Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine.

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient's tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine.

Outpatient photodynamic-guided diagnosis of carcinoma in situ with flexible cystoscopy: an alternative to conventional inpatient photodynamic-guided bladder biopsies in the operating theatre?

OBJECTIVE: The aim of this prospective open comparative study was to establish whether conventional photodynamic-guided bladder biopsies using general anaesthesia and rigid resectoscopes (inpatient) can be replaced with less traumatic flexible cystoscopy in non-sedated patients (outpatient), without compromising the diagnosis of carcinoma in situ (CIS). MATERIALS AND METHODS: Thirty-one patients were included. After BCG instillation for CIS, bladder biopsies were obtained using photodynamic-guided flexible cystoscopy. Two weeks later, patients underwent the conventional inpatient procedure. An external pathologist reviewed the biopsy samples. Pain and quality of life (QoL) symptom score were recorded. RESULTS: Post-BCG biopsies showed only CIS in 10 patients; high-grade Ta or T1 tumour in three patients, who were referred for cystectomy; and normal or low-grade tumour tissue in 18 patients. There was a high agreement of identification of high-grade disease in biopsies and cytology using the two methods (κ = 0.93, 95% confidence interval 0.8-1.0). The outpatient procedure identified four high-grade patients diagnosed as 'normal/low-grade' in the inpatient procedure. The opposite was observed in two patients. Quality of biopsies did not differ between the two procedures. Pain scores for outpatients were low, and median QoL symptom score was significantly lower than for inpatients (24 vs 33, p = 0.02). Hospital length of stay was significantly longer for inpatients. CONCLUSIONS: Outpatient photodynamic-guided flexible cystoscopy is less traumatic than the conventional inpatient procedure in the diagnosis of CIS. It is safe and cost-effective, and may be an alternative to conventional inpatient biopsy procedures in patients with malignant urine cytology and normal white-light cystoscopy.

Clinical characteristics and primary management of patients diagnosed with prostate cancer between 2007 and 2013: status from a Danish primary referral center.

BACKGROUND: The Danish Cancer Registry holds information on all prostate cancers (PCa) cases, including diagnostic TNM. However, stratification according to contemporary risk classification is not possible because histopathological grading and prostate-specific antigen (PSA) level are not registered. The objective of the study was to report clinical characteristics and primary management of men diagnosed with PCa from a primary referral center in Denmark. MATERIAL AND METHODS: Records on all men diagnosed with PCa at the Department of Urology, Frederiksberg Hospital, 1 January 2007 - 31 December 2013, were reviewed. Clinical characteristics and primary treatment were recorded. The National Comprehensive Cancer Network risk group classification was used. RESULTS: A total of 1934 men with a median age of 69 years (interquartile range 65-75) were diagnosed with PCa in the study period resulting in an incidence rate (World Standard Population) of 84/100 000. Overall, 18% were classified as low-risk, 34% as intermediate-risk, 23% as high-risk, 8% as very high-risk and 17% had metastatic disease at diagnosis. Among men age <65 years 70% had low- or intermediate-risk disease, while this was the case for 58% of men aged 65-75 and 22% of men aged >75. Metastatic disease was found in 11% of men <65 years, 17% of men 65-75 years and 23% of men >75 years. In total 73% of men with low-risk PCa were managed on watchful waiting or active surveillance. Curatively intended treatment was performed in 56% of men with intermediate-risk and 61% of men with high-risk PCa, while hormonal therapy was used in 90% of men with very high-risk and 98% of men with metastatic PCa. CONCLUSION: In a population without systematic PSA testing we found a large proportion of patients presenting with advanced PCa at diagnosis. Elderly patients presented with more advanced disease. Curative treatment was primarily used in younger men with clinically localized PCa.

Plasma levels of OLFM4 in normals and patients with gastrointestinal cancer.

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 µg/ml, mean 0.028 µg/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 µg/ml, mean 15 µg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.

Medullary carcinoma of the colon: can the undifferentiated be differentiated?

Medullary carcinoma of the colon is a rare variant of colorectal cancer claimed to have a more favorable prognosis than conventional adenocarcinomas. The histopathologic appearance may be difficult to distinguish from poorly differentiated adenocarcinoma. The study aimed to evaluate the diagnostic interobserver agreement and to characterize the immunohistochemical and molecular differences between these two subgroups. Fifteen cases initially classified as medullary carcinoma and 30 cases of poorly differentiated adenocarcinomas were included. Two pathologists reviewed the slides independently without knowledge of the original diagnosis and subgrouped the tumors into the two entities. Agreement was reached in 31 of 45 cases (69 %) with kappa = 0.32. An extensive immunohistochemical panel was performed, and KRAS, NRAS, and BRAF mutational status was assessed. Of the 31 cases with diagnostic agreement, the expression of only MLH-1 along with corresponding expression of PMS-2 differed significantly (p = 0.04). A high rate of BRAF mutations was detected in both subgroups without significant differences. Expression of MLH-1 was superior in dividing the tumors into two separate entities with significant differences in CK20 (p = 0.005) expression and in the rate of BRAF mutations (p = 0.0035). In conclusion, medullary carcinomas of the colon are difficult to discriminate from poorly differentiated adenocarcinoma even with the help of immunohistochemical and molecular analyses. This raises the question whether these morphological subtypes should be maintained or whether an alternative classification of poorly differentiated colorectal adenocarcinomas based on MLH-1 status rather than morphology should be suggested.

ProHNPs are the principal α-defensins of human plasma.

BACKGROUND: Human neutrophil peptides (HNPs) were discovered as abundant antimicrobial peptides of azurophil granules. Later studies revealed that most HNPs were produced by myelocytes and metamyelocytes and secreted into the bone marrow plasma as the inert proforms, proHNPs. Despite the vast amounts of proHNPs released into bone marrow plasma, little has been done to characterize these. Numerous studies have investigated HNPs in plasma, linking them to a variety of diseases, but without distinguishing between HNPs and their proforms. MATERIALS AND METHODS: We used an antibody with specificity against the propiece of proHNPs to investigate proHNPs in plasma and tissue. RESULTS: In contrast to previous studies using HNP antibodies, we found proHNPs to be many-fold more abundant than HNPs in plasma with a mean concentration of 2 µg/mL. The concentration was substantially higher in bone marrow plasma in accordance with the bone marrow being the site of origin of plasma proHNPs. ProHNPs were not bound to high molecular weight plasma proteins. Accordingly, proHNPs were filtered in the kidneys and resorbed in the proximal tubules. CONCLUSIONS: Most HNPs in plasma are in fact proHNPs, which is important given the differences in their origin and biological activities.

Outpatient diagnostic of bladder tumours in flexible cystoscopes: evaluation of fluorescence-guided flexible cystoscopy and bladder biopsies.

OBJECTIVE: The aim of this study was to evaluate photodynamic diagnosis (PDD) in flexible cystoscopes and the diagnostic quality of biopsies for diagnosis of non-muscle-invasive bladder cancer in the outpatients department (OPD). MATERIAL AND METHODS: Seventy-three patients (aged 36-91 years) with recurrent non-muscle-invasive bladder cancer and a medium to high risk of recurrence had a flexible PDD cystoscopy performed in the OPD. The bladder was first examined in standard white light followed by PDD. RESULTS: PDD was superior to white light diagnosis; PDD was positive in 16 patients (22%) where white light showed a normal bladder mucosa. Four of these patients had bladder tumour [4/73, 6%; two carcinoma in situ (CIS), two Ta]. The diagnosis was verified by transurethral resection of the bladder tumour in the operating room. In 20 patients (20/73, 27%) PDD identified additional tumour lesions that were not identified in white light (five CIS, 15 Ta). The false-positive detection rate of PDD was 0.41. False positivity was significantly reduced by simultaneous flex biopsies disproving malignancy. Biopsies were obtained from 57 patients and diagnosis of stage and grade were possible in 55 of these (97%). In two patients (4%) the tissue material was too small for diagnostic evaluation. Biopsies from 47 patients (83%) included muscularis mucosa and from 20 patients (35%) muscularis propria. In 30 patients all but one diagnosis from the OPD was confirmed by biopsy in rigid scopes in the operating room. CONCLUSIONS: PDD-guided cystoscopy and bladder biopsy in flexible cystoscopes can be performed in an OPD setting and with reliable results for diagnosis of tumour stage Ta, CIS and T1a bladder cancer.

Evidence-based medicine: the time has come to set standards for staging.

For international communication in cancer, staging systems such as TNM are essential; however, the principles and processes used to decide about changes in every new edition of TNM need to be subject to debate. Changes with major impact for patient treatment are introduced without evidence. We think that TNM should be a continual reactive process, rather than a proactive process. Changes should only occur after extensive discussion within the community, and before the introduction of any changes these should be tested for reproducibility and compared to the currently used gold standard. TNM should not be used to test hypotheses. It should introduce established facts that are beneficial to predicting patient prognosis. TNM should thus be restructured on a basis equivalent to evidence-based guidelines. The strength of the evidence should be explicitly stated and the evidence base given. It is time for the principles of staging to be widely debated and new principles and processes to be introduced to ensure that we are not in the same situation in the future. The disparity between therapeutic decision making and TNM staging is marked and we would appeal for the radical overhaul of TNM staging to make it fit for the twenty-first century. TNM is central to the management of cancer patients and we must protect and enhance its reputation.

[Acute ischemic proctitis following an epileptic attack]. / Akut iskaemisk proktitis efter et epileptisk anfald.

Acute ischemic proctitis is a rare diagnosis mainly because the rectum is supplied by an extensive arterial network. Consequently, in more than 90% of patients with ischemic colitis the rectum is spared. Previously reported cases are related to severe vascular insufficiency of the rectal circulation caused by systemic atherosclerosis, usually following aortic or aortoiliac operations. We report one case of acute ischemic proctitis following an epileptic attack.