Genomic alterations accompanying tumour evolution in colorectal cancer: tracking the differences between primary tumours and synchronous liver metastases by whole-exome sequencing.

BACKGROUND: Colorectal cancer (CRC) patients with metastatic disease can become cured if neoadjuvant treatment can enable a resection. The search for predictive biomarkers is often performed on primary tumours tissue. In order to assess the effectiveness of tailored treatment in regard to the primary tumour the differences in the genomic profile needs to be clarified. METHODS: Fresh-frozen tissue from primary tumours, synchronous liver metastases and adjacent normal liver was collected from 21 patients and analysed by whole-exome sequencing on the Illumina HiSeq 2500 platform. Gene variants designated as 'damaging' or 'potentially damaging' by Ingenuity software were used for the subsequent comparative analysis. BAM files were used as the input for the analysis of CNAs using NEXUS software. RESULTS: Shared mutations between the primary tumours and the synchronous liver metastases varied from 50 to 96%. Mutations in APC, KRAS, NRAS, TP53 or BRAF were concordant between the primary tumours and the metastases. Among the private mutations were well-known driver genes such as PIK3CA and SMAD4. The number of mutations was significantly higher in patients with right- compared to left-sided tumours (102 vs. 66, p = 0.004). Furthermore, right- compared to left-sided tumours had a significantly higher frequency of private mutations (p = 0.023). Similarly, CNAs differed between the primary tumours and the metastases. The difference was mostly comprised of numerical and segmental aberrations. However, novel CNAs were rarely observed in specific CRC-relevant genes. CONCLUSION: The examined primary colorectal tumours and synchronous liver metastases had multiple private mutations, indicating a high degree of inter-tumour heterogeneity in the individual patient. Moreover, the acquirement of novel CNAs from primary tumours to metastases substantiates the need for genomic profiling of metastases in order to tailor metastatic CRC therapies. As for the mutational status of the KRAS, NRAS and BRAF genes, no discordance was observed between the primary tumours and the metastases.

Do risk factors for epithelial ovarian cancer have an impact on prognosis? Focus on previous pelvic surgery and reproductive variables.

OBJECTIVES: The prognostic impact of risk factors for ovarian cancer development is sparsely explored, but previous sterilisation has been shown to have a negative impact on survival. METHODS: Ovarian cancer cases were from the Danish MALOVA study. Information on previous pelvic surgery as well as reproductive variables was obtained from a personal interview conducted closely after primary surgery. Cox regression models were used to estimate adjusted hazard ratios (HR) and 95% confidence intervals (95% CI) for ovarian cancer specific death in relation to previous pelvic surgery and reproductive variables including lifetime number of ovulation years. RESULTS: A total of 295 women with Stage III ovarian carcinomas were identified and followed to death or for a median of 7.3 years (range 5.4-9.5 years). Previously sterilised or hysterectomised women seemed to have a slightly decreased risk of ovarian cancer death (HR = 0.62; 95% CI: 0.36-1.08 and HR = 0.82; 95% CI: 0.55-1.21), although none of these associations reached statistical significance. The prognostic impacts of the individual reproductive variables followed the same pattern as the impact of the variables on ovarian cancer development, although significance was only reached for age at menarche (HR = 0.91 per year; 95% CI: 0.84-0.99). By accumulation of the possible minor effects of the reproductive variables in calculation of the total lifetime number of ovulation years, we found that survival decreased significantly with increasing number of ovulations (HR = 1.53 per 10 years; 95% CI: 1.09-2.14). CONCLUSION: Increasing lifetime number of ovulations was a negative prognostic factor for ovarian cancer specific survival. Previous sterilisation or hysterectomy seemed to be associated with improved survival.

Pre-treatment prediction of chemoresistance in second-line chemotherapy of ovarian carcinoma: value of serological tumor marker determination (tetranectin, YKL-40, CASA, CA 125).

OBJECTIVE: To examine if the determination of the levels of serological tumor markers at time of relapse had any predictive value for chemoresistance in the second-line treatment of ovarian cancer patients. METHODS: From a registry of consecutive single-institution patients with epithelial ovarian carcinoma pretreated with paclitaxel plus platinum, we selected 82 patients with (a) solid tumor recurrence, and (b) second-line chemotherapy consisting of topotecan (platinum-resistant disease) or paclitaxel plus carboplatin (platinum-sensitive disease). Stored serum samples were analyzed for the biochemical tumor markers tetranectin, YKL-40, CASA (cancer-associated serum antigen), and CA 125. The serum tumor marker levels at time of relapse were correlated with response status at landmark time after 4 cycles of second-line chemotherapy. Univariate and multivariate logistic regression analyses (chemoresistant vs non-chemoresistant disease) were performed. RESULTS: At landmark time, 26% of patients had progression according to the GCIG (Gynecologic Cancer Intergroup) progression criteria. In univariate logistic regression analysis, the tumor markers tetranectin (OR 0.4; 95% CI: 0.2-0.8; p=0.008), YKL-40 (OR 1.8; 95% CI: 1.0-3.3; p=0.045), and CASA (OR 1.8; 95% CI: 1.2-2.7; p=0.007) had predictive value for second-line chemoresistance, whereas serum CA 125 had no predictive value. In a multivariate logistic regression analysis, serum tetranectin and CASA both had independent predictive value for chemoresistance. The combined determination of tetranectin and CASA had a specificity of 90% with 33% sensitivity for the prediction of chemoresistance (area under the receiver operating characteristic curve = 0.78; 95% CI: 0.66-0.91; p=0.001). CONCLUSION: Low serum levels of tetranectin, or high serum levels of CASA or YKL-40, are associated with increased risk of second-line chemoresistance in patients with ovarian cancer.

Increased risk of ovarian cancer in integrin beta3 Leu33Pro homozygotes.

We previously demonstrated that integrin beta(3) Leu33Pro homozygotes have an increased risk of cancer, possibly most pronounced for ovarian cancer. We now test the latter hypothesis in case-control and prospective studies. We genotyped 463 Danish women with ovarian cancer, and 4291 women from the Danish general population. Calculation of odds ratios by conditional logistic regression was performed in the case-control study (n = 463 + 3543), and of ovarian cancer incidence, log-rank statistics and hazard ratios by Cox regression in the prospective study (n = 4291) with 9.5-year follow-up. In the case-control study matched for age and marital status, the odds ratio for ovarian cancer in homozygotes versus non-carriers was 1.6 (95% confidence interval: 1.0-2.6). In the prospective study with 28 incident ovarian cancers, non-carriers and homozygotes had incidences of 7 (4-11) and 30 (10-92) per 10 000 person-years (log-rank P = 0.02). The age-adjusted hazard ratio for ovarian cancer in homozygotes versus non-carriers was 3.9 (1.1-13). Risk of ovarian cancer did not differ between heterozygotes and non-carriers in either study. Integrin beta(3) Leu33Pro homozygotes have an increased risk of ovarian cancer.

Cancer-associated serum antigen level: a novel prognostic indicator for survival in patients with recurrent ovarian carcinoma.

The aim was to examine the value of the pretherapeutic serum cancer-associated serum antigen (CASA) level as a prognostic factor for survival in patients with recurrent epithelial ovarian carcinoma. Serum levels of CASA and cancer antigen (CA)125 were prospectively determined in 70 consecutive patients with recurrent ovarian cancer before the start of second-line chemotherapy. Univariate and multivariate analyses of survival were performed. The median level of serum CASA was 6.5 U/mL (range: 0.2-1437 U/mL). Univariate analysis showed that patients with a CASA level >10.0 U/mL had significantly shorter survival than patients with CASA level < or =10.0 U/mL (P= 0.002). Using different CASA cutoff levels (6.0, 6.5, and 10.0 U/mL), multivariate Cox analyses identified CASA as an independent prognostic factor for survival at every cutoff level. The strongest prognostic function for CASA was found at a cutoff level of 10.0 U/mL (>10 vs < or =10 U/mL; hazard ratio, 2.7; 95% confidence interval, 1.6-4.7; P < 0.001). The pretreatment CA125 level was not found to be significantly associated with survival by any of the cutoffs (35, 65, 132, and 339 U/mL). A pretreatment elevated level of the tumor marker CASA is an adverse prognostic factor for survival in patients with ovarian cancer relapse.

Do CA125 response criteria overestimate tumour response in second-line treatment of epithelial ovarian carcinoma?

Recent studies indicate that cancer antigen 125 (CA125) response criteria tend to overestimate a tumour reduction measured by standard WHO response criteria in recurrent epithelial ovarian carcinoma. The aim of the study was to validate the recently introduced GCIG (The Gynaecological Cancer Intergroup) CA125 response criteria in predicting a tumour response measured by WHO (World Health Organization) criteria. Changes in CA125 levels (GCIG criteria) were retrospectively compared with alterations in the tumour load (WHO criteria) during second-line chemotherapy with topotecan or paclitaxel-platinum in 124 consecutive patients with recurrent or refractory disease. In patients assessable by both response criteria (n=72), the overall response rate using GCIG CA125 criteria was 57% (95% confidence interval (CI): 45-69%) and significantly higher than the response rate of 39% (95% CI: 28-51%) using WHO response criteria (P=0.045). The GCIG CA125 criteria had a sensitivity of 96% (95% CI: 82-100%), a specificity of 68% (95% CI: 52-81%) and an accuracy of 79% (95% CI: 68-88%) in predicting a response measured by WHO criteria. In conclusion, the GCIG CA125 response criteria seem to overestimate a tumour response by WHO criteria when monitoring the efficacy of second-line chemotherapy with topotecan or paclitaxel-platinum in patients with epithelial ovarian carcinoma.

Evaluation of sample handling in relation to levels of tissue inhibitor of metalloproteinases-1 measured in blood by immunoassay.

BACKGROUND: The possible effect of preanalytical conditions such as blood sample preparation and handling on TIMP-1 levels in blood needs thorough investigation. MATERIALS AND METHODS: Blood was collected in dry tubes and tubes containing EDTA and kept at 4 degrees C or 20 degrees C for 1, 3, 8, 24 or 72 hours before processing into serum or EDTA plasma. In addition, serum and EDTA plasma samples were frozen and thawed 1-8 times. TIMP-1 was measured by ELISA. RESULTS: Time to processing for up to 72 hours did not significantly affect TIMP-1 levels in serum. In EDTA plasma, TIMP-1 levels were stable for up to eight hours; however, if samples were kept for 24 hours or longer the TIMP-1 levels increased (p < 0.0001). Repeated freezing and thawing had a significant effect on TIMP-1 levels in serum (p = 0.04). In plasma, repeated freezing and thawing for up to six times did not influence TIMP-1. However, in plasma samples exposed to seven or eight freeze/thaw cycles TIMP-1 levels decreased, although not significantly (p = 0.23). CONCLUSIONS: Handling and processing of blood samples is crucial for TIMP-1 measurement by immunoassay. In serum, TIMP-1 levels are unaffected by time to processing. Plasma samples should be processed within eight hours to avoid a TIMP-1 increase. For the measurement of TIMP-1 in archival material, serum should not be used because TIMP-1 levels are significantly affected by repeated freezing and thawing; archival plasma can readily be used provided that samples have not been frozen and thawed more than six times.

Predictive values of serum tumour markers tetranectin, OVX1, CASA and CA125 in patients with a pelvic mass.

Our objective was to compare the predictive value of the well-established tumour marker CA125 with the newer tumour markers tetranectin (TN), OVX1 and CASA in distinguishing benign and malignant pelvic masses in women. Participants included 185 women, 19 years or older, with a pelvic mass planned for surgical exploration. Significantly different CA125 levels were found between benign tumours and localised ovarian cancer (OC), advanced OC and other non-OCs. Significantly different TN levels were found between benign tumours and advanced OC (stage III/IV), between benign tumours and other cancers and between all OCs and other cancers. For CASA, significant differences were found between benign tumours and all OCs as well as advanced OC. No significant differences could be demonstrated for OVX1. Significant correlations for the 44 OC patients were found between CA125, TN and CASA. No significant correlations were found for OVX1, possibly because of the method used for collection and handling of serum samples. None of the new markers had any additional predictive value compared to CA125. TN and CASA levels correlated with FIGO stage and could be used to discriminate between benign and advanced OC. However, in comparison to the performance of CA125, the additional discriminative value of TN and CASA was minor.

The prognostic value of pretherapeutic tetranectin and CA-125 in patients with relapse of ovarian cancer.

OBJECTIVE: The aim of the study was to examine the prognostic values of, respectively, tetranectin (TN) and CA-125 measured in serum from patients presenting with relapse of ovarian cancer (OC). METHODS: TN and CA-125 were measured in serum samples from 75 patients with relapse of OC before the start of second-line chemotherapy. The endpoint used was death of OC. The variables were analyzed by univariate life table analysis and multivariate Cox analysis. RESULTS: A significantly shortened survival was found for patients with low serum TN values compared to patients with serum TN levels above one of the cutoff levels. The survivals are illustrated by life tables. No prognostic function was found for CA-125. TN and relapse

Stability of YKL-40 concentration in blood samples.

The stability of YKL-40, a mammalian member of the family of 18 glycosylhydrolases, in blood samples handled under different temperatures and different time intervals before centrifugation was studied in paired serum and plasma samples from 25 healthy premenopausal Danish women. Significant elevations of YKL-40 were found in 8 paired serum samples left on the clot for more than 3 h at room temperature compared to paired serum samples left on the clot for 3 h or less. Significant elevations of YKL-40 were found in 8 paired plasma (EDTA) samples left on the blood cells for more than 8 h at room temperature compared to paired plasma (EDTA) samples left on the blood cells for 8 h or less. No elevations were found in YKL-40 levels in serum samples left on the clot at 4 degrees C for 24 h or in plasma (EDTA) samples left on the blood cells for 72 h before centrifugation. Significantly lower concentrations of YKL-40 were measured in plasma (EDTA) compared with paired serum samples with a serum/plasma ratio of 1.4 in samples left on the clot or on blood cells at 4 degrees C for up to 24 h. Repetitive freezing and thawing had no significant effect on the measured YKL-40 concentrations. In conclusion, we have shown that YKL-40 is very dependent on the handling procedures. All the blood samples must be processed into plasma (EDTA) within 8 h at room temperature or into serum in less than 3 h at room temperature. If this is not possible, the blood samples must be stored at 4 degrees C until processed.

Alpha-fetoprotein in human fetal cerebrospinal fluid.

Alpha-fetoprotein (AFP) is a fetal glycoprotein. It has been ascribed a regulatory function of growth factor responses and immune functions. The concentrations of AFP and albumin (ALB) are highly variable in fetal serum and CSF and change with gestational age. The AFP index=[AFP(CSF)/AFP(SERUM)]/[ALB(CSF)/ALB(SERUM)] was determined in six normal fetuses at gestational age 17-23 weeks and found to be independent of gestational age and close to unity, mean 0.90+/-0.11 (S.D.). The ratio of CSF-serum concentrations of AFP and ALB both decreased significantly (p<0.05) with gestational age. The mean fraction of AFP being non-reactive with concanavalin A was 1.7% in serum and 1.9% in CSF, suggesting a common hepatic origin of AFP in both compartments. In conclusion, the concentration of AFP in CSF seems to be determined largely by the serum-CSF concentration gradient in normal fetuses. This finding, combined with the remarkable constancy of the AFP index compared to the highly variable absolute concentrations of AFP in both serum and CSF should make the AFP index the marker of choice when analyzing for intrathecal AFP synthesis during development and in pathological conditions.

Structure and tissue-specific expression of genes encoding bovine copper amine oxidases.

A cDNA coding for an enzyme belonging to the family of copper amine oxidases was cloned from a bovine lung cDNA library using a PCR approach. The nucleotide sequence of this cDNA was found to be different from that of the previously published liver cDNA encoding bovine serum amine oxidase, another copper amine oxidase. Analyses using reverse transcription followed by PCR of RNA extracted from different bovine tissues confirmed that the copper amine oxidase gene expressed in bovine liver is closely related to, but different from, the copper amine oxidase gene expressed in bovine lung, kidney, spleen and heart. Northern blotting data showed that the level of copper amine oxidase expression in liver is considerably higher than in the other tissues tested. Southern blotting analyses of bovine chromosomal DNA suggested the existence of at least three copper amine oxidase genes. Two of these genes are apparently expressed in a tissue-specific manner as outlined above. A fragment of a third copper amine oxidase gene is identified. The exon-intron organization of the bovine copper amine oxidase genes analyzed is similar to that of the related human diamine oxidase gene, except that no intron in the position equivalent to that of the third intron in the human gene is found. In the third gene, a complete replacement of the third intron of the bovine copper amine oxidase gene (equivalent to the fourth intron of the human gene) has occurred.

Lactoferrin: similarity to diamine oxidase and purification by aminohexyl affinity chromatography.

A protein reacting with a monoclonal antibody against human placental diamine oxidase was purified from the specific granules of human neutrofil granulocytes using affinity chromatography on aminohexyl-divinylsulfonyl-agarose. The protein had an M(r) determined by SDS/PAGE, corresponding to diamine oxidase, but had other properties which indicated that it might be a different protein. A combination of protein chemical techniques, including N-terminal sequencing, identified the protein as lactoferrin, an iron-containing protein with an M(r) of approximately 800000, a high isoelectric point and ferroxidase activity. Purified commercial lactoferrin was shown to bind to aminohexyl-divinylsulfonyl-agarose, and to be eluted in a heterogenous way from the matrix by amines and salt. Alignment of the sequences of diamine oxidase and lactoferrin showed that they are similar, indicating a common ancestry for these two different classes of metallo-oxidases.

Use of tetranectin, CA-125 and CASA to predict residual tumor and survival at second- and third-look operations for ovarian cancer.

Tetranectin (TN), CA-125 and CASA were measured in serum prior to 63 second-look and 5 third-look operations for ovarian cancer. Patients with residual tumor had significantly lower levels of TN and higher levels of CASA and CA-125 compared with tumor-free patients. The predictive values PVPos = 100% and PVNeg = 50.9% were found for TN at 9.3 mg/l. For CASA, a predictive value PVPos = 100% was found at 10 U/ml with a corresponding PVNeg = 52.7%. At the cut-off 35 U/ml for CA-125, the PVPos was 100% and the PVNeg = 53.6%. By combining the markers, PVNeg increased to 61.7% with a PVPos on 100%. Significantly differences in survival were found by lifetable analysis between patients tested as positive and negative respectively for any of the markers. Using multivariate Cox analyses, it was found that every marker had an independent prognostic function for survival.

[Screening of newborn infants for cystic fibrosis. A combined analysis of immunoreactive trypsin and delta F508 mutation--a screening without false positive results]. / Screening af nyfødte for cystisk fibrose. En kombineret analyse af immunreaktivt trypsin og delta F508-mutationen--screening uden falsk positive.

A total of 1081 blood screening cards taken from newborn babies, were anonymously selected for cystic fibrosis (CF) screening by quantitation of immunoreactive trypsin (IRT, Delfia) and by delta F508 mutational analyses using polymerase chain reaction followed by a time resolved fluorescence hybridization assay (Delfia). The IRT values showed a log normal distribution and were significantly higher in girls than boys and in 28 carriers compared with 1052 normals. In 12 newborns, corresponding to 1.02%, an IRT concentration greater than 70 micrograms/l was found. One of these was a delta F508 homozygote with an IRT concentration of 380 micrograms/l. delta F508 mutational analyses showed 1052 normals, 28 heterozygotes, and one homozygote, i.e., a carrier frequency of this mutation for delta F508 of 1:39. In future newborn CF-screening programmes we therefore recommend Delfia IRT followed by Delfia delta F508 analyses for IRT values greater than 70 micrograms/l.

Pre-operative plasma tetranectin as a prognostic marker in ovarian cancer patients.

Plasma tetranectin (TN) was tested as a biochemical prognostic marker in ovarian cancer on 39 patients. In stage I + II the 5-year survival was 33% (2/6) if plasma TN was < or = 6.7 mg l-1 and 100% (15/15) with plasma TN > 6.7 mg l-1. For stage III + IV the survival was 0% (0/11) at 26 months for patients with plasma TN < or = 6.7 mg l-1 and 29% (2/7) after 5 years with plasma TN > 6.7 mg l-1. By multivariate testing the relative hazard (RH) of death was found to be 73 times higher in patients with plasma TN < or = 6.7 mg l-1 compared to patients with values above 6.7 mg l-1 (p < 0.001). For comparison, the maximal RH for the other tested variables were: 15 for advanced stage, 2.5 for grade, four for residual tumour and 2.5 for younger age.

Changes in plasma tetranectin following hip surgery with or without thrombotic complications.

The fibrinolytic system seems to play a role in the development of postoperative thromboembolic complications (DVT). The newly described tetrameric protein tetranectin (TN), which has been found to enhance the activation of plasminogen to plasmin, therefore was studied in 55 patients who had total hip replacement and solely elastic stockings as physical thromboprophylaxis. No significant difference in plasma TN was found between the 5 patients with DVT and those without DVT, neither preoperatively or postoperatively at day 0, 1, 3, 7 or 10. A significant decrease in plasma TN was found from preoperative to postoperative values, indicating that TN may be a possible marker for other postoperative events. Because of the observed postoperative decrease it is important to consider the sampling time in the future research with TN.

CA-125 as a maternal serum marker for Down's syndrome in the first and second trimesters.

CA-125, alpha-fetoprotein (AFP), and human chorionic gonadotropin (HCG) were determined in maternal serum in the first trimester from 14 women with a Down's syndrome fetus and 61 women with a healthy fetus. In the second trimester, 15 and 60 serum samples were determined from women with a Down's syndrome and a healthy fetus respectively. In both trimesters, maternal serum CA-125 was found to be elevated in Down's syndrome pregnancies compared with controls. Using discrimination functions, our preliminary results indicate that CA-125 is a better marker than AFP and HCG respectively for a Down's syndrome fetus in the first trimester and improves the detection rate in the second trimester.