicion of a myeloproliferative disease. The trephine biopsy showed morphological features that were consistent with an essential thrombocythaemia (ET). The diagnosis of a myeloproliferative disease could be corroborated by demonstration of the V617F mutation of JAK2. Besides the histological features of ET, the marrow showed a peculiar infiltrate that consisted of multivacuolated cells that were immunohistochemically identified as brown adipose tissue with a hibernoma-like picture. To the best of our knowledge, this is the first report on brown adipose tissue in the bone marrow.

Significant high expression of CD23 in follicular lymphoma of the inguinal region.

AIMS: Inguinal lymph nodes are considered to be problematic for the diagnosis of lymphoma due to architectural changes resulting from previous inflammatory processes. The aim was to investigate the morphology and immunophenotype of follicular lymphomas (FL) in order to clarify whether FL presenting in inguinal nodes differs from FL biopsies from other sites. METHODS AND RESULTS: A total of 219 FLs were studied, comprising 78 biopsy specimens of inguinal lymph nodes and 141 from other sites. All samples were assessed for growth pattern, grade, sclerosis and immunophenotype (Bcl-2, CD10, CD23, Mib-1). Cases negative for Bcl-2 were analysed by polymerase chain reaction and fluorescence in situ hybridization. In comparison with the biopsies from other regions, we found a significantly increased number of CD23+ FLs in samples of inguinal lymph nodes (38% versus 21%). Expression of CD23 was more frequently detected in grade 1 FLs than in other grades (grade 1, 37%; grade 2, 18%; grade 3, 23%; transformed, 6%). Other immunohistochemical parameters, however, did not differ between the two groups. CONCLUSION: There is an unexpectedly high frequency of CD23 expression in FL in general, which is even more pronounced in inguinal nodes.

Expression of MMP-2, MMP-7, MMP-9, MMP-10 and MMP-11 in human astrocytic and oligodendroglial gliomas.

The members of the matrix metalloproteinase family (MMP) have the ability to degrade macromolecules of the extracellular matrix and are responsible for tumor invasion and infiltration, limiting the effectiveness of the neurosurgical resection of brain tumors. Among the glial brain tumors, astrocytomas and oligodendrogliomas are the most important tumor entities and require a different therapeutic approach. To determine the pattern of MMP expression in astrocytic and oligodendroglial tumors, sections of astrocytic and oligodendroglial differentiated glioblastomas (WHO grade IV), as well as of anaplastic oligodendrogliomas (WHO grade III) and anaplastic gemistocytic astrocytomas (WHO grade III) were immunostained for MMP-2, MMP-7, MMP-9, MMP-10 and MMP-11. MMP-7, MMP-10 and MMP-11 were strongly expressed by neoplastic gemistocytic astrocytes while oligodendrocytic tumor regions showed only a low immunoreaction. In contrast, MMP-2 and MMP-9 mainly immunolabeled vascular structures. These data indicated that MMP-7, MMP-10 and MMP-11 contribute to the worse prognosis of astrocytic tumors when compared to oligodendrogliomas, while MMP-2 and MMP-9 might play an important role in neo-angiogenesis and tumor vascularization.

VEGF-, KIT protein- and neutral endopeptidase (NEP/CD10)-positive myofibroblasts-precursors of angiogenesis in chorioangiomas?

Chorioangiomas are benign angiomatous tumours of the placenta occurring with a frequency of approximately one per cent of all examined placentae. Hypoxia and genetic factors are discussed to be predisposing factors for chorioangiomas. However, not much is known about the tumorigenesis of these benign tumours. Screening with various antibodies in a rare case of chorangiomatosis, we found disseminated spindle cells coexpressing vascular epithelial growth factor (VEGF), neutral endopeptidase 24.11 (NEP/CD10), and KIT protein (CD117) within the tumour stroma. A possible involvement of such factors in angiogenesis and tumorigenesis of chorioangiomas/chorangiomatosis has not been studied so far.Seven placentae with chorioangiomas (n=6) or chorangiomatosis (n=1), six normal placentae, and four cutaneous haemangiomas were analysed immunohistochemically (ABC and APAAP methods) using antibodies against VEGF, NEP, KIT protein, as well as endothelial markers like PECAM-1 (CD31), CD34, v. Willebrand factor (factor VIII), and ulex europaeus. In addition, analysis of c-kit 'gain of function' mutation Asp 816 to Val by means of Hinfl digestion and direct sequencing of semi-nested polymerase chain reaction products was performed. All chorioangiomas and haemangiomas strongly expressed the endothelial markers CD34, CD31, and FVIII, while only weak expression of ulex lectin was noted. Disseminated groups of VEGF-, NEP-, and KIT protein-positive spindle cells, which coexpressed vimentin and smooth-muscle actin were identified as myofibroblasts in the stroma of four chorioangiomas. These spindle cells were quantified as numerous in two and as rare in two other cases. No VEGF-positive myofibroblasts, however, were detected in the villous stroma of normal control placentae and haemangiomas. Only scattered perivascular myofibroblasts expressing KIT protein and NEP were detected in early gestational placenta controls. In all chorioangiomas and chorangiomatosis PCR analysis failed to unveil c-kit 'gain of function' mutation Asp 816 to Val in KIT protein-positive spindle cells. Moreover, a significant increase in mast cells was observed only in the haemangiomas. As expected, endothelial origin of chorioangiomas/chorangiomatosis was verified by CD31, CD34, FVIII expression. Myofibroblastic spindle cells expressing VEGF and NEP may be precursor cells in these peculiar angiomatous tumours. Although activating c-kit mutation Asp 816 to Val was not detected by PCR, the presence of KIT protein (CD117)-positive intratumoral myofibroblastic spindle cells in chorioangiomas and chorangiomatosis might suggest involvement of the stem cell factor (SCF)-receptor in pathologically enhanced angiogenesis.

Matrix-metalloproteinases in Hodgkin lymphoma.

BACKGROUND: Classical Hodgkin lymphomas are characterized by relatively few tumour cells and prominent proliferation of plasma cells, histiocytes, lymphocytes and eosinophils. In addition there is a varying degree of sclerosis, which is especially prominent in nodular sclerosis. These morphological peculiarities led to the idea that the interaction between tumour cells and bystander cells as well as the extracellular matrix may be important in Hodgkin lymphomas. MATERIALS AND METHODS: Thirty-four classical Hodgkin lymphomas (CHL) were analysed regarding the expression of EMMPRIN, MMP-2, -7, -9, -10 and-11 using immunohistochemistry. RESULTS: The tumour cells were positive for EMMPRIN in 100% of the cases. In 82% of CHL the Hodgkin and Reed-Sternberg cells (HRS) were negative for MMP-2. In contrast the surrounding non-neoplastic cells were MMP-2-positive in 71% of the cases. The HRS cells stained positive for MMP-7 in 68% of CHL, whereas only a few surrounding cells were positive for this marker. In all but one case (97%) the HRS cells were negative for MMP-9. However, the surrounding cells stained positive in 32%, thus resembling the staining pattern for MMP-2. Only scattered cells of both populations, HRS cells as well as bystander cells, stained for MMP-10 and -11, and no specific staining pattern was observed. CONCLUSION: Our data indicate a complex interaction between tumour cells and bystander cells with regard to metalloproteinases. The expression of EMMPRIN in the tumour cells may induce the expression of MMP-2 in the surrounding non-neoplastic cells. MMP-2 can be activated by MMP-7, which is expressed in the tumour cells. It is tempting to speculate that an interruption of this cycle could be of therapeutic benefit.

[Mycosis fungoides in childhood and adolescence with clonal T-cell receptor gamma gene rearrangement. Two cases]. / Mycosis fungoides im Kindes- und Jugendalter mit klonaler Rekombination der gamma-Kette des T-Zell-Rezeptorgens. Zwei Fallberichte.

Mycosis fungoides (MF) is a cutaneous T-cell lymphoma (CTCL) characterized by its typical progress in three stages: the patch-, the plaque- and the tumour-stage. The incidence of mycosis fungoides rises with age and the average age at presentation is about 50. Children and adolescents are rarely affected and there are only few reports in the literature. We report a 12- and a 15-year-old boy showing refractory skin lesions not typical for mycosis fungoides. The histo- and immunohistological investigations and the detection of clonal T-cell receptor gamma gene rearrangements confirmed the diagnosis of early onset mycosis fungoides in both cases.

Prognostic significance of a grading system considering tumor heterogeneity in muscle-invasive urothelial carcinoma of the urinary bladder.

The fact that urothelial carcinomas (UC) often contain areas with different histologic grades has been recently shown to bear some prognostic relevance. Here we examined the prognostic significance of a grading system considering tumor heterogeneity in muscle-invasive bladder carcinomas. 151 UC treated by radical cystectomy were included. According to the World Health Organization/International Society of Urological Pathology (WHO/ISUP) classification, histologic grade was low-grade (LG) in 8 and high-grade (HG) in 143 cases. 65 HG tumors which focally harbored LG areas were assigned to mixed-type (MT) carcinomas. Mean follow-up was 50 months. While the WHO/ISUP classification showed no significant correlation with disease-specific survival (p = 0.3995 by log-rank test), stratification into LG/MT and HG tumors had a significant prognostic relevance (p = 0.0404). Nodal status was identified as the only independent prognostic factor (p = 0.0001 by multivariate analysis). In this respect, stratification into LG/MT and HG tumors missed the level of statistical significance by a norrow margin (p = 0.07 by multivariate analysis), but it turned out better than tumor category (p = 0.08). In conclusion, a grading system considering tumor heterogeneity may improve the predictive power of the WHO/ISUP classification in muscle-invasive UC of the urinary bladder. Although the two-tired grading system proposed in this study was not identified as an independent prognostic factor, it may help to obtain additional prognostic information on patients with advanced bladder cancer treated by radical cystectomy.

Anti-CD20 monoclonal antibody (Rituximab) and Cidofovir as successful treatment of an EBV-associated lymphoma with CNS involvement.

BACKGROUND: Epstein-Barr virus(EBV)-associated posttransplant lymphoproliferative disease (PTLD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). Especially in cases with involvement of the central nervous system (CNS) treatment is difficult because the efficacy of most chemotherapeutic agents as well as EBV-specific cytotoxic donor T cells in liquor is uncertain. In the last years the anti-CD20 monoclonal antibody Rituximab was intensively investigated in the treatment of EBV-PTLD. However, only 8 patients with B-cell lymphoma and CNS involvement treated with Rituximab were reported. CASE REPORT: A 24-year-old female patient with acute T-lymphoblastic leukemia in second complete remission had received allogeneic, unrelated, T-cell depleted HSCT. 10 months later an EBV-associated PTLD was diagnosed. Beside peripheral lymphomas and B symptoms the patient showed neurological symptoms. Examination of the cerebrospinal fluid (CSF) revealed a meningeosis lymphoblastica caused by the EBV lymphoma. Treatment with Rituximab and the antiviral drug Cidofovir led to complete remission with regression of the peripheral lymphomas and disappearance of the neurological symptoms. In addition, the PCR control on EBV DNA became negative in the plasma as well as in CSF. CONCLUSION: The combination of Rituximab and Cidofovir appears as an interesting alternative treatment in patients with EBV-associated PTLD and CNS involvement.

[Anatomy of the shoulder joint]. / Anatomie des Schultergelenkes.

The shoulder joint and its associated joints form one of the most complex joint systems of the human locomotor apparatus. Its large range of motion is made possible by the interplay of 5 joints: sternoclavicular-joint, acromioclavicular-joint, glenohumeral joint, thoracoscapular joint and subacromial joint. The rotator cuff works mostly as an active stabilizer of the shoulder joint. The supraspinatus muscle causes a compression of the humerus in the glenoid mainly, furthermore it effects synergistic the abduction with the delta muscle. On the basis of its lever-arm the supraspinatus works between 0 and 60 degrees abduction the most optimally. With failure of the supraspinatus, the deltoideus can almost completely take its function. The inferior glenohumeral ligament-complex is the main passive stabilizer. The blood supply of the humerus head is ensured mainly by the a. circumflexa anterior and its rami ascendents, by several small branches from the a. circumflexa posterior and over intraosseous anastomoses. The most important vessel of the cap is the intraosseous a. arcuata out of the ramus ascendens lateralis of the a. circumflexa anterior.