Sensitive and Specific Droplet Digital PCR Assays for Circulating Tumor HPV DNA: Development, Validation, and Clinical Application in HPV-Associated Cancers.

BACKGROUND: Human papillomavirus (HPV) has emerged as a significant contributor to cancer incidence globally, particularly in the context of oropharyngeal squamous cell carcinoma (OPSCC) and cancer of unknown primary (HNCUP). This study aimed to develop and validate droplet digital PCR (ddPCR) assays for the detection of circulating tumor HPV DNA (ctHPV-DNA) in plasma, focusing on high-risk HPV genotypes associated with these cancers. METHODS: ddPCR assays for HPV16, 18, 33, 35, 56, and 59 were developed and tested using gBlocks, HPV cell-free DNA, fragmented tumor HPV+ DNA, and plasma samples from patients with HPV+ OPSCC (n = 110) and HNCUP (n = 9). RESULTS: Assays demonstrated robust technical sensitivity across all tested HPV genotypes. Clinical application of the assays on a cohort of patients with HPV+ OPSCC and HNCUP revealed high sensitivity (91.6%) and wide variability in ctHPV-DNA levels. Analyses revealed correlations between ctHPV-DNA levels and TNM stage and tumor viral load. The association between ctHPV-DNA and tumor viral load persisted even after adjusting for TNM stage. At posttreatment, 72.5% of samples had reached undetectable ctHPV-DNA levels. Having detectable ctHPV-DNA posttreatment was associated with a higher ctHPV-DNA level at diagnosis and higher viral load at diagnosis. CONCLUSION: The findings underscore the potential of ctHPV-DNA as a biomarker for monitoring HPV+ cancers and offer insights into tumor dynamics. Implementation of these assays in clinical practice could enhance no-invasive treatment monitoring and recurrence detection in HPV-associated cancers. CLINICAL TRIALS: NCT05904327.

Tissue microarray validation in cervical carcinoma studies. A methodological approach.

Tissue microarrays (TMAs) are a cost-effective tool to study biomarkers in clinical research. Cervical cancer (CC) is one of the most prevalent in women worldwide, with the highest prevalence in low-middle-income countries due to a lack of organized screening. CC is associated with persistent high-risk human papillomavirus infection. Several biomarkers have been studied for diagnostic, therapeutic, and prognostic purposes. We aimed to evaluate and validate the effectiveness of TMA in CC compared to whole slide images (WSs). We selected and anonymized twenty cases of CC. P16, cytokeratin 5 (CK5), cytokeratin 7 (CK7), programmed death-ligand 1 (PD-L1), and CD8 expression were immunohistochemically investigated. All WS were scanned and 10 representative virtual TMA cores with 0.6 mm diameter per sample were selected. Ten random combinations of 1-5 cylinders per case were assessed for each biomarker. The agreement of scoring between TMA and WS was evaluated by kappa statistics. We found that three cores of 0.6 mm on TMA can accurately represent WS in our setting. The Kappa value between TMA and WS varied from 1 for p16 to 0.61 for PD-L1. Our study presents an approach to address TMA sampling that could be generalized to TMA-based research, regardless of the tissue and biomarkers of interest.

PD-L1 expression in squamous cervical carcinomas of Mozambican women living with or without HIV.

Programmed death-ligand 1 (PD-L1) is overexpressed in squamous cervical cancer (SCC) and can be used for targeted immunotherapy. The highest mortality rates of SCC are reported in sub-Saharan Africa, where Human immunodeficiency virus (HIV) prevalence is high. In Mozambique most SCC patients present at advanced stages. Thus, there is a need to introduce new treatment options. However, immunocompromised patients were frequently excluded in previous clinical trials. Our aim was to determine if PD-L1 expression in SCC is as prevalent among women living with HIV (WLWH) as among other patients. 575 SCC from Maputo Central Hospital were included. HIV status was available in 266 (46%) cases PD-L1 expression was scored through tumour proportion score (TPS) and combined positive score (CPS). PD-L1 was positive in 20.1% of the cases (n = 110), TPS (score ≥ 25%) and in 26.3% (n = 144), CPS (score ≥ 1). Stratifying according to the HIV status, WLWH were TPS positive in 16.7%, compared to 20.9%, p = 0.43, and concerning CPS 21.1% versus 28.7%, p = 0.19, respectively. PD-L1 status was not influenced by stage, Ki-67 or p16, CD8 expression influenced only CPS status. Our data indicates that the documented effect of PD-L1 therapy on SCC should be confirmed in randomized clinical trials in an HIV endemic milieu.

Visual Counting and Automated Image-analytic Assessment of Ki-67 and their Prognostic Value in Synovial Sarcoma.

Background: Ki-67 is a widely used proliferation marker reflecting prognosis in various tumors. However, visual assessment and scoring of Ki-67 suffers from marked inter-observer and intra-observer variability. We aimed to assess the concordance of manual counting and automated image-analytic scoring methods for Ki-67 in synovial sarcoma. Patients and Methods: Tissue microarrays from 34 patients with synovial sarcoma were immunostained for Ki-67 and scored both visually and with 3DHistech QuantCenter. Results: The automated assessment of Ki-67 expression was in good agreement with the visually counted Ki-67 (r Pearson =0.96, p<0.001). In a Cox regression model automated [hazard ratio (HR)=1.047, p=0.024], but not visual (HR=1.063, p=0.053) assessment method associated high Ki-67 scores with worse overall survival. Conclusion: The automated Ki-67 assessment method appears to be comparable to the visual method in synovial sarcoma and had a significant association to overall survival.

Detection of High- and Low-Risk HPV DNA in Archived Breast Carcinoma Tissues from Ethiopian Women.

BACKGROUND: Human papilloma virus (HPV) is involved in the development of cancer of the cervix, mouth and throat, anus, penis, vulva, or vagina, but it has not been much considered as a cause of breast cancer. Recently, a number of investigations have linked breast cancer to viral infections. High-risk HPV types, predominantly HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, are established as carcinogens in humans. In this study we aimed to detect 19 high-risk and 9 low-risk HPVs from archived breast tumor tissue among Ethiopian women. METHODS: In this study, 75 breast cancer patients from Tikur Anbassa Specialized Hospital in Addis Ababa (Ethiopia) were included. HPV detection and genotyping were done using the novel Anyplex™ II HPV28 Detection Assay at the Orebro University Hospital, Sweden. The Anyplex™ II PCR System detects 19 high-risk HPV types (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82) and 9 low-risk HPV types (6, 11, 40, 42, 43, 44, 54, 61, and 70). IHC for p16 was done using an automated system, the Dako Autostainer Link. RESULTS: Out of the 75 valid tests, two were found to be positive (2.7%) for HPV. One of the cases was positive for the high-risk HPV16 genotype while the other was positive both for the high-risk HPV39 and the low-risk HPV6. The cell cycle protein p16 was highly expressed in the case positive for the high-risk HPV16, but it was not expressed in the case positive for HPV39. CONCLUSION: The prevalence of HPV is low in Ethiopian breast cancer patients, but the role played by HPV in breast carcinogenesis among Ethiopian breast cancer patients cannot be commented based on these observations.

Liquid biopsy as an option for predictive testing and prognosis in patients with lung cancer.

BACKGROUND: The aim of this study was to investigate the clinical value of liquid biopsy as a primary source for variant analysis in lung cancer. In addition, we sought to characterize liquid biopsy variants and to correlate mutational load to clinical data. METHODS: Circulating cell-free DNA was extracted from plasma from patients with lung cancer (n = 60) and controls with benign lung disease (n = 16). Variant analysis was performed using the AVENIO ctDNA Surveillance kit and the results were correlated to clinical and variant analysis data from tumor tissue or cytology retrieved from clinical routine diagnostics. RESULTS: There were significantly more variants detected in lung cancer cases compared to controls (p = 0.011), but no difference between the histological subgroups of lung cancer was found (p = 0.465). Furthermore, significantly more variants were detected in patients with stage IIIb-IV disease compared to patients with stage I-IIIa (median 7 vs 4, p = 0.017). Plasma cfDNA mutational load was significantly associated with overall survival (p = 0.010). The association persisted when adjusted for stage and ECOG performance status (HR: 3.64, 95% CI 1.37-9.67, p = 0.009). Agreement between tumor and plasma samples significantly differed with stage; patients with stage IIIb-IV disease showed agreement in 88.2% of the cases with clinically relevant variants, compared to zero cases in stage I-IIIa (p = 0.004). Furthermore, one variant in EGFR, two in KRAS, and one in BRAF were detected in plasma but not in tumor samples. CONCLUSION: This study concludes that in the vast majority of advanced NSCLC patients a reliable variant analysis can be performed using liquid biopsy from plasma. Furthermore, we found that the number of variants in plasma is associated with prognosis, possibly indicating a strategy for closer follow up on this crucial patient group.

A randomized porcine study of the hemodynamic and metabolic effects of combined endovascular occlusion of the vena cava and the aorta in normovolemia and in hemorrhagic shock.

BACKGROUND: Mortality from traumatic retrohepatic venous injuries is high and methods for temporary circulatory stabilization are needed. We investigated survival and hemodynamic and metabolic effects of resuscitative endovascular balloon occlusion of the aorta (REBOA) and vena cava inferior (REBOVC) in anesthetized pigs. METHODS: Twenty-five anesthetized pigs in normovolemia or severe hemorrhagic shock (controlled arterial bleeding in blood bags targeting systolic arterial pressure of 50 mm Hg, corresponding to 40-50% of the blood volume) were randomized to REBOA zone 1 or REBOA+REBOVC zone 1 (n = 6-7/group) for 45 minutes occlusion, followed by 3-hour resuscitation and reperfusion. Hemodynamic and metabolic variables and markers of end-organ damage were measured regularly. RESULTS: During occlusion, both the REBOA groups had higher systemic mean arterial pressure (MAP) and cardiac output (p < 0.05) compared with the two REBOA+REBOVC groups. After 60 minutes reperfusion, there were no statistically significant differences between the two REBOA groups and the two REBOA+REBOVC groups in MAP and cardiac output. The two REBOA+REBOVC groups had higher arterial lactate and potassium concentrations during reperfusion, compared with the two REBOA groups (p < 0.05). There was no major difference in end-organ damage markers between REBOA and REBOA+REBOVC. Survival after 1-hour reperfusion was 86% and 100%, respectively, in the normovolemic REBOA and REBOA+REBOVC groups, and 67% and 83%, respectively, in the corresponding hemorrhagic shock REBOA and REBOA+REBOVC groups. CONCLUSION: Acceptable hemodynamic stability during occlusion and short-term survival can be achieved by REBOA+REBOVC with adequate resuscitation; however, the more severe hemodynamic and metabolic impacts of REBOA+REBOVC compared with REBOA must be considered. LEVEL OF EVIDENCE: Prospective, randomized, experimental animal study. Basic science study, therapeutic.

Distribution and characteristics of androgen receptor (AR) in breast cancer among women in Addis Ababa, Ethiopia: A cross sectional study.

Evaluation of the role of androgen receptor (AR) in the biology of breast cancer is an emerging area of research. There are compelling evidences that AR expression may be used to further refine breast cancer molecular subtyping with prognostic and therapeutic implications. Many studies indicated co-expression of AR with the hormonal receptors in breast cancer has a favorable prognosis. AR is also investigated by many researchers as a potential therapeutic target in treatment of breast cancer. Studies on the frequency and distribution of AR in breast cancer among Africans is barely available. Given the heightened interest to understand its role in breast cancer, we determined AR expression and assessed its association with clinicopathological parameters among Ethiopian women. In this study, 112 newly diagnosed patient with invasive breast cancer at Tikur Anbessa Specialized Hospital were enrolled. Immunohistochemical assessment of AR, ER, PR, Ki67 and HER2 were performed using tissue microarrays (TMA) constructed from their primary tumor block. Out of the 112 participants, 91 (81%) were positive for AR expression and the remaining 21 participants (19%) were negative for AR expression. Expression of AR in ER+, HER2+ and TNBC cases were 93%, 83% and 48% respectively. Our study reveals AR is expressed in a significant number of breast cancers patients and this may indicate that breast cancers cases in Ethiopia have favorable prognosis and could benefit from progresses in AR targeted treatments. Since AR expression has important consequences on the prognosis and treatment of breast cancer, further studies with an increased number of participants is necessary to confirm our reports.

Total resuscitative endovascular balloon occlusion of the aorta causes inflammatory activation and organ damage within 30 minutes of occlusion in normovolemic pigs.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) causes physiological, metabolic, end-organ and inflammatory changes that need to be addressed for better management of severely injured patients. The aim of this study was to investigate occlusion time-dependent metabolic, end-organ and inflammatory effects of total REBOA in Zone I in a normovolemic animal model. METHODS: Twenty-four pigs (25-35 kg) were randomized to total occlusion REBOA in Zone I for either 15, 30, 60 min (REBOA15, REBOA30, and REBOA60, respectively) or to a control group, followed by 3-h reperfusion. Hemodynamic variables, metabolic and inflammatory response, intraperitoneal and intrahepatic microdialysis, and plasma markers of end-organ injuries were measured during intervention and reperfusion. Intestinal histopathology was performed. RESULTS: Mean arterial pressure and cardiac output increased significantly in all REBOA groups during occlusion and blood flow in the superior mesenteric artery and urinary production subsided during intervention. Metabolic acidosis with increased intraperitoneal and intrahepatic concentrations of lactate and glycerol was most pronounced in REBOA30 and REBOA60 during reperfusion and did not normalize at the end of reperfusion in REBOA60. Inflammatory response showed a significant and persistent increase of pro- and anti-inflammatory cytokines during reperfusion in REBOA30 and was most pronounced in REBOA60. Plasma concentrations of liver, kidney, pancreatic and skeletal muscle enzymes were significantly increased at the end of reperfusion in REBOA30 and REBOA60. Significant intestinal mucosal damage was present in REBOA30 and REBOA60. CONCLUSION: Total REBOA caused severe systemic and intra-abdominal metabolic disturbances, organ damage and inflammatory activation already at 30 min of occlusion.

Estrogen receptor beta expression correlates with proliferation in desmoid tumors.

BACKGROUND AND OBJECTIVES: Estrogen receptor signaling and cyclin D1 have a major role in tumor cell proliferation in breast cancer. Desmoid tumors are rare neoplasms that may respond to endocrine treatment. The present study aimed to investigate the expression levels and the clinical relevance of estrogen receptor beta (ERß) and cyclin D1 in desmoid tumors. METHODS: This study consists of 83 patients with a surgically treated desmoid tumor. ERß and cyclin D1 expression was examined by immunohistochemistry in tissue microarrays. Cyclin A and Ki67 were studied in our previous work. RESULTS: Median ERß expression was 10.8%. ERß expression correlated with expression of the proliferation antigens Ki67 (rp = 0.35, P = 0.003), cyclin D1 (rp = 0.34, P = 0.004), and cyclin A (rp = 0.34, P = 0.004). ERß immunoexpression showed a trend towards predictive impact for recurrence as a continuous variable. Further explorative analysis indicated that very high ERß expression was related to high risk of relapse (hazard ratio [HR] 2.6; P = 0.02). Median cyclin D1 expression was 15.6%. High cyclin D1 expression was associated with high Ki67 and cyclin A expression. Cyclin D1 was not associated with time to recurrence. CONCLUSIONS: ERß and cyclin D1 immunopositivity correlated with high proliferation in desmoid tumors. High ERß expression might be predictive for postoperative recurrence.

High cyclin A expression, but not Ki67, is associated with early recurrence in desmoid tumors.

BACKGROUND AND OBJECTIVES: Desmoid tumors are soft-tissue tumors originating from myofibroblasts with a tendency to recur after surgery. High expression of proliferation markers is associated with shortened progression-free and/or overall survival in many neoplasms, including soft-tissue sarcomas. We investigated the prognostic role of cyclin A and Ki67 in desmoid tumors by immunohistochemistry. METHODS: The study included 76 patients with desmoid tumor operated at Helsinki University Hospital between 1987 and 2011. A tissue micro array (TMA) was constructed and the TMA sections were immunostained with cyclin A and Ki67 antibodies. A computer-assisted image analysis was performed. RESULTS: Cyclin A expression was evaluable in 74 and Ki67 in 70 patients. Cyclin A immunopositivity varied from 0% to 9.9%, with a mean of 1.9%. Cyclin A expression correlated significantly with Ki67. Cyclin A expression was associated with recurrence-free survival (HR 1.9, 95% CI = 1.1-3.2, P = .02), as were positive margin (HR 6.0, 95% CI = 1.6-22.5, P = .008) and extremity location (HR 5.3, 95% CI = 1.7-16.8, P = 0.005). Ki67 immunopositivity varied from 0.33% to 13.8%, with a mean of 4.6%, but had no significant prognostic impact (HR 1.1, P = .2). CONCLUSIONS: Our study indicates that cyclin A may be a new prognostic biomarker in surgically treated desmoid tumors.

Breast cancer in Ethiopia: evidence for geographic difference in the distribution of molecular subtypes in Africa.

BACKGROUND: Breast cancer is a heterogeneous disease with several morphological and molecular subtypes. Widely accepted molecular classification system uses assessment of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and proliferation marker Ki67. Few studies have been conducted on the incidence and molecular types of breast cancer in Sub-Saharan Africa. Previous studies mainly from Western and Central Africa, showed breast cancer to occur at younger ages and to present with aggressive features, such as high-grade, advanced stage and triple-negative phenotype (negative for ER, PR and HER2). Limited data from East Africa including Ethiopia however shows hormone receptor negative tumors to account for a lower proportion of all breast cancers than has been reported from elsewhere in Africa. METHODS: In this study from Tikur Anbessa Specialized Hospital, 114 breast cancer patients diagnosed between 2012 and 2015 were enrolled. ER, PR, Ki67 and HER2 receptor status were assessed using immunohistochemistry from tissue microarrays. FISH was used for assessment of gene amplification in all equivocal tumor samples and for confirmation in HER2-enriched cases. RESULTS: The distribution of molecular subtypes was: Luminal A: 40%; Luminal B: 26%; HER2-enriched: 10%; TNBC: 23%. ER were positive in 65% of all tumors and 43% the cases were positive for PR. There was statistically significant difference in median age at diagnosis between the molecular subtypes (P < 0.05). There was a bimodal distribution of molecular subtypes in different age ranges with Luminal B subtype being more common at younger ages (median = 36) and Luminal A subtype more prevalent at older ages (median = 42). There were no statistically significant differences in tumor grade, histology, and stage between the molecular subtypes of breast cancer. CONCLUSION: The present study detected Luminal A breast cancer to be the most common subtype and reveals a relatively low rate of hormone receptor negative and TNBC. Our findings and results from other East African studies suggest geographic variability in the distribution of the molecular subtypes of breast cancer in Africa and hence have important clinical and policy implications for breast cancer control and treatment in Ethiopia.

Inconsistent results in the analysis of ALK rearrangements in non-small cell lung cancer.

BACKGROUND: Identification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. Although fluorescence in situ hybridization (FISH) is regarded as the gold standard for detection of ALK rearrangements, ALK immunohistochemistry (IHC) is often used as screening tool in clinical practice. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data. METHODS: ALK rearrangements were detected using FISH on tissue microarrays (TMAs), including tissue from 851 NSCLC patients. In parallel, ALK protein expression was detected using IHC, applying the antibody clone D5F3 with two different protocols (the FDA approved Ventana CDx assay and our in house Dako IHC protocol). Gene expression microarray data (Affymetrix) was available for 194 patients. RESULTS: ALK rearrangements were detected in 1.7 % in the complete cohort and 2.0 % in the non-squamous cell carcinoma subgroup. ALK protein expression was observed in 1.8 and 1.4 % when applying the Ventana assay or the in house Dako protocol, respectively. The specificity and accuracy of IHC was high (> 98 %), while the sensitivity was between 69 % (Ventana) and 62 % (in house Dako protocol). Furthermore, only 67 % of the ALK IHC positive cases were positive with both IHC assays. Gene expression analysis revealed that 6/194 (3 %) tumors showed high ALK gene expression (≥ 6 AU) and of them only three were positive by either FISH or IHC. CONCLUSION: The overall frequency of ALK rearrangements based on FISH was lower than previously reported. The sensitivity of both IHC assays was low, and the concordance between the FISH and the IHC assays poor, questioning current strategies to screen with IHC prior to FISH or completely replace FISH by IHC.

Prognostic impact of COX-2 in non-small cell lung cancer: a comprehensive compartment-specific evaluation of tumor and stromal cell expression.

Cyclooxygenase-2 (COX-2) is an enzyme that has been extensively investigated as a prognostic marker in cancer. In non-small cell lung cancer (NSCLC) previous results regarding the prognostic impact of COX-2 expression are inconsistent. Therefore we evaluated the association between transcript levels and overall survival in nine publicly available gene expression data sets (total n = 1337) and determined in situ compartment-specific tumor and stromal cell protein expression in two independent cohorts (n = 616). Gene expression did not show any correlation with clinical parameters or with overall survival. Protein expression in tumor and stromal cells did not correlate with any clinical parameter or with overall survival in one of the analyzed cohorts, while a significant association of high stromal expression with longer survival was observed in both univariate and multivariate analysis in the other cohort. Stromal expression of COX-2 has not been separately evaluated in NSCLC previously and may be a subject of further investigation, whereas the presented findings from this comprehensive compartment specific evaluation clearly reject the hypothesis of COX-2 tumor cell expression having a prognostic value in NSCLC.

Alterations of INPP4B, PIK3CA and pAkt of the PI3K pathway are associated with squamous cell carcinoma of the lung.

The aim of the study was to investigate how alterations in the PI3K pathway correlate with non-small cell lung cancer subtypes squamous cell carcinoma (SSC) and adenocarcinoma (ADCA). We analyzed copy number variation and protein expression of INPP4B, protein expression of pAkt, PDPK1, and PTEN and mutational status of PIK3CA and PTEN in 180 cases. Nineteen% displayed loss of INPP4B copy, whereas 47% lacked expression, both showing correlation with SCC. Elevated pAkt expression was seen in 63% of all cases, also correlating to SCC. PDPK1 was expressed in 70%, more in male than female patients. Regarding PTEN, 50% displayed loss of expression, of which seven were identified with mutations in the phosphatase domain. We detected nine cases (5%) of PIK3CA mutations, all identified as the E545K hot spot mutation in the helical domain, all except one in SCC. When analyzing all PI3K pathway components together, we show that patients with at least one alteration in the PI3K pathway are twice as likely to have SCC, than ADCA. Interestingly, we also found a strong correlation between high pAkt expression and PTEN expression. As comparison, we also analyzed mitogen-activated protein kinase (MAPK) pathway genes, where we identified fifteen KRAS mutations (8%) and one BRAF mutation (1%), significantly associated to ADCA. No association was found to the Gly972Arg polymorphism of IRS-1, involved in activation of both PI3K and MAPK pathways. In conclusion, we show here that several components of the PI3K pathway, alone and in combination, are correlated to development of SCC of the lung.

Biomarker discovery in non-small cell lung cancer: integrating gene expression profiling, meta-analysis, and tissue microarray validation.

PURPOSE: Global gene expression profiling has been widely used in lung cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis and therapy response. So far, the value of these multigene signatures in clinical practice is unclear, and the biologic importance of individual genes is difficult to assess, as the published signatures virtually do not overlap. EXPERIMENTAL DESIGN: Here, we describe a novel single institute cohort, including 196 non-small lung cancers (NSCLC) with clinical information and long-term follow-up. Gene expression array data were used as a training set to screen for single genes with prognostic impact. The top 450 probe sets identified using a univariate Cox regression model (significance level P < 0.01) were tested in a meta-analysis including five publicly available independent lung cancer cohorts (n = 860). RESULTS: The meta-analysis revealed 14 genes that were significantly associated with survival (P < 0.001) with a false discovery rate <1%. The prognostic impact of one of these genes, the cell adhesion molecule 1 (CADM1), was confirmed by use of immunohistochemistry on tissue microarrays from 2 independent NSCLC cohorts, altogether including 617 NSCLC samples. Low CADM1 protein expression was significantly associated with shorter survival, with particular influence in the adenocarcinoma patient subgroup. CONCLUSIONS: Using a novel NSCLC cohort together with a meta-analysis validation approach, we have identified a set of single genes with independent prognostic impact. One of these genes, CADM1, was further established as an immunohistochemical marker with a potential application in clinical diagnostics.

Oestrogen receptor ß in NSCLC - prevalence, proliferative influence, prognostic impact and smoking.

In non-small-cell lung carcinoma (NSCLC) there are gender differences. The female gender is associated with more adenocarcinomas (ADCA), among both smokers and non-smokers compared to men. Women with NSCLC have a better prognosis compared to men, regardless of other factors. A possible role for oestrogen receptor (ER) signalling has been proposed. The role for ERß in NSCLC is still not clear, especially concerning the impact of smoking. In a material of NSCLC (n = 262), ERß and cyclins A1 and A2 were studied by immunohistochemistry on formalin-fixed paraffin embedded tissue. In 137 of those cases, frozen material was available, on which expression analysis of ESR2 (ERß) and cyclin A1 were performed. Data were correlated to histology, gender, smoking habits, stage and clinical outcome. ERß was expressed in 86% of the cases. ERß was most frequently expressed in Stage I ADCAs, especially in male subjects. A correlation between ERß expression and cyclins was observed in ADCA, also with a male predominance. ERß transcripts had a positive prognostic impact in ADCA. ERß transcripts were increased in NSCLC among smokers compared to non-smokers. In conclusion, our data support a role for ERß in lung ADCAs, proposing a role for ERß in lungcarcinogenesis, especially among smokers.

Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides.

Storage of tissue slides has been claimed to induce dramatically reduced antigen detection particularly for immunohistochemistry (IHC). With tissue microarrays, the necessity to serially cut blocks in order to obtain as much material as possible is obvious. The presumed adverse effect of storage might hamper such an approach. The authors designed an experimental setting consisting of four different storage conditions with storage time of tissue slides of up to 1 year. Detection of proteins, DNA, and mRNA was performed using IHC and in situ hybridization techniques. Slight but significant changes in IHC occurred over time. The most important factor is the primary antibody used: four showed no significant changes, whereas limited decreases in 8 antibodies could be detected by image analysis. Whether the antigen was nuclear or cytoplasmic/membranous did not matter. No major differences between different storage conditions could be shown, but storage at 4C was overall the best procedure. Furthermore, gene copy number aberrations, chromosomal translocations, and the presence of mRNA could be detected on slides stored up to 1 year. In conclusion, in tissues optimally formalin fixed and using modern histological techniques, only minute changes in tissue antigenicity are induced by long-term storage.

Dignified end-of-life care in the patients' own homes.

Nowadays it is increasingly common that the patients in the end of life phase choose to be cared for in their own home. Therefore it is vital to identify significant factors in order to prevent unnecessary suffering for dying patients and their families in end-of-life homecare. This study aimed to describe 10 nurses' perceptions of significant factors that contribute to good end-of-life care in the patients own home. The transcribed texts from the interviews' were analyzed using phenomenological hermeneutical method, which focuses on the life-world of human beings. The results demonstrate that good end-of-life care presupposes that the aim of the caring staff is to provide safety, autonomy and integrity for the patient and family in order to create the respect required for as good and dignified a death as possible.

Tissue microarray validation: a methodologic study with special reference to lung cancer.

BACKGROUND: Although tissue microarray (TMA) studies of histopathologic material have been frequently reported in studies of malignant diseases, the question of sample size (i.e., the diameter and the number of tissue cylinders investigated) has been rarely discussed. This study addresses the methodologic question of sample size in a variety of tumor types. MATERIAL AND METHODS: Material from 29 cases of lung carcinoma (small cell, squamous cell, and adenocarcinomas) was examined immunohistochemically for Ki-67 and p53 expression in virtually constructed cylinders of different diameters. The influence of tissue sample size (i.e., different numbers of virtual cylinders) was also investigated. Results from Ki-67 evaluation were analyzed as a continuous variable, whereas p53 expression was scored. p53 evaluations based on scoring in cylinders versus scoring of whole sections were also compared. Furthermore, 10 cases of endometrial and breast carcinomas were evaluated for estrogen receptor, Ki-67, and HER2 by scoring up to five cylinders. RESULTS AND CONCLUSIONS: Tissue cylinders of 0.6 and 1.0 mm diameters were compared and found equally informative about Ki-67 expression (intraclass correlation, 0.96). A statistical approach considering intraindividual and interindividual variation data is presented, indicating that in this specific setting three cylinders per case is an adequate sample size for TMA studies. Further sampling yields only a small gain in accuracy as determined by Ki-67 quantification and p53 scoring (kappa-coefficient, 0.9). For endometrial and breast tissues, TMA scoring of three cylinders yielded excellent agreement (kappa, >0.75) compared with whole-section scoring.