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Introduction: Treatment and surveillance of hepatocellular carcinoma (HCC) is associated with significant costs, both financially and personally for the patient. Therefore, it is crucial to evaluate the clinical response to treatment thoroughly. We have yet to identify a biomarker able to detect progression, relapse or stable disease in HCC, but circulating tumor DNA (ctDNA) may be such a biomarker. Potential advantages are; easy accessibility, safety/non-invasive sampling, high specificity, possibility of sequential testing and circumvention of problems with tumor heterogeneity. The mutational landscape of HCC is dependent of the etiology of any preexisting liver disease, which varies substantially among different parts of the world. Previous studies on ctDNA in HCC arises from areas with a high prevalence of chronic hepatitis B. In Denmark, the etiology of liver cirrhosis and HCC is primarily alcohol consumption (ALC), hepatitis C (HCV) and non-alcoholic steatohepatitis (NASH). Additionally, an increasing number of elderly patients are diagnosed with HCC without underlying cirrhosis. CTNNB1, a gene in the β-catenin pathway, harbors mutations in up to 10-25 % of HCC tissue samples. It has yet to be established how this affects the prognosis. Until now, no targeted treatment against CTNNB1 mutations has been approved. Methods: Blood samples were obtained from HCC patients before treatment start (radiofrequency ablation (31%), resection (10%), transarterial chemoembolization (19%), Sorafenib (30%) or selective internal radiation therapy (4%)), 1 month and every 6 months after treatment. Some patients discontinued treatment before follow up (8.4%). Cell free DNA was purified from 4 mL plasma. The droplet digital PCR technique was used for detection of CTNNB1 c. 121A>G (p.T41A) mutated DNA fragments. Results: The study is ongoing. So far, we have included 84 HCC patients, 63 males (75%), mean age 69 (range 35-85). Etiology of cirrhosis and HCC is ALC alone n=31 (37%), ALC and HCV n=6 (8%), HCV alone n=7 (8%), NASH n=2 (2%), autoimmune n=4 (5%), hemochromatosis n=1 (1%), undetermined n=6 (7%) and non-cirrhosis n=27 (32%). Child Pugh stage: 26 stage A, 27 stage B and 4 stage C, and 27 non-cirrhotic patients. Barcelona Clinic Liver Cancer stage: 11 stage 0, 13 stage A, 9 stage B, 47 stage C, 4 stage D. Nine patients (11%) had detectable p.T41A mutation in CTNNB1 at baseline. One patient underwent resection and in follow up samples the mutation was undetectable and with no signs of relapse on simultaneous diagnostic imaging. One patient received radiofrequency ablation and had no detectable mutations in follow up samples. One patient remained positive after treatment with Sorafenib. One patient received transarterial chemoembolization and had a decline in amount of mutated DNA in follow up samples but the mutation were still detectable. The remaining five patients terminated treatment before follow up samples were obtained. Conclusion: ctDNA can be detected in a Danish cohort of patients with HCC with alcohol as primary cirrhosis etiology. The frequency of patients harboring the CTNNB1 p.T41A mutation in their plasma corresponds to the preexisting literature and databases on genetic features of HCC tissue samples. The first steps towards an individualized surveillance and possible treatment has been taken. Further studies on larger cohorts and longer time of follow up is needed in order to determine whether ctDNA and the CTNNB1 p.T41A mutation can be used to predict the clinical course of HCC.

Forty-two cases of Chinese T-cell lymphoma were studied for expression of Epstein-Barr virus (EBV) encoded RNA (EBER-1) and EBV latent membrane protein-1 (LMP-1) using in situ hybridization and immunohistochemistry, respectively. EBV was detected in tumour cells in 24/39 peripheral T-cell lymphomas (62%), comprising 18/27 pleomorphic, medium and large cell lymphomas (67%), 4/6 angioimmunoblastic lymphadenopathy-like lymphomas (67%), 2/2 Lennert's lymphomas, 0/2 anaplastic large cell lymphomas, and 0/2 T-zone lymphomas. EBV was not found in three T-lymphoblastic lymphomas. EBV was associated with 12/24 nodal (50%) compared with 12/15 extranodal (80%) peripheral T-cell lymphomas. In EBV positive nodal lymphomas, 9/12 cases (75%) contained less than 10% EBER positive tumour cells. In EBV positive extranodal lymphomas, 9/11 cases (82%) showed EBV gene expression in more than 50% of the tumour cells, and in five of these almost all tumour cells were positive. Lymphomas of the nasopharynx (mainly midline granuloma-type) showed EBER-1 expression in nearly all tumour cells. LMP-1 was detected in 19/23 EBER positive peripheral T-cell lymphomas (83%). Our results show that EBV is strongly associated with peripheral T-cell lymphomas in Chinese. An important role for the virus is suggested in lymphomas of the nasopharynx. The significance of EBV in T-cell lymphomas that contain only a minor population of virally infected tumour cells is currently unclear.

Abstract 269 Introduction: Markers in the human leukocyte antigen (HLA) class I locus and infectious mononucleosis (IM) have both been associated with Epstein-Barr (EBV)-positive Hodgkin lymphoma (HL). IM is caused by late infection with EBV. To further explore associations between HLA class I genes and EBV-positive HL and to investigate potential interactions between genetic (HLA) and environmental risk factors in the development of HL, we performed a case-only study of 934 HL cases. Methods: HLA-A and B typing were performed by PCR sequence specific oligonucleotide assay. Logistic regression was used to estimate odds ratios (ORs) of EBV-positive HL relative to EBV-negative HL for HLA-A and B genotypes and other known risk factors. Results: Increasing age, male sex, HLA-A*01 and a previous history of IM were all associated with a statistically significant increased odds of EBV-positive HL while HLA-A*02 and HLA-B*07 were associated with a protective effect. Significant interactions were detected between HLA-A*01 and HLA-B*07 and between prior IM and HLA-A*02, such that HLA-B*07 abolished the increased odds associated with HLA-A*01 and HLA-A*02 abrogated the increased odds associated with prior IM. In our final model of EBV-positive HL, HLA-B*07 was associated with a dominant protective effect (OR=0.57, 95% confidence interval (CI): 0.34–0.94); HLA-A*02 was associated with a protective effect which was additive (for individuals with no prior IM, ORper allele =0.69, 95% CI: 0.50–0.97); and HLA-A*01 was associated with an increased odds in patients who were HLA-B*07-negative (ORper allele=2.15, 95% CI: 1.54–3.01). Prior IM was independently associated with EBV-positive HL (OR=3.35, 95% CI: 1.67–6.73). This resulted in a >32-fold higher odds of EBV-positive HL in HLA-A*01 homozygotes with IM compared to HLA-A*02 homozygotes without IM. Conclusions: We have identified strong associations between both genetic and environmental risk factors and EBV-positive HL. Our findings suggest that HLA class I-restricted, cell-mediated responses to EBV and a prior history of IM play critical roles in the pathogenesis of EBV-positive HL. Disclosures: No relevant conflicts of interest to declare.

Udgivelsesdato: 2003-Feb 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 (

Background: Many suggestions of how and when to assess the regenerated liver in animal models have been proposed. The aim of the present study was to evaluate the natural history of liver regeneration in healthy rats, regarding size, weight and functional capacity. The study was meant as a precursor for further interventional studies in healthy and cirrhotic animals.Material and Methods: Partial hepatectomy (PHx) of 70% was performed on 64 rats. After PHx the animals were randomised into 8 groups for evaluation. The animals in each group were evaluated on same postoperative day (POD) from POD 1 to 8. Results: At PHx the animals had a mean body weight of 238.4 g (211.4;286.5). Mean weight of resected liver was 6.25 g (5.2;7.3) and an estimated total liver weight of 8.9 g. Liver weight analysis showed an ascending curve, with max slope POD 1-3, reaching a steady state of ca 9 g on day 5-8. When correlated to body weight, through regeneration rate, a similar pattern was seen, with a RR of 100% reached day 5-8.Interleukin-6 (Il-6) peaked POD 1 and was falling to undetectable levels day 4. Alfa-2-Macroglobulin (α-2-M) levels were high POD 1-3 before falling to low levels. Tumor-necrosis-factor-alpha (Tnf-α) was undetectable throughout the period. At the moment functional capacity of the regenerating liver, is analysed by the enzymatic method BTR (Ratio of Branched-Chain Amino Acids to Tyrosine) Stereological aspects are looked into regarding KI-67 analysis and blood vessel density estimation, as is more serological markers (Alanin-amino-transferase, Bilirubin, Alkaline-phosfatase and Hepatocyte-growth-factor).Conclusion: Liver regeneration, regarding size and weight, were at top speed day 1-3. Full liver regeneration reached a plateau after 5-8 days, and should after this point be considered practically at end.Liver regeneration should in further studies be evaluated on postoperative day 1, 3 and 8.

Undifferentiated salivary gland carcinomas may be divided into small cell and large cell types. Among large cell undifferentiated carcinomas, lymphoepithelial carcinomas have to be distinguished, the latter of which are endemic in the Arctic regions and southern China where virtually all cases of these tumors are associated with the Epstein-Barr virus (EBV). Association with EBV may also be observed in sporadic cases, and detection of EBV gene products may aid their diagnosis. Immunohistology may be employed to resolve the differential diagnosis of undifferentiated salivary gland carcinomas, comprising malignant lymphomas, amelanotic melanomas, Merkel cell carcinomas, and adenoid cystic carcinomas, in particular in small biopsy materials. Because of the rarity of undifferentiated salivary gland carcinomas, the differential diagnosis should always include metastases of undifferentiated carcinomas arising at other primary sites, particularly when expressing the thyroid transcription factor-1 (TTF-1) Udgivelsesdato: 2004

We investigated the association of Epstein-Barr virus (EBV) with African cases of Burkitt's lymphoma (BL) and high grade B cell non-Burkitt's lymphoma (non-BL) occurring in areas where BL is endemic. The presence of EBV genomes was analysed in 24 cases using in situ hybridization with a 35S-labelled EBV probe applied to paraffin sections. EBV DNA was detected in each of 10 cases of BL in which technically satisfactory results were obtained, the virus being homogeneously distributed in all identifiable tumour cells. Two other cases of BL could not be evaluated because of technical problems. In contrast, EBV DNA was not detected in any case of high-grade non-BL (10 centroblastic and two immunoblastic lymphomas). These results confirm previous reports of the strong association of EBV with endemic BL, but suggest that the virus is not important in the pathogenesis of other types of African high-grade B cell lymphoma from regions where BL is endemic.

Some acquired immunodeficiency syndrome (AIDS)-related lymphomas (ARLs) are infected with Epstein-Barr virus (EBV), although the frequency and importance of this association is disputed. Using paraffin section RNA in situ hybridization (ISH) with digoxigenin-labeled riboprobes, we screened 16 central nervous system (CNS) non-Hodgkin's lymphomas (NHLs), 101 systemic NHLs, and 11 Hodgkin's disease cases arising in human immunodeficiency virus-seropositive individuals for EBV-encoded small RNA (EBER 1) expression, an EBV gene product transcribed in abundance during latent infection. Tumor cells contained EBV in 85 of 128 ARLs (66%), but infection rates differed with lymphoma type. EBER 1 was expressed in tumor cells in 11 of 11 Hodgkin's disease cases (100%), 15 of 16 CNS NHLs (94%), and 46 of 60 systemic immunoblast- rich/large-cell lymphomas (77%), but in only 12 of 35 Burkitt-type (small noncleaved cell) (34%) and 1 of 6 monomorphic centroblastic (diffuse large noncleaved cell) (17%) lymphomas. In most EBV-positive ARLs, all recognizable viable tumor cells expressed EBER 1. We conclude that (1) EBV infects tumor cells in all AIDS-related Hodgkin's disease cases, in virtually all primary CNS ARLs, and in most systemic immunoblast-rich/large-cell ARLs; (2) only a minority of Burkitt-type and monomorphic centroblastic lymphomas are associated with EBV; and (3) EBER-ISH is ideal for the histopathologic detection of latent EBV in routine tissue specimens.

Biological tissues (in particular those affected by disease) are inherently complex mixtures of different cell types and matrices. This heterogeneity can complicate the interpretation of molecular biological studies performed on whole-tissue extracts if the precise cellular origin of the molecules being tested is not known. Laser-assisted microdissection (LAM) has emerged as a leading histological technique for obtaining samples enriched for specific target cell populations or tissue components for subsequent molecular (especially polymerase chain reaction-based) analysis. This method allows the identification and study of target-specific molecular alterations in heterogeneous specimens, and enables more accurate detection and quantification of target molecules. In this chapter, we focus on tissue microdissection performed with an ultraviolet laser system and describe protocols for the basic procedure and for handling of the samples.

Melanoma metastasis burden: Let the computer do the counting.

To investigate the range of pathology shown by acquired immune deficiency syndrome (AIDS)-related lymphomas arising in an epidemiologically well-defined group of patients, all cases of lymphoma recognized in Danish human immunodeficiency virus (HIV)-infected individuals up to the end of 1988 were studied. Twenty-seven cases (26 high-grade non-Hodgkin's lymphoma [NHL], 1 Hodgkin's disease) were found, to give a cumulative incidence rate of 8% among Danish AIDS patients. Morphologically most NHL patients were classified into two groups: 1) high-grade tumors with a predominant population of immunoblasts, either monomorphic or more often polymorphic with plasmacytic differentiation; 2) Burkitt-type. Of 26 NHLs, 22 had a B-cell paraffin-section immunophenotype and 4 were non-B, non-T. Epstein-Barr virus (EBV) DNA was demonstrated in tumor cells of 12 of 24 cases (50%) using in situ nucleic acid hybridization with a 35S-labeled probe in paraffin sections. Epstein-Barr virus DNA was found in 65% of group 1 and 20% of group 2 tumors. This study suggests the existence of two main groups of AIDS-related lymphoma with different pathogeneses. First there are immunoblast-rich lesions, which usually are associated with EBV and morphologically resemble lymphomas described in immunosuppressed organ-transplantation patients. Second there are Burkitt-type tumors in which EBV sequences are less common and that may be pathogenetically analogous to sporadic Burkitt's lymphoma.

Infectious mononucleosis, which is caused by the Epstein-Barr virus, has been associated with an increased risk for Hodgkin's disease. Little is known, however, about how infectious mononucleosis affects long-term risk of Hodgkin's disease, how this risk varies with age at infectious mononucleosis diagnosis, or how the risk for Hodgkin's disease varies in different age groups. In addition, the general cancer profile among patients who have had infectious mononucleosis has been sparsely studied.Population-based cohorts of infectious mononucleosis patients in Denmark and Sweden were followed for cancer occurrence. The ratio of observed-to-expected numbers of cancers (standardized incidence ratio [SIR]) served as a measure of the relative risk for cancer. SIRs of Hodgkin's disease in different subsets of patients were compared with the use of Poisson regression analysis. All statistical tests including the trend tests were two-sided.A total of 1381 cancers were observed during 689 619 person-years of follow-up among 38 562 infectious mononucleosis patients (SIR = 1. 03; 95% confidence interval [CI] = 0.98-1.09). Apart from Hodgkin's disease (SIR = 2.55; 95% CI = 1.87-3.40; n = 46), only skin cancers (SIR = 1.27; 95% CI = 1.13-1.43; n = 291) occurred in statistically significant excess. In contrast, the SIR for lung cancer was reduced (SIR = 0.71; 95% CI = 0.58-0.86; n = 102). The SIR for Hodgkin's disease remained elevated for up to two decades after the occurrence of infectious mononucleosis but decreased with time since diagnosis of infectious mononucleosis (P: for trend.001). The SIR for Hodgkin's disease tended to increase with age at diagnosis of infectious mononucleosis (P: for trend =.05). Following infectious mononucleosis, the SIR for Hodgkin's disease at ages 15-34 years was 3.49 (95% CI = 2.46-4.81; n = 37), which was statistically significantly higher than the SIR for any other age group (P: for difference =.001).The increased risk of Hodgkin's disease after the occurrence of infectious mononucleosis appears to be a specific phenomenon.

Background: Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is used to accelerate growth of the future liver remnant. We investigated alternative methods for increasing the future liver remnant. Methods: A total of 152 rats were randomized as follows: (1) sham; (2) portal vein ligation; (3) portal vein ligation/surgical split (ALPPS); (4) portal vein ligation/split of the liver with a radiofrequency ablation needle; (5) portal vein ligation/radiofrequency ablation of the deportalized liver (portal vein ligation/radiofrequency ablation necrosis in the deportalized liver); (6) portal vein ligation/radiofrequency ablation of the future liver remnant (portal vein ligation/radiofrequency ablation-future liver remnant); and (7) controls. Animals were evaluated on postoperative days 2 and 4. Bodyweight, liver parameters, hepatic regeneration rate, proinflammatory cytokines, hepatocyte proliferation, and gene expression were measured. Results: Hepatic regeneration rate indicated a steady increase in all intervention groups compared with sham rats (P < .001). At postoperative day 2, the hepatic regeneration rate was significantly higher in the portal vein ligation/radiofrequency ablation necrosis in the deportalized liver group than in the portal vein ligation group (P = .039). On postoperative day 4, we found significant differences between the portal vein ligation group and the ALPPS (P = .015), portal vein ligation/split of the liver with a radiofrequency ablation needle (P = .010), and portal vein ligation/radiofrequency ablation necrosis in the deportalized liver (P = .046) groups. Hepatocyte proliferation was significantly higher at all times compared with sham rats. On postoperative day 4, we found a significantly higher proliferation in groups 3, 4, 5, and 6 compared to portal vein ligation. Gene analysis revealed upregulation of genes involved in cellular proliferation and downregulation of genes involved in cellular homeostasis in all intervention groups. Between the intervention groups, gene expression was nearly identical. Biochemical markers and proinflammatory cytokines were comparable between groups. Conclusion: The surplus liver regeneration after ALPPS is probably mediated through parenchymal damage and subsequent release of growth stimulators, which again upregulates genes involved in cellular regeneration and downregulates genes involved in cellular homeostasis. We also demonstrate that growth of the future liver remnant, comparable to that seen after ALPPS, could be achieved by radiofrequency ablation treatment of the deportalized liver, that is, a procedure in which the initial step in humans can be performed percutaneously.

Udgivelsesdato: april

The Epstein-Barr virus (EBV) is associated with an increasing range of reactive and neoplastic lesions. There is a need for a sensitive and specific method for detecting latent EBV in routine histological sections. We report the use of a highly sensitive paraffin section RNA/RNA in situ hybridization (ISH) technique using digoxigenin-labelled antisense riboprobes for demonstrating EBV encoded small RNAs (EBERs), EBV gene products that are transcribed in abundance during latent EBV infection. We applied EBER-ISH to 846 paraffin embedded specimens, including cases of reactive lymphoid hyperplasia (n = 28), infectious mononucleosis (16), Burkitt's lymphoma (44), immunodeficiency-associated lymphomas in transplant recipients (9) and AIDS patients (128), Hodgkin's disease (130), CD30 antigen positive lymphomas (106), peripheral T-cell lymphomas (104), sporadic B-cell non-Hodgkin's lymphomas (162), undifferentiated nasopharyngeal carcinoma (86), salivary gland lymphoepithelioma (11), and oral hairy leukoplakia (5). Strong, reproducible EBER staining was seen in EBV latently infected cells in archival surgical biopsy and autopsy specimens. EBER-ISH is specific, has a sensitivity comparable to that of the polymerase chain reaction, and is now the method of choice for the in situ detection of latent EBV infection.

Udgivelsesdato: januar

8528 Background: Classical Hodgkin lymphoma (cHL) is characterized by a minority of neoplastic cells surrounded by a heterogeneous background of non-neoplastic cells. A possible prognostic role of LAMs in cHL and follicular lymphomas has been suggested. CD163 is a haemoglobin scavenger receptor expressed by monocytes/macrophages. A high CD163 expression has been observed in macrophages belonging to the “alternative activation” pathway suggested to have pro-tumoral effect. The aim of the present study was to correlate the expression of the tumor-infiltrating CD163+ LAMs with clinico-pathological features and prognosis in a cohort of previously untreated HL patients (pts). Methods: A tissue microarray (TMA) was constructed with paraffin embedded tissue specimens from 286 cHL cases. CD163 expression was assessed immunohistochemically and the degree of intratumoral LAM infiltration was scored semi-quantitatively. Clinical data were obtained from the Danish population-based lymphoma registry and clinical records. All pts were homogeneously treated with either chemo-radiotherapy (localised disease) or ABVD chemotherapy (advanced disease). Results: The median age was 37 yrs (range: 6–86 yrs). The M:F ratio was 1.2. The histological subtypes were: nodular sclerosis (NS)-type I, 167 cases (59 %); NS-type II, 71 (25%); mixed cellularity (MC), 44 (15 %); lymphocyte-rich, lymphocyte-depleted and cHL-NOS, each one case. Of 253 pts with assessable International Prognostic Score (IPS), 204 had a low-risk (≤ 2) and 49 a high risk (>2) profile. A high number of tumour-infiltrating CD163+ cells was associated with a significantly poorer overall and event-free survival (p=0.029 and p=0.018, respectively). Furthermore, a high expression of CD163 strongly correlated to stage IV disease (p=0.035), presence of B-symptoms (p=0.008), lymphocytopenia (p=0.003), hypersedimentation (p=0.009). In a multivariate analysis, IPS score > 2 remained the strongest predictor. Conclusions: In cHL, a high number of intratumoral CD163+ monocytes/macrophages correlates with adverse outcome and with clinical parameters reflecting underlying aggressive disease biology. No significant financial relationships to disclose.

A post-mortem study of 17 AIDS-related brain lymphomas was carried. These were B-cell lymphomas of the immunoblastic-type. They expressed activation and adhesion markers, and EBV latent gene products.

Galectin-1 (Gal-1) has been associated with prognosis in several cancers including lymphomas. CD30-dependent cell signaling mediated by Gal-1 has been proposed in CD30-positive lymphomas and cell lines. Still, Gal-1 expression has not been systematically assessed as a possible prognosticator in nodal peripheral T-cell lymphomas (PTCL). Tissue micro arrays with pre-therapeutic tumor specimens from 169 patients with nodal PTCL (n=29 AITL, n=37 ALK-negative ALCL, n=14 ALK-positive ALCL, and n=83 PTCL-NOS) were assessed for intratumoral Gal-1 expression by immunohistochemistry and quantified using digital image analysis. For outcome analysis Gal-1 expression in tumors was categorized in two groups i.e. i) the quartile of patients with highest expression values (Gal-1 high) and ii) the lower three quartiles taken as one group (Gal-1 low). Overall survival (OS) in high and low Gal-1 groups was compared in the entire cohort and in CD30-positive subsets of PTCL. Gal-1 stains were evaluable in 163 cases (96%). The Gal-1 expression level was significantly different between PTCL subtypes (P