Analysis of molecular intra-patient variation and delineation of a prognostic 12-gene signature in non-muscle invasive bladder cancer; technology transfer from microarrays to PCR.

BACKGROUND: Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade. METHODS: We measured the intra-patient variation of an 88-gene progression signature using 39 metachronous tumours from 17 patients. For delineation of the optimal quantitative reverse transcriptase PCR panel of markers, we used 115 tumour samples from patients in Denmark, Sweden, UK and Spain. RESULTS: Analysis of intra-patient variation of the molecular markers showed 71% similar classification results. A final panel of 12 genes was selected, showing significant correlation with outcome. In multivariate Cox regression analysis, we found that the 12-gene signature was an independent prognostic factor (hazard ratio=7.4 (95% confidence interval: 3.4-15.9), P<0.001) when adjusting for stage, grade and treatment. Independent validation of the 12-gene panel and the determined cut-off values is needed and ongoing. CONCLUSION: Intra-patient marker variation in metachronous tumours is present. Therefore, to increase test sensitivity, it may be necessary to test several metachronous tumours from a patient's disease course. A PCR-based 12-gene signature significantly predicts disease progression in patients with non-muscle invasive bladder cancer.

The lesson from cancer pain.

The success of the WHO guidelines for the treatment of cancer pain indicates that cancer pain was previously undertreated. At the heart of these guidelines lies the three-step analgesic ladder, the last two steps of which consist of prescribing opioids. Today, the use of opioids in cancer patients is generally accepted, but there are still some concerns over the risks of addiction and adverse reactions, and opioids are sometimes withheld from patients who would otherwise benefit from them. However, it has been shown that such concerns are misplaced: the risks of severe adverse reactions and addiction are low when opioids are used correctly in patients with chronic pain. The use of opioids to treat benign pain is even less widely accepted in many countries, despite recommendations that they should be prescribed. It is emphasized that the use of opioids is a valid option for treating benign pain, and they should not be withheld from patients who need them. Opioids are always indicated when other therapeutic options, including NSAIDs, have failed or are contraindicated. When opioids are prescribed, procedures should be followed to provide the patient with maximum benefit and minimum risk.

Hepatocarcinogenesis (Z#2)/mutagenesis during initiation stage.

Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human alpha-1-antitrypsin (alpha1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10(-5)-10(-4)) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (< 2x) when compared to age matched negative controls. In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.

[Strong opioids for treatment of chronic pain: a meta-analysis]. / Stark wirksame Opioide zur Therapie chronischer Schmerzen. Metaanalyse.

In the following presentation 59 German and English articles on the use of strong opioids in chronic pain syndromes are analysed. Studies concerning the epidural, intrathecal, intracerebroventricular and transdermal application of opioids were excluded. The articles were attributed to different study levels according to their contents. The majority of articles (n=39, 66.1%) consisted of case presentations, while only 33.9% of the articles presented randomised (n=15) or non-randomised (n=5) study designs. The analgesic effect of the strong opioids morphine, buprenorphine and methadone in oral, rectal, subcutaneous and intravenous forms of application was confirmed for cancer-associated and non-cancer-associated chronic pain. Altogether there is a lack of controlled clinical studies.

[Supply of opioid analgesics to outpatients with cancer pain]. / Die Versorgung von ambulanten Tumorpatienten mit Opioidanalgetika.

INTRODUCTION: Treatment of chronic cancer pain with strong opioids is indicated in about 60-70 % of patients. Surprisingly, these very potent analgesics are prescribed with great reservations in many countries, including Germany. The aim of our investigation was to analyse the supply of opioid analgesics to outpatients with cancer pain in the region of Hannover, which has about 1.1 million inhabitants. METHODS: In Germany special prescription forms, i. e. triplicate forms, have to be used for the prescription of strong opioids. At the time when our investigation took place prescriptions for outpatients had to be renewed every 7 days. For two observation periods of 6 months lasting from January to June 1988 and from January to June 1991 all of the opioid prescription forms that had been issued by general practitioners for clients of the AOK Hannover (one of the major medical insurance companies) were evaluated. The reasons for prescribing opioids were analysed by recording the diagnosis. The individual treatment period on an outpatient basis during these 6 months was determined, excluding, e. g. days of hospitalization. RESULTS: During the two observation periods only 16.2 % (1988) and 19.5 % (1991) of the practitioners in the region of Hannover prescribed strong opioids to outpatients. The majority of the practitioners consisted of general practitioners and specialists in internal medicine. Although these two groups mainly function as family doctors who are responsible for the basic therapeutical needs of their patients, only 22.6-33.8 % of these doctors prescribed opioids to outpatients. In two-thirds of the patients, cancer pain was the reason for prescribing the drug. The total number of patients with a prescription for cancer pain was 164 in 1988 and 196 in 1991. Altogether 1002 prescription forms in 1988 and 1065 prescription forms in 1991 had been issued for these patients. Applied to the individual treatment period as an outpatient, only 36.0 % of the patients in 1988 and 32.1 % in 1991 received a regular prescription of strong opioids every 7 days. The mean time interval between separate prescriptions was 16.8 +/- 25.4 days in 1988 and 19.4 +/- 29.1 days in 1991. Accordingly, the majority of patients with chronic cancer pain had been supplied with opioids only occasionally. CONCLUSION: Our data indicate a significant undertreatment of outpatients suffering from cancer pain. Taking into account the estimated total number of patients suffering from cancer, only 14.5 % (1988) and 19.0 % (1991) of all outpatients in need of strong opioids were supplied sufficiently with those analgesics. Comparing the results from the observation period in 1988 with the results from 1991 it becomes obvious that the situation has not changed. There are different reasons for the insufficiency of opioid treatment: many physicians as well as their patients are still afraid of the side effects of strong opioids. Therefore, it is necessary to improve education concerning this issue. The legal restrictions on the use of narcotics and their complexity are another important reason for doctors not to prescribe strong opioids. In 1993 the regulations were simplified; nevertheless, this has not led to a profound change in the attitude of the prescribing practitioners. Thus, further changes in legislation seem to be necessary so that the requirements for the prescription of strong opioids do not differ from other drugs.

Creating seamless junctions independent of restriction sites in PCR cloning.

A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5'-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyldeoxycytosine (m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5' overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event.

Carcinomas metastatic to follicular adenomas of the thyroid gland. Report of two cases.

Metastases to the thyroid gland are an uncommon occurrence, and metastasis to a preexisting thyroid neoplasm is even more rare. We report two cases of tumor-to-tumor metastasis where a prostatic and a breast carcinoma metastasized to follicular adenoma of the thyroid gland. The metastatic process in case 1 was initially diagnosed by fine-needle aspiration biopsy and later confirmed with the hemithyroidectomy. Immunostaining for prostate-specific antigen and prostatic acid phosphatase in case 1 and estrogen and progesterone receptors in case 2 demonstrated strong immunoreactivity in the metastatic tumor cells. Flow cytometric DNA analysis of the primary and the metastatic tumors in both cases demonstrated stemline fidelity that supported their association. Our cases exemplify why attention should be given to the possibility of metastasis when distinctly different morphologic features are seen in an otherwise typical tumor and the utility of ancillary tests that may assist in establishing the diagnosis.

Hepatocarcinogenesis is the sequel to hepatitis in Z#2 alpha 1-antitrypsin transgenic mice: histopathological and DNA ploidy studies.

Z mutant-associated alpha 1-antitrypsin deficiency in human beings leads to hepatitis and, in some cases, hepatocellular carcinoma. To begin to delineate the molecular basis for the development of hepatocellular carcinoma in alpha 1-antitrypsin deficiency, we previously developed transgenic mice using human alpha 1-antitrypsin M and Z genomic clones. High-copy Z lineage mice (12 gene copies/haploid mouse genome; "Z#2") had hepatocytes distended with human alpha 1-antitrypsin deficiency globules. Hepatitis was present, and the morphological changes mimicked those observed in human alpha 1-antitrypsin deficiency-related liver disease. The numbers of hepatocytes containing alpha 1-antitrypsin globules decreased with age, and alpha 1-antitrypsin-negative nodular aggregates of hepatocytes increased in number and size. Hepatocytic dysplasia occurred as early as 6 wk and was almost universally present at 1 yr. Nodules of dysplastic cells demonstrating aneuploidy were seen as early as 10 wks. These became persistent, proliferative lesions. Dysplasia and aneuploidy distinctly increased with time and advancing microscopic stage as lesions progressed to malignancy. Tumors were seen after 1 yr as adenomas, which are aneuploid and most likely well-differentiated hepatocellular carcinoma, and borderline malignant lesions; and, in 82% of Z#2 mice 16 to 20 mo old, as invasive hepatocellular carcinoma. These observations suggest but do not conclusively prove that hepatocellular carcinoma in alpha 1-antitrypsin deficiency and other hepatic disorders arises as a result of a common, endogenously stimulated pathway for hepatocellular carcinogenesis.

Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage.

We have combined the efficiency and ease of use of bacteriophage lambda vectors with the power of phage display screening technology to create SurfZAP. The use of bacteriophage lambda allows the construction of large lambda expression libraries, which are rapidly and efficiently converted to stable plasmid libraries by mass excision. In SurfZAP, clones are expressed as fusions with amino acids 198-406 of the M13 minor coat protein (cpIII) and are displayed on the surface of filamentous phage. When produced with helper phage proteins, the fusion proteins are incorporated into the surface of phagemid particles. We demonstrate the utility of biopanning by isolating tetanus toxoid-binding mouse Fab clones from SurfZAP libraries. Approximately 10-100-fold enrichment of specific clones was observed after each panning round. The ability to create a large library of genotypes and screen the phenotypes by activity may be a potent methodology for basic research and drug discovery.

A bacteriophage lambda vector for the cloning and expression of immunoglobulin Fab fragments on the surface of filamentous phage.

We have combined the high cloning efficiency of the lambda bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles. The utility of the herein described ImmunoZAP 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library. The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda library to a phagemid library, and preparation of phagemid particles displaying Fabs. Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively. Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.

[Revision of the legal prescription requirements for strong opioids in the federal republic of Germany-an initiative of the German association for the study of pain.]. / Zur Novellierung des Betäubungsmittelgesetzes (BtMG) und der Betäubungsmittel-Verschreibungsverordnung (BtMVV)-Die Initiative der Deutschen Gesellschaft zum Studium des Schmerzes.

In the Federal Republic of Germany, strict regulations complicate the prescription of strong opioids to patients suffering from severe pain. In summer 1991 the German Association for the Study of Pain launched an initiative directed at simplification of the prescription requirements for such analgesics and submitted the suggestions to the ministries responsible. In the meantime a draft bill has been presented by the Ministry of Health, with due consideration for the main recommendations. For example, substantial increases in the permitted quantities of the individual narcotic analgesics and the prescription periods are planned. Moreover, the prescriptions are to be simpler to make out and certain exemptions have been specified by law. Acceptance of the bill by the political committees and its realization are expected in autumn 1992.

[Not Available]. / Medikamentöse Schmerztherapie bei rheumatischen Erkrankungen : Bericht über ein symposium bei der 16. Jahrestagung der Deutschen Gesellschaft zum Studium des Schmerzes e.V., Berlin, 24.-27. Oktober 1991.

Different therapeutic modalities are available for the treatment of rheumatic pain. The most important one, besides physiotherapy, is medication with analgesics and adjuvant drugs. Analgesics are given orally and by a stepwise approach in keeping with the principles of cancer pain therapy. In the first step nonopioid analgesics are prescribed, especially non-steroid anti-inflammatory drugs (NSAID) if pain is caused by inflammation. Other nonopioid analgesics, which can be used as alternatives for patients with non-inflammatory pain, are metamizol and paracetamol. Weak or even strong opioids must be administered to patients with rheumatic diseases when pain relief is insufficient or side-effects occur during medication with non-opioids. Long-term treatment of rheumatic pain even with strong opioids such as oral morphine involves only a small risk of severe side-effects such as respiratory depression or the development of tolerance and drug abuse. Patients often suffer from constipation, nausea and vomiting, but these side-effects can be treated with laxatives and antiemetic drugs. There is no reason to differentiate between opioid medication in a cancer patient with pain and in a patient with "non-malignant" rheumatic pain. Centrally acting muscle relaxants may be helpful as adjuvant medication in patients with myalgia for example, and tricyclic antidepressants can also be beneficial, especially in neuropathic pain and for patients with psychiatric distress associated with pain.

Expression of a heterodimeric Fab antibody protein in one cloning step.

A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.

[Continuous ambulatory intravenous morphine infusion for pain therapy in advanced ovarian cancer]. / Ambulante intravenöse Morphin-Dauerinfusion zur Schmerztherapie bei fortgeschrittenem Ovarialkarzinom.

We report on a female outpatient with cancer of the ovary, who received continuous intravenous morphine infusion for terminal pain control. The patient was treated over a period of 48 days with a morphine dosage ranging from 10 to 60 mg/h, which kept her free of pain. With treatment, she was alert, communicative with her relatives and moved freely. At a later stage, we complemented the treatment with Diazepam and Haloperidol. No side-effects were observed over the whole period of morphine infusion.

Bacteriophage cloning and Escherichia coli expression of a human IgM Fab.

We have combined the molecular biology methods of the polymerase chain reaction and recombinant DNA cloning in bacteriophage lambda to express a human IgM Fab in Escherichia coli using genes derived from an Epstein-Barr virus transformed cell line. This method comprises three cDNA amplifications and a single cloning step, culminating in the stable overexpression of mammalian heterodimeric recombinant protein in a prokaryotic host.