TERT promoter mutated circulating tumor DNA as a biomarker for prognosis in hepatocellular carcinoma.

BACKGROUND AND AIMS: Plasma circulating tumor DNA (ctDNA) with tumor-specific mutations is an attractive biomarker. The telomerase reverse transcriptase (TERT) C228T promoter mutation is the most prevalent tumor-associated mutation in hepatocellular carcinoma (HCC). We evaluated the presence and prognostic value of the TERT C228T mutation in plasma and tissue in a Danish HCC cohort. METHODS: We analyzed ctDNA from 95 HCC patients and 45 liver cirrhotic patients without HCC for the TERT mutation using droplet digital polymerase chain reaction. We also analyzed DNA from the corresponding primary tumor tissues in 34 HCC patients. RESULTS: The plasma TERT C228T mutation was detected in 42/95 HCC patients (44%) but in none of the non-HCC patients. The TERT mutation was detected in 23/34 tumor samples (68%). The TERT mutation was associated with increased mortality when detected in plasma (adjusted HR 2.16 (1.20-3.88), p = .010) but not in tumor tissue (adjusted HR 1.11 (0.35-3.56), p = .860). There was a positive correlation between the presence of the TERT mutation in plasma and an advanced TNM stage (p < .0001) and vascular invasion (p = .005). Analysis of the TERT mutation in plasma and tumor DNA from the same patient was concordant in 21/34 samples (62%; kappa value 0.31, p = .014). Non-concordance was associated with an early TNM stage. CONCLUSION: The plasma TERT mutation was detected in 44% of HCC patients and in none of non-HCC cirrhotic patients; and was associated with increased mortality. We propose the TERT C228T mutation in ctDNA as a promising HCC biomarker for prognosis.

Cytochrome P-450 2D6 (CYP2D6) Genotype and Breast Cancer Recurrence in Tamoxifen-Treated Patients: Evaluating the Importance of Loss of Heterozygosity.

Tamoxifen therapy for estrogen receptor-positive breast cancer reduces the risk of recurrence by approximately one-half. Cytochrome P-450 2D6, encoded by the polymorphic cytochrome P-450 2D6 gene (CYP2D6), oxidizes tamoxifen to its most active metabolites. Steady-state concentrations of endoxifen (4-hydroxy-N-desmethyltamoxifen), the most potent antiestrogenic metabolite, are reduced in women whose CYP2D6 genotypes confer poor enzyme function. Thirty-one studies of the association of CYP2D6 genotype with breast cancer survival have yielded heterogeneous results. Some influential studies genotyped DNA from tumor-infiltrated tissues, and their results may have been susceptible to germline genotype misclassification from loss of heterozygosity at the CYP2D6 locus. We systematically reviewed 6 studies of concordance between genotypes obtained from paired nonneoplastic and breast tumor-infiltrated tissues, all of which showed excellent CYP2D6 genotype agreement. We applied these concordance data to a quantitative bias analysis of the subset of the 31 studies that were based on genotypes from tumor-infiltrated tissue to examine whether genotyping errors substantially biased estimates of association. The bias analysis showed negligible bias by discordant genotypes. Summary estimates of association, with or without bias adjustment, indicated no clinically important association between CYP2D6 genotype and breast cancer survival in tamoxifen-treated women.

Increased Risk of Colorectal Cancer Development Among Patients With Serrated Polyps.

BACKGROUND & AIMS: Sessile serrated adenomas/polyps (SSA/Ps) and traditional serrated adenomas (TSAs) are now distinguished from hyperplastic polyps and recognized as precursors to colorectal cancer (CRC). We studied CRC risks associated with serrated polyps. METHODS: By using Danish databases (1977-2009), we conducted a nationwide population-based, case-control study nested within individuals who had received colonoscopies (n = 272,342), and identified 2045 CRC cases and 8105 CRC-free individuals (controls). For each case and control, we identified the first colorectal polyp(s) that underwent a biopsy or were excised during or after the initial colonoscopy, and obtained tissue blocks for hyperplastic lesions. Four expert pathologists reviewed these lesions using current terminology for serrated polyps. We used logistic regression to compute odds ratios (ORs) to associate the risk of CRC with polyp type and estimated the absolute risks by multiplying the risk in patients with no polyps by these ORs. RESULTS: Seventy-nine cases and 142 controls had SSA/Ps (OR, 3.07; 95% confidence interval [CI], 2.30-4.10). SSA/Ps with cytology markers of dysplasia were associated with a particularly high OR (4.76; 95% CI, 2.59-8.73). Women with SSA/P had a higher risk for CRC than men with SSA/P (OR for women, 5.05; 95% CI, 3.05-8.37 vs OR for men, 2.18; 95% CI, 1.24-3.82); patients with SSA/P proximal to the splenic flexure had the highest risk for CRC (OR, 12.42; 95% CI, 4.88-31.58). The OR for CRC was 4.84 in the 14 cases vs 17 controls with TSAs (95% CI, 2.36-9.93), 2.51 in the 757 cases vs 1698 controls with conventional adenomas (95% CI, 2.25-2.80), and 1.30 in the 55 cases vs 235 controls with hyperplastic polyps (95% CI, 0.96-1.77). The 10-year risk for CRC was 4.4% for patients with SSA/P with dysplasia, 4.5% for patients with TSAs, and 2.3% for patients with conventional adenomas. CONCLUSION: Patients with SSA/P or TSA are at increased risk for CRC; their level of risk is similar to or higher than that of patients with conventional adenomas.

Pak1, adjuvant tamoxifen therapy, and breast cancer recurrence risk in a Danish population-based study.

Background Adjuvant tamoxifen therapy approximately halves the risk of estrogen receptor-positive (ER+) breast cancer recurrence, but many women do not respond to therapy. Observational studies nested in clinical trial populations suggest that overexpression or nuclear localization of p21-activated kinase 1 (Pak1) in primary tumors predicts tamoxifen failure. Material and methods We measured the association between Pak1 expression and breast cancer recurrence in a Danish population-based case-control study. Pak1 cytoplasmic expression level and nuclear positivity were determined by immunohistochemical staining of primary breast tumors from recurrence cases and matched controls from two breast cancer populations; women diagnosed with ER-positive tumors who received at least one year of tamoxifen therapy (ER+/TAM+), and women diagnosed with ER-negative tumors who survived for at least one year (ER-/TAM-). Pak1 staining was assessed by a single, blinded pathologist, and associations were estimated with conditional logistic regression models. Results We included 541 recurrence cases and 1:1 matched controls from the ER+/TAM + group and 300 recurrence cases and 1:1 matched controls from the ER-/TAM - group. Pak1 cytoplasmic intensity was not associated with breast cancer recurrence in either group (ER+/TAM + ORadj for strong vs. no cytoplasmic staining = 0.91, 95% CI 0.57, 1.5; ER-/TAM - ORadj for strong vs. no cytoplasmic staining = 0.74, 95% CI 0.39, 1.4). Associations between Pak1 nuclear positivity and breast cancer recurrence were similarly near null in both groups. Conclusion Pak1 positivity in primary breast tumors was neither predictive nor prognostic in this prospective, population-based study.

High intratumoral macrophage content is an adverse prognostic feature in anaplastic large cell lymphoma.

AIMS: Macrophage infiltration has been associated with prognosis in several cancers, including lymphoma, but has not been assessed systematically in anaplastic large cell lymphoma (ALCL). The aim of the study was to correlate expression of the macrophage-associated antigens CD68 and CD163 with pre-therapeutic parameters and outcome in a cohort of treatment-naive ALCL patients. METHODS AND RESULTS: Pre-therapeutic tumour specimens from 52 patients with ALCL were included in a tissue microarray. The intratumoral macrophage content was assessed by immunohistochemical staining for CD68 and CD163, and quantified using digital image analysis. Anaplastic lymphoma kinase (ALK)-positive patients were significantly younger and had a favourable outcome compared with ALK-negative ALCL patients (median age: 42 versus 59 years; P = 0.008). However, ALK expression was not a significant predictor when adjusting for age. Although classical risk factors were distributed evenly between the compared groups, high intratumoral content of CD68 and/or CD163 correlated with poor outcome, in both univariate and multivariate analyses. High intratumoral CD163 content showed the strongest adverse association with both overall and progression-free survival in ALK-negative patients (P < 0.001). CONCLUSIONS: A high content of intratumoral CD68- and/or CD163-positive macrophages correlates with an adverse outcome in ALK-negative ALCL.

Automated digital volume measurement of melanoma metastases in sentinel nodes predicts disease recurrence and survival.

AIMS: Total metastatic volume (TMV) is an important prognostic factor in melanoma sentinel lymph nodes (SLNs) that avoids both the interobserver variation and unidirectional upstaging seen when using semi-quantitative size estimates. However, it is somewhat laborious for routine application. Our aim was to investigate whether digital image analysis can estimate TMV accurately in melanoma SLNs. METHODS AND RESULTS: TMV was measured in 147 SLNs from 95 patients both manually and by automated digital image analysis. The results were compared by Bland-Altman plots (numerical data) and kappa statistics (categorical data). In addition, disease-free and melanoma-specific survivals were calculated. Mean metastatic volume per patient was 10.6 mm(3) (median 0.05 mm(3); range 0.0001-621.3 mm(3)) and 9.62 mm(3) (median 0.05 mm(3); range 0.00001-564.3 mm(3)) with manual and digital measurement, respectively. The Bland-Altman plot showed an even distribution of the differences, and the kappa statistic was 0.84. In multivariate analysis, both manual and digital metastasis volume measurements were independent progression markers when corrected for primary tumour thickness [manual: hazard ratio (HR): 1.21, 95% confidence interval (CI): 1.07-1.36, P = 0.002; digital: HR: 1.21, 95% CI: 1.06-1.37, P = 0.004]. CONCLUSIONS: Stereology-based, automated digital metastasis volume measurement in melanoma SLNs predicts disease recurrence and survival.

XIAP-mediated caspase inhibition in Hodgkin's lymphoma-derived B cells.

The malignant Hodgkin and Reed-Sternberg cells of Hodgkin's lymphoma (HL) and HL-derived B cell lines were previously shown to be resistant to different apoptotic stimuli. We show here that cytochrome c fails to stimulate caspases-9 and -3 activation in cytosolic extracts of HL-derived B cells, which is due to high level expression of X-linked inhibitor of apoptosis (XIAP). Coimmunoprecipitation studies revealed that XIAP, apoptosis protease-activating factor-1, and caspase-3 are complexed in HL-derived B cell lysates. Even after stimulation with exogenous cytochrome c and dATP, XIAP impairs the proteolytic processing and activation of caspase-3. In cytosolic extracts, inhibition of XIAP by the second mitochondria-derived activator of caspases (Smac)/DIABLO, or immunodepletion of XIAP restores cytochrome c-triggered processing and activation of caspase-3. Smac or a Smac-derived agonistic peptide also sensitized intact HL-derived B cells for the apoptotic action of staurosporine. Finally, Hodgkin and Reed-Sternberg cells of primary tumor HL tissues also constitutively and abundantly express XIAP. The results of this paper suggest that high level XIAP expression is a hallmark of HL, which may play a crucial role in resistance to apoptosis.

Metastatic melanoma volume in sentinel nodes: objective stereology-based measurement predicts disease recurrence and survival.

AIMS: Sentinel lymph node (SLN) status is the most important prognostic factor in intermediate thickness melanoma. The amount of metastatic disease in positive SLNs varies greatly between patients, and this tumour burden appears to influence the prognosis of node-positive patients. The aim was to use objective stereological techniques to correlate accurately total SLN tumour burden with recurrence and patient survival. METHODS AND RESULTS: SLNs from 327 patients were examined by complete step sectioning and immunohistochemistry. The total metastasis volume (TMV) of 156 positive SLNs from 99 patients (30.3%) was measured using stereological methods based on the 2D-nucleator and Cavalieri's principle. The maximum metastasis diameter was also measured. These two measurements were correlated with disease recurrence and patient survival. The mean TMV for SLN+ patients was 10.5 mm(3) (median 0.05 mm(3); range 0.0001-623.7 mm(3)). Median follow-up was 26.3 months. On multivariate analysis, TMV was an independent predictor of recurrence when corrected for primary tumour thickness (P = 0.001) and was a stronger prognosticator compared with the maximum metastasis diameter (P < 0.0001 versus P = 0.01). CONCLUSIONS: Combining total step sectioning of SLNs with stereological assessment of metastases, we found metastasis volume to be a highly significant predictor of disease recurrence and survival.

Quantification of microRNA-21 and microRNA-125b in melanoma tissue.

Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series of benign melanocytic nevi. Using three different quantification methods [array analysis, quantitative PCR (qPCR) and in-situ hybridization (ISH) quantified by digital image analysis], we found considerable miRNA dysregulation in tumours. Using array analysis, samples mainly clustered according to their biological group (benign vs. malignant) and 77 miRNAs differed significantly between nevi and melanoma samples. Increase of miR-21 and miR-142, and decrease of miR-125b, miR-211, miR-101 and miR-513c in the melanomas were verified in both cohorts using qPCR, whereas the decrease of miR-205 observed with array analysis could not be confirmed using qPCR. ISH with digital quantification showed expression of miR-21 and miR-125b in the melanocytic lesions. miR-21 ISH was increased in melanomas, whereas quantification of miR-125b showed uniform ISH expression across nevi and melanomas. Our results support the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi.

Pathologic assessment of melanoma sentinel nodes: a role for molecular analysis using quantitative real-time reverse transcription-PCR for MART-1 and tyrosinase messenger RNA.

PURPOSE: Molecular analysis of melanoma sentinel nodes (SN) is sensitive, but poorly specific because metastases cannot be distinguished from benign nevus inclusions (BNI). We investigated whether quantitative reverse transcription-PCR (RT-PCR) detection of MART-1 and tyrosinase mRNAs could improve this specificity and contribute to SN assessment. EXPERIMENTAL DESIGN: Two hundred twenty SNs from 95 melanoma patients analyzed by extensive immunohistopathology and real-time quantitative RT-PCR. RESULTS: Using histopathology, SNs and patients were allotted to three diagnostic groups: (a) metastasis positive, (b) BNI positive, and (c) melanocyte-free. Median MART-1 and tyrosinase mRNA levels in SNs were significantly different in patients with metastasis compared with patients with BNIs (P < 0.05) and patients without melanocytic lesions (P < 0.001). However, a "gray-zone" was observed where distinction, based on mRNA levels, could not be made between the three groups. For both genes, the highest mRNA level recorded in each RT-PCR-positive patient was positively correlated with Breslow's tumor thickness. For SNs with metastases, tumor burden was significantly correlated to the mRNA level. Using the presence of a MART-1 RT-PCR signal to detect patients with metastases, a sensitivity of 100% and a negative predictive value of 100% were achieved when extensive immunohistology was used as reference. CONCLUSIONS: Quantitative RT-PCR MART-1 and tyrosinase mRNA analysis cannot be used alone for SN diagnosis because of its poor specificity for melanoma metastasis. However, in approximately one third of cases without RT-PCR evidence of MART-1 expression, extensive histopathologic SN investigation is not necessary, thus substantially reducing the cost of SN analysis. The level of melanocyte-associated mRNA is associated with both tumor thickness and tumor burden as measured histopathologically, suggesting that this may be of prognostic value.

Loss of B cell identity correlates with loss of B cell-specific transcription factors in Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma.

In classical Hodgkin lymphoma the malignant Hodgkin/Reed-Sternberg (HRS) cells characteristically constitute only a small minority of the tumour load. Their origin has been debated for decades, but on the basis of rearrangement and somatic hypermutations of their immunoglubulin (Ig) genes, HRS cells are now ascribed to the B-cell lineage. Nevertheless, phenotypically HRS cells have lost their B cell identity: they usually lack common B cell-specific surface markers such as CD19 and CD79a as well as Ig gene transcripts. Here we demonstrate that Ig promoters as well as both intronic and 3' enhancer sequences are transcriptionally inactive in HRS cell lines. This inactivity correlates with either reduced levels or even a complete lack of several B cell-specific transcription factors required for their expression: Oct-2, OBF-1, PU.1, E47/E12, PAX-5 and EBF. Moreover, we demonstrate that PU.1 and PAX-5 are significantly down-regulated in HRS cells in pathological specimens from primary tumour tissues. However, forced expression of these transcription factors can activate regulatory sequences of silenced B cell marker genes, and in one instance also transcription from a silenced endogenous locus. Thus, HRS cells are dedifferentiated B cells with global down-regulation of B cell-specific genes.

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction.

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

Human papillomavirus and the incidence of nonmelanoma and melanoma skin cancer using cervical conization as a surrogate marker: a nationwide population-based Danish cohort study.

PURPOSE: Human papillomavirus' (HPV's) role in skin cancer is controversial. To examine whether an individual is prone to develop a chronic oncogenic infection, we conducted a nationwide population-based cohort study of the risk of skin cancer after another HPV-related neoplasia-that is, cervical high-grade dysplasia or carcinoma-using cervical conization as a surrogate marker. METHODS: Using Danish registries, we identified all women who underwent conization from 1978 to 2011 (n = 87,164) and followed them until first-time skin cancer diagnosis, death, emigration, or 31 December 2011, whichever came first. We calculated standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) for basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and malignant melanoma (MM) according to national incidence rates. RESULTS: The 1-year absolute risks were 0.0012%, 0.045%, and 0.029% for SCC, BCC, and MM, respectively. Conization was clearly associated with increased incidence of SCC (SIR = 1.37; 95% CI: 1.13-1.65), but not MM (SIR = 1.00; 95% CI: 0.91-1.11). BCC risk was slightly increased (SIR = 1.08; 95% CI: 1.02-1.13). CONCLUSIONS: The association between conization and cutaneous SCC provides evidence for conization as a marker of underlying general susceptibility to oncogenic HPV.

Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction: a methodological study on lymph nodes from melanoma patients.

Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, beta-actin, and beta(2)-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

Quantification of melanoma mRNA markers in sentinel nodes: pre-clinical evaluation of a single-step real-time reverse transcriptase-polymerase chain reaction assay.

Although reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of melanocyte-associated mRNA can detect sentinel node melanoma metastases, most published assays are semi-quantitative methods of unknown sensitivity and precision, unsuitable for clinical use. We describe a single-step real-time quantitative RT-PCR assay for MART-1 and tyrosinase mRNAs, suitable for sentinel node analysis in a clinical setting. Using serial dilutions of melanoma cell line SK-MEL-28 RNA in water as a calibrator, we obtained linear calibration curves covering the range 0.5 to 10,000 arbitrary units (SK-MEL-28 melanoma cell equivalents). The sensitivity limit was 0.32 (MART-1) and 5 (tyrosinase) arbitrary units. Analytical imprecision was between 11% and 34%. MART-1 PCR efficiency was unaffected when samples were diluted with negative lymph node RNA rather than water, whereas tyrosinase PCR efficiency was halved. To evaluate the clinical suitability of our assay, we quantified melanocyte mRNAs in sentinel nodes with histologically verified micrometastases (n = 10) and benign nevus inclusions (n = 10), and in sentinel nodes without evidence of intranodal melanocytes (n = 10). We found significant differences in median melanocyte-derived mRNA levels comparing the three types of lymph nodes, suggesting that this quantitative molecular protocol may increase assay precision and be useful for the clinical evaluation of sentinel nodes.

The influence of immunohistochemistry on mRNA recovery from microdissected frozen and formalin-fixed, paraffin-embedded sections.

Laser-assisted microdissection (LAM) is now widely used to obtain specific cell populations from heterogeneous tissues. A major disadvantage of LAM is poor tissue morphology during microscopy, in part because coverslips are not used. Immunohistochemical labeling can improve identification of target cells but may affect the subsequent analysis of the microdissected tissue. We studied the effect of immunohistochemistry (IHC) on mRNA recovery from labeled cells after microdissection from both frozen and formalin-fixed, paraffin-embedded (FFPE) sections, using Melan-A and Ki-67 staining in lymph nodes with metastatic melanoma as a model. We developed rapid protocols for immunostaining in an attempt to limit loss of mRNA during procedures. A sensitive real-time quantitative reverse transcription-PCR was used to measure mRNA. We found a marked decrease in the mRNA yield from 500 microdissected cells from frozen and paraffin sections after immunostaining for both markers. Recovery of mRNA decreased by up to 89%, comparing the immunostained with the routinely stained sections. Interestingly, the ratio between mRNA for the two markers was similar in all stains, indicating that immunostained sections may be used for mRNA analysis. We also investigated the effect of storing membrane-mounted sections for microdissection under different conditions. Slides mounted with paraffin sections could be stored at room temperature for up to 90 days with no significant decrease in mRNA recovery.

Nodal distribution, stage migration due to diameter measurement and the prognostic significance of metastasis volume in melanoma sentinel lymph nodes: a validation study.

To validate metastasis location, maximum metastasis diameter (MMD) measurement induced stage migration and the prognostic significance of total metastatic volume (TMV) in melanoma sentinel lymph nodes (SLNs). A new 227 patient SLN cohort examined by complete step sectioning (250 µm) was tested for TMV and MMD prognostic significance; metastasis location by three different three-level protocols (Central vs Peripheral vs Global); and potential treatment changing down-staging by two restricted five-level protocols [Reduced Central (250 µm) vs Reduced Even (500 µm) sections]. Both TMV and MMD independently predicted recurrence (TMV, hazard ratio (HR): 1.21; 95% confidence interval (CI) 1.07-1.37; p = 0.003; and MMD, HR: 1.46; 95% CI 1.08-1.98, p = 0.02) and melanoma-specific death (TMV, HR: 1.30; 95% CI 1.11-1.51; p = 0.001; and MMD, HR: 1.82; 95% CI 1.25-2.66, p = 0.002). The Central, Peripheral and Global protocols detected 72%, 76% and 83% of metastases found by complete step sectioning. Based on MMD, using the Reduced Central or Reduced Even protocols, potential treatment changing down-staging occurred in 20 (20%) or 13 (13%) of SLN-positive patients. This validation study establishes that: (i) metastases are globally located in melanoma SLNs; (ii) MMD but not TMV leads to uni-directional stage migration; and (iii) TMV analysis is of prognostic significance.

Digital pathology for the validation of tissue microarrays in peripheral T-cell lymphomas.

Systematic validation of construction and analysis parameters when using tissue microarray (TMA) in rare, morphologically heterogenous entities such as peripheral T-cell lymphoma (PTCL) is not reported. We describe a tissue-saving virtual TMA to predetermine the number of cores needed to represent whole tissue sections (WTS) from the same biopsies, using automated and traditional manual methods for the quantification of immunohistochemical stains. Whole paraffin hematoxylin and eosin- and immunohistochemical (CD2, CD30, and Ki-67)-stained sections from 30 PTCLs were digitalized. A virtual TMA with six 1-mm cores per slide was designed to compare agreements in the immunohistochemical scoring. Using digital image analysis and manual stereological counting, immunohistochemical positivity was quantified. Associations were analyzed using the Bland-Altman and correlation plots. In PTCL, we report that 4 cores are required to represent WTS results (ie, agreement within ±10%). High concordance was demonstrated between digital results obtained with WTS compared with 4-core virtual TMA (correlation coefficients: 0.89-0.98), and in the comparative evaluation of 4-core virtual TMA by digital image analysis versus manual stereology (correlation coefficients: 0.91 to 0.99). Virtual TMAs provide an efficient tool for optimizing and validating TMA construction parameters when planning a study. The method can be applied to the same tissues used in a subsequent formal study, without wasting scarce tissue resources. In PTCL, TMAs constructed with four 1-mm cores are representative of WTS. In parallel tests using TMAs and WTS from PTCLs, there is a high level of agreement comparing automated digital with manual stereological methods for the quantification of immunohistochemical biomarker staining.

Interactions between immunity, proliferation and molecular subtype in breast cancer prognosis.

BACKGROUND: Gene expression signatures indicative of tumor proliferative capacity and tumor-immune cell interactions have emerged as principal biology-driven predictors of breast cancer outcomes. How these signatures relate to one another in biological and prognostic contexts remains to be clarified. RESULTS: To investigate the relationship between proliferation and immune gene signatures, we analyzed an integrated dataset of 1,954 clinically annotated breast tumor expression profiles randomized into training and test sets to allow two-way discovery and validation of gene-survival associations. Hierarchical clustering revealed a large cluster of distant metastasis-free survival-associated genes with known immunological functions that further partitioned into three distinct immune metagenes likely reflecting B cells and/or plasma cells; T cells and natural killer cells; and monocytes and/or dendritic cells. A proliferation metagene allowed stratification of cases into proliferation tertiles. The prognostic strength of these metagenes was largely restricted to tumors within the highest proliferation tertile, though intrinsic subtype-specific differences were observed in the intermediate and low proliferation tertiles. In highly proliferative tumors, high tertile immune metagene expression equated with markedly reduced risk of metastasis whereas tumors with low tertile expression of any one of the three immune metagenes were associated with poor outcome despite higher expression of the other two metagenes. CONCLUSIONS: These findings suggest that a productive interplay among multiple immune cell types at the tumor site promotes long-term anti-metastatic immunity in a proliferation-dependent manner. The emergence of a subset of effective immune responders among highly proliferative tumors has novel prognostic ramifications.

Internal gallbladder drainage prevents development of acute cholecystitis in a pig model: a randomized study.

BACKGROUND: Acute cholecystitis can be the result of retention of bile in the gallbladder with possible secondary infection and ischaemia. The aim of the present study was to investigate whether internal drainage of the gallbladder could protect against the development of acute cholecystitis in a pig model. MATERIALS AND METHODS: Twenty pigs were randomized to either internal drainage (drained) or not (undrained). Day 0 acute cholecystitis was induced by ligation of the cystic artery and duct together with inoculation of bacteria. Four days later the pigs were killed and the gallbladders were removed and histologically scored for the presence of cholecystitis. Bile and blood samples were collected for bacterial culturing and biochemical analyses. RESULTS: The histological examination demonstrated statistical significant differences in acute cholecystitis development between groups, the degree of inflammation being highest in undrained pigs. There were no differences in bacterial cultures between the two groups. CONCLUSION: Internal drainage of the gallbladder protected against the development of acute cholecystitis in the present pig model. These findings support the theory that gallstone impaction of the cystic duct plays a crucial role as a pathogenetic mechanism in the development of acute cholecystitis and suggest that internal drainage may be a way to prevent and treat acute cholecystitis.