Neuroblastoma cells injected into experimental mature teratoma reveal a tropism for embryonic loose mesenchyme.

Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem­cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell­induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non­random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86.

BACKGROUND: Radiotherapy is central in the treatment of cervical cancer. The formation of DNA double-strand breaks is considered to be critical for the radiotherapeutic effect. The non-homologous end joining (NHEJ) proteins DNA-PKcs, Ku70 and Ku86 have a major role in repairing DNA lesions. The objective of this study was to analyse if the expression of DNA-PKcs, Ku70 and Ku86 and their downstream signalling molecules p53, p21 and Mdm-2 are altered in residual cervical tumours after radiotherapy. METHODS: Retrospective analysis of 127 patients with cervical cancer stage IB-IIA treated with preoperative radiotherapy and radical surgery, revealed residual tumour in the cervical specimen in 30 patients. In 22 cases tumour material from residual and corresponding primary tumour were retrieved and the expression of DNA-PKcs, Ku86, Ku70, p53, p21 and Mdm-2 were assessed by immunohistochemistry. RESULTS: Residual tumours showed increased frequency of DNA-PKcs (P=0.037), Ku70 (P=0.018), Ku86 (P=0.008) positive cells. A correlation in DNA-PKcs expression between primary and residual tumours was found. The frequency of p21-positive cells was decreased (P=0.007) in residual tumours whereas no change in p53 or Mdm-2-positive cells were observed. CONCLUSION: Our results show that cervical carcinoma surviving radiotherapy have an increased DNA-PK expression. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of DNA-PK function may be part of a radioresistance mechanism within this tumour type.

Analysis of HLA class I-II haplotype frequency and segregation in a cohort of patients with advanced stage ovarian cancer.

In solid tumors, human leucocyte antigen (HLA)-A2 has been suggested to be a risk factor and a negative prognostic factor. The HLA-A2 allele in Scandinavia has a high prevalence; it decreases with latitude and also with ovarian cancer mortality in Europe. Furthermore, an association of the HLA-A2 allele with severe prognosis in serous adenocarcinoma of the ovary in stages III-IV was found. Thirty-two unrelated Swedish women with relapsing or progressive ovarian cancer were analysed for the genotypes at the HLA-A, HLA-B, HLA-Cw, and HLA-DRB1 loci by the polymerase chain reaction/sequence-specific primer method. The frequencies of HLA alleles of healthy Swedish bone marrow donors provided by the coordinating centre of the Bone Marrow Donors Worldwide Registries, Leiden, the Netherlands were used as controls. When this cohort of epithelial ovarian cancer patients was compared with healthy Swedish donors, the frequency of HLA-A1 and HLA-A2 gene/phenotype appears, although not statistically significant, to be increased in patients with ovarian carcinoma, while HLA-A3 was decreased. HLA-A2 homozygotes were twofold higher in patients. The A2-B8 haplotype was significantly increased (corrected P value). A2-B5, A2-B15, A2-DRB1*03, A2-DRB1*04, A2-B15-Cw3, and A2-B8-DRB1*03 had odds ratio as well as the level of the lower confidence interval above 1 and significant P value only when considered as single, non-corrected analysis. HLA-B15 and HLA-Cw3 were only present in HLA-A2-positive patients showing that the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes were segregated. In this selected cohort with advanced disease, there are indications of an unusual overrepresentation of HLA class I and II genes/haplotypes as well as segregation for the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes. These findings are presented as a descriptive analysis and need further investigations on a larger series of ovarian cancer patients to establish prognostic associations.

Expression of DNA damage response proteins and complete remission after radiotherapy of stage IB-IIA of cervical cancer.

The primary aim of this study was to investigate if the expression of the DNA damage identifying protein DNA-PKcs known to be involved in DNA repair after treatment with ionising radiation can be used as a predictive marker for radiotherapy (RT) response in cervical cancer. Formalin-fixed primary tumour biopsies from 109 patients with cervical cancer, FIGO-stage IB-IIA, treated with preoperative brachytherapy followed by radical surgery were analysed by immunohistochemistry. In addition, correlation studies between early pathological tumour response to radiation and expression of Ku86, Ku70, Mdm-2, p53 and p21 in primary tumours were also performed. We found that tumour-transformed tissue shows positive immunostaining of DNA-PKcs, Ku86 and Ku70, while non-neoplastic squamous epithelium and tumour-free cervix glands show negative immunoreactivity. Expression of DNA-PKcs positively correlated with both Ku86 and Ku70, and a statistically significant correlation between the Ku subunits was also found. After RT, 85 patients demonstrated pathologic complete remission (pCR), whereas 24 patients had residual tumour in the surgical specimen (non-pCR). The main finding of our study is that there was no correlation between the outcome of RT and the expression of DNA-PK subunits. Positive p53 tumours were significantly more common among non-pCR cases than in patients with pCR (P=0.031). Expression of p21 and Mdm-2 did not correlate with the outcome of RT.

Expression of the insulin-like growth factor 1 receptor (IGF-1R) in breast cancer cells: evidence for a regulatory role of dolichyl phosphate in the transition from an intracellular to an extracellular IGF-1 pathway.

In this study we provide evidence that the low expression of IGF-1R at the cell surface of estrogen-independent breast cancer cells is due to a low rate of de novo synthesis of dolichyl phosphate. The analyses were performed on the estrogen receptor-negative breast cancer cell line MDA231 and, in comparison, the melanoma cell line SK-MEL-2, which expresses a high number of plasma membrane-bound IGF-1R. Whereas the MDA231 cells had little or no surface expression of IGF-1R, they expressed functional (i.e., ligand-binding) intracellular receptors. By measuring the incorporation of [3H]mevalonate into dolichyl phosphate, we could demonstrate that the rate of dolichyl phosphate synthesis was considerably lower in MDA231 cells than in SK-MEL-2 cells. Furthermore, N-linked glycosylation of the alpha-subunit of IGF-1R was 8-fold higher in the melanoma cells. Following addition of dolichyl phosphate to MDA231 cells, N-linked glycosylation of IGF-1R was drastically increased, which in turn was correlated to a substantial translocation of IGF-1R to the plasma membrane, as assayed by IGF-1 binding analysis and by Western blotting of plasma membrane proteins. The dolichyl phosphate-stimulated receptors were proven to be biochemically active since they exhibited autophosphorylation. Under normal conditions MDA231 cells, expressing very few IGF-1R at the cell surface, were not growth-arrested by an antibody (alphaIR-3) blocking the binding of IGF-1 to IGF-1R. However, after treatment with dolichyl phosphate, leading to a high cell surface expression of IGF-1R, alphaIR-3 efficiently blocked MDA231 cell growth. Taken together with the fact that the breast cancer cells produce IGF-1 and exhibit intracellular binding, our data suggest that the level of de novo -synthesized dolichyl phosphate may be critical for whether the cells will use an intracellular or an extracellular autocrine IGF-1 pathway.

Reverse transcriptase polymerase chain reaction on fine needle aspirates for rapid detection of translocations in synovial sarcoma.

OBJECTIVE: To evaluate the utilization of fine needle aspiration (FNA) biopsy to obtain material for reverse-transcriptase polymerase chain reaction (RT-PCR) in the detection of the t(X;18)(p11.2;q11.2) translocation in synovial sarcomas. STUDY DESIGN: We applied RT-PCR to detection of synovial sarcoma fusion gene transcripts on fine needle aspirates. Five clinical samples were first analyzed: one was a tumor previously diagnosed as malignant hemangiopericytoma, one was a poorly defined tumor, and three were suspected synovial sarcomas. FNA material was transferred directly to the RT-PCR reaction tube without RNA extraction. RESULTS: The t(X;18) translocation could be detected on the limited amount of material that FNA provides. In each of the cases studied the representivity of the tumor samples was confirmed microscopically. CONCLUSION: Our protocol permits analysis directly on representative samples without extraction of RNA. The results imply that RT-PCR offers reliable detection of sarcoma fusion gene transcripts on fine needle aspirates. The procedure, apart from being applicable to outpatients, is rapid and sensitive.

Prevalence of sensitization to aeroallergens in California patients with respiratory allergy. Allergy Skin Test Project Team.

BACKGROUND: The number of allergy skin tests required to evaluate patients with respiratory allergy has recently been challenged by the managed care community. OBJECTIVES: The purpose of this study was to determine which aeroallergens are prevalent in patients with respiratory allergy (allergic rhinitis and bronchial asthma) in California. METHODS: Utilizing aeroallergens thought to be relevant from recent aerobiologic and botanic data, 141 allergic and 17 asymptomatic control subjects were tested for the prevalence of 103 allergens. A standardized prick puncture technique and standardized interpretation of wheal/flare responses were utilized using the same lot of allergen for 13 allergy practices distributed throughout California. Frequency curves based on prevalence were established to determine the number of tests required to give up to 90% of positive responses for tree, weed and grass pollen, mold spores, and miscellaneous allergens which included house dust mite, cat, dog, and cockroach allergens. RESULTS: Positive responses in allergic subjects for grasses ranged from 46% to 54%, for weeds 19% to 37%, and for trees 10% to 42%. For molds the range was from 11% to 22%. The response rate for Dermatophagoides pteronyssinus was 53%, for Dermatophagoides farinae 42%, for cat pelt 39% and cat hair 37%, for cockroach 23% and dog dander 19%. Asymptomatic control subjects responded to only 4% of all allergens tested. Ninety percent of all positive tests required three miscellaneous allergens (house dust mite, cat, and cockroach), 9 molds, 2 grasses, 16 weeds, and 27 trees for a total of 57 allergens (56% of total tested). There was no clear relationship between locale and specific allergen response, probably related to the limited number of subjects tested and variability within the same geographic region. Several seldom tested tree and weed allergens showed a higher prevalence rate than several commonly tested for allergens. CONCLUSIONS: This preliminary study suggests that approximately 57 aeroalleroens might be adequate to detect 90% of all positive responses in patients with respiratory allergy in California. This study was limited by subject number and variability between study sites. It is hoped a standardized model can be developed from this pilot study to definitively determine which aeroallergens are relevant in the United States.

Jump distance of dance landings influencing internal joint forces: I. Axial forces.

Knowledge of the magnitude and rate of applying axial forces (AF) during actual dance movements is necessary for understanding the etiology of chronic injuries and osteoarthritis. The purpose of this study was to investigate the effect of jumping distance on component ankle and knee joint AFs generated during the landing phase of traveling jumps. Six female dancers performed 10 jumps each at 30, 60, and 90% maximum jump distance (JD) and 15 jumps ranging from 35 to 100% JD. A sagittal view of the right leg landing onto a force platform was filmed. Greater ground reaction force maxima, knee flexion, knee and ankle flexion velocity, tibial landing angle, net ankle and knee joint moment maxima, ankle and knee joint reaction AFs, and quadriceps AFs (QuadAF) peak magnitudes and rates of AF application (dFmax/dt) were observed (P < 0.05) at increased JD. The QuadAF was a more important determinant of knee AF than joint reaction AF. Increased quadriceps force was useful for accommodating impact forces but served to increase its contribution to Knee AF, particularly during the later portion of the impact phase. High impact situations create significant magnitudes (e.g., 14 BW) and dFmax/dt of muscle AFs which could contribute to excessive joint wear.

Paneth cell-rich flat adenoma of the rectum: report of a case.

A patient having familiar adenomatosis polyposis and an ileo-rectal anastomosis developed a flat mucosal lesion in the rectum. A punch biopsy revealed a villous adenoma with high-grade dysplasia. The subsequent surgical specimen indicated that the flat villous adenoma was rich in Paneth cells. Special stains included lysozyme muramidase (to visualize Paneth cells), MIB1 proliferation monoclonal antibody and single and multilabel immunohistochemistry for Paneth cells. Other methods included transmission electron microscopy and quantification with an image quantifier (Program Optilab 2.1) of lysozyme-stained Paneth cells. The subjective evaluation of hematoxylin-eosin-stained preparations demonstrated that the Paneth cells were mainly located in the lower half of the villi. Sections labeled with a specific stain (lysozyme muramidase) revealed more Paneth cells in the villi and electron microscopy showed even more in lysozyme-negative areas. Obviously some migrating dysplastic Paneth cells had retained their characteristic granules on their way towards the tip of the villi. Quantitative studies indicated that the lysozyme muramidase-positive material accounted for 41% of the adenomatous tissue. MIB1 revealed intense cell proliferation at the base of the adenoma and in the entire slopes of the villi. Despite the wide distribution of Paneth cells in intestinal metaplasia of the stomach, in the normal small intestine and in the large bowel with chronic inflammatory diseases, it is surprising that tumors arising in Paneth cells are extremely rare. The causes of the apparent natural resistance of Paneth cells to tumor development deserve to be investigated. This is the first case of Paneth cell-rich flat adenoma of the rectum in the literature.

Utility of a proliferation marker in distinguishing between benign naevocellular naevi and naevocellular naevus-like lesions with malignant properties.

In the present study we have investigated the utility of the proliferation marker MIB 1 in distinguishing between benign naevocellular naevi and naevocellular naevus-like lesions with malignant potential. Percentages of MIB 1 immunoreactivity in the intradermal portion of the lesions were determined. In benign congenital and acquired naevi, as well as in dysplastic naevi, there was no or only a slight intradermal melanocytic proliferation (0-2%), whereas vertical growth phase melanomas exhibited a substantial proliferative activity (11-48%). In five cases of naevus-lke lesions, which had all relapsed as unmistakable malignant melanomas (locally or metastatically) after primary surgery, there was also clear proliferative activity (9-67%). Our findings suggest that MIB 1 may be a useful tool in the routine histopathological examination of problematic naevocellular lesions.

Left ventricular wall movement in heart failure.

Of 34 patients admitted to hospital with left ventricular failure seven died before echocardiograms could be repeated after treatment and in three no echocardiograms could be obtained owing to chronic obstructive lung disease. In the remaining 24 patients echocardiograms were taken soon after admission and compared with echocardiograms taken later, after clinical improvement. The results show that in most patients both anterior and posterior motion of the posterior left ventricular wall increased. The rate of backward diastolic motion was appreciably less before and after treatment of heart failure compared with that in a small group of normal younger healthy men. This technique is a quick and apparently reliable way to assess ventricular function. The rate of diastolic motion is probably a reflection of left ventricular wall compliance.