MR Imaging Characteristics Associate with Tumor-Associated Macrophages in Glioblastoma and Provide an Improved Signature for Survival Prognostication.

BACKGROUND AND PURPOSE: In glioblastoma, tumor-associated macrophages have tumor-promoting properties. This study determined whether routine MR imaging features could predict molecular subtypes of glioblastoma that differ in the content of tumor-associated macrophages. MATERIALS AND METHODS: Seven internally derived MR imaging features were assessed in 180 patients, and 25 features from the Visually AcceSAble Rembrandt Images feature set were assessed in 164 patients. Glioblastomas were divided into subtypes based on the telomere maintenance mechanism: alternative lengthening of telomeres positive (ALT+) and negative (ALT-) and the content of tumor-associated macrophages (with [M+] or without [M-] a high content of macrophages). The 3 most frequent subtypes (ALT+/M-, ALT-/M+, and ALT-/M-) were correlated with MR imaging features and clinical parameters. The fourth group (ALT+/M+) did not have enough cases for correlation with MR imaging features. RESULTS: Tumors with a regular margin and those lacking a fungating margin, an expansive T1/FLAIR ratio, and reduced ependymal extension were more frequent in the subgroup of ALT+/M- (P < .05). Radiologic necrosis, lack of cystic component (by both criteria), and extensive peritumoral edema were more frequent in ALT-/M+ tumors (P < .05). Multivariate testing with a Cox regression analysis found the cystic imaging feature was additive to tumor subtype, and O6-methylguanine methyltransferase (MGMT) status to predict improved patient survival (P < .05). CONCLUSIONS: Glioblastomas with tumor-associated macrophages are associated with routine MR imaging features consistent with these tumors being more aggressive. Inclusion of cystic change with molecular subtypes and MGMT status provided a better estimate of survival.

p53 promotes adenoviral replication and increases late viral gene expression.

The tumor suppressor protein, p53, plays a critical role in viro-oncology. However, the role of p53 in adenoviral replication is still poorly understood. In this paper, we have explored further the effect of p53 on adenoviral replicative lysis. Using well-characterized cells expressing a functional p53 (A549, K1neo, RKO) and isogenic derivatives that do not (K1scx, RKOp53.13), we show that virus replication, late virus protein expression and both wtAd5 and ONYX-015 virus-induced cell death are impaired in cells deficient in functional p53. Conversely, by transfecting p53 into these and other cells (IIICF/c, HeLa), we increase late virus protein expression and virus yield. We also show, using reporter assays in IIICF/c, HeLa and K1scx cells, that p53 can cooperate with E1a to enhance transcription from the major late promoter of the virus. Late viral protein production is enhanced by exogenous p53. Taken together, our data suggest that functional p53 can promote the adenovirus (Ad) lytic cycle. These results have implications for the use of Ad mutants that are defective in p53 degradation, such as ONYX-015, as agents for the treatment of cancers.

p53 and disease: when the guardian angel fails.

The p53 tumor suppressor gene (TP53) is mutated more often in human cancers than any other gene yet reported. Of importance, it is mutated frequently in the common human malignancies of the breast and colorectum and also, but less frequently, in other significant human cancers such as glioblastomas. There is also one inherited cancer predisposing syndrome called Li-Fraumeni that is caused by TP53 mutations. In this review, we discuss the significance of p53 mutations in some of the above tumors with a view to outlining how p53 contributes to malignant progression. We also discuss the usefulness of TP53 status as a prognostic marker and its role as a predictor of response to therapy. Finally, we outline some evidence that abnormalities in p53 function contribute to the etiology of other non-neoplastic diseases.

Nm23 protein expression in ductal in situ and invasive human breast carcinoma.

BACKGROUND: Mortality associated with human breast carcinoma is almost entirely due to subsequent metastatic disease, but the molecular basis of this metastasis is not understood. Elucidation of the genetic control of metastatic propensity of a tumor is important in determining prognosis and choice of therapy. Expression of nm23, a putative metastasis suppressor gene, has been detected in human breast cancers, but studies have not consistently shown high levels of the Nm23 messenger RNA or protein to be associated with better histological differentiation. This inconsistency suggests that Nm23 protein may act independently as a metastasis suppressor. PURPOSE: The purpose of this retrospective study was to investigate the relationship of Nm23 protein expression with 1) histology in ductal breast carcinoma in situ and 2) the variables considered to be the major prognostic indicators in invasive breast carcinoma. METHODS: We obtained formalin-fixed biopsy specimens of breast tissue excised from 128 patients with breast lesions detected by mammography. Of these patients, 35 had been diagnosed with benign breast disease, 26 with ductal carcinoma in situ (DCIS), and 67 with invasive carcinoma. Tissue sections were embedded in paraffin blocks, and immunohistochemical staining was used to determine Nm23 expression. Specimens were rated positive if all lesional epithelium was stained and negative if any lesional epithelium was unstained. Statistical analysis was performed by multiple regression analysis because of nonorthogonality of the data. RESULTS: All 35 examples of benign breast disease showed uniform epithelial cell staining. The seven cases of comedo DCIS were negative for Nm23 protein; all 18 noncomedo types were positive. Nm23 negativity was significantly associated with worsening invasive ductal carcinoma grade and advancing lymph node stage but not with tumor diameter or vascular invasion. Despite the putative antimetastatic role of the nm23 gene, no statistically significant association was found between Nm23 protein expression and vascular invasion. CONCLUSIONS: The precise role of the nm23 gene remains to be established, but our simplified immunohistochemical rating system shows an association between Nm23 protein expression and the two most significant prognostic factors relating to histologic grade and stage. Nm23 negativity distinguished comedo ductal carcinoma in situ from the other histological types, a finding consistent with the fact that comedo histology is known to have a higher likelihood of becoming invasive and of having higher cell proliferation rates and higher expression of growth factor (c-erb B2) receptor.

p53 and p21waf-1 expression correlates with apoptosis or cell survival in poorly differentiated, but not well-differentiated, retinoblastomas.

In human retinoblastomas, rare genetic mutations of the retinoblastoma gene cause massive cell proliferation, altered differentiation, and tumor formation; but paradoxically, this is accompanied by extensive apoptotic cell loss. We quantified the immunohistochemical distribution of p53, its downstream effector p21 (WAF-1), and apoptotic cells in 50 human retinoblastomas, within three concentric zones of sleeves of tumor cells surrounding blood vessels. In poorly differentiated retinoblastomas, both p53 expression and apoptosis increase toward the outer zone of tumor sleeves, whereas p21 expression occurs primarily within the inner zone. This staining pattern of p53 expression is reversed in well-differentiated tumors, whereas p21 staining and apoptotic cell distributions are unchanged. We detected no p53 mutations in four retinoblastomas and two retinoblastoma cell lines. We postulate that oxygen and cell "survival/growth factors" delivered via blood vessels protect retinoblastoma cells from apoptosis. In poorly differentiated tumors, apoptosis is spatially associated with increased p53 expression and may be p53 mediated, but in well-differentiated tumors, apoptosis does not colocalize with p53 and may be p53 independent. In retinoblastomas, p21 is involved not in cell death by apoptosis but in cell survival. Thus, p53 varies its expression (and by implication its function) with altered differentiation in retinoblastomas.

Correlation of VEGF production with IL1 alpha and IL6 secretion by human pituitary adenoma cells.

OBJECTIVES: Vascular endothelial growth factor (VEGF) is considered to be the most important angiogenic factor involved in the neovascularisation of solid tumours. Regulatory molecules include cytokines and growth factors. Interleukin (IL)1 and IL6 have both been shown to regulate VEGF levels in a variety of tissues. The role of cytokines in the pathogenesis of pituitary tumours remains unclear. We have examined the expression of VEGF and its relationships with IL1 and IL6 in the human pituitary tumour cell line HP75 and a series of human pituitary tumours. We have also looked at the relationship of tumour volume and invasive status to VEGF secretion. METHODS: Surgically resected tumours were routinely cultured in single-cell suspension at 200 K/well (standard unit for culture of dispersed primary pituitary adenoma cells). We measured VEGF, IL1 alpha and IL6 levels by ELISA. Tumour volume and invasion grade were assessed by preoperative magnetic resonance imaging. RESULTS: VEGF was detected in conditioned medium of HP75 cells (900+/-52 pg/ml) and in 82% of tumours tested (range 26-16 464 pg/ml). Tumour volume and secretion of VEGF were significantly associated with levels of IL6 (volume, P = 0.056; VEGF, P < 0.001 (P values based on Spearman's test)) and IL1 alpha produced (volume, P < 0.005; VEGF, P < 0.001). Invasive tumours showed a higher basal secretion of VEGF that that of the non-invasive type; however, this difference was not significant. Addition of exogenous IL1 alpha, but not IL6, significantly increased VEGF production. CONCLUSIONS: The significant associations between VEGF and the levels of IL6 and IL1 alpha suggest an important role for these cytokines in the development of these tumours.

Childhood leukemia: cerebrospinal fluid enolase isoenzymes in central nervous system infiltration.

Enolase isoenzymes were measured in the cerebrospinal fluid of seven consecutive children with lymphoblastic leukemia who developed meningeal (CNS) leukemia. Assays were performed at the time CNS disease was discovered and during the subsequent 4 weeks. Three of the seven were also examined 1-3 months before CNS relapse was confirmed. Fourteen children on similar systemic therapy without CNS infiltration served as controls. Prior to and at the onset of CNS disease alpha enolase was elevated in all patients studied. The gamma form was raised in only one beforehand and only three at the time of relapse. The alpha isoenzyme was related to the blast cell count and fell during therapy in all but one child, whereas the gamma was not and showed no significant change. The three patients with raised gamma enolase were the only children with other than common lymphoblastic leukemia. There was no clear indication whether either enzyme concentration had any importance in terms of disease outcome, although one child developed a further CNS relapse 10 months later. He was the only patient whose alpha enolase rose following intrathecal methotrexate. Neuronal disruption due to common lymphoblastic leukemia in the CNS appears to be minimal. Other types of leukemia may give rise to more neuronal damage. The alpha isoenzyme, from glial tissue and malignant cells, may be elevated even in the absence of detectable blasts in the cerebrospinal fluid and may be a sensitive marker of CNS infiltration in such circumstances.

Δ122p53, a mouse model of Δ133p53α, enhances the tumor-suppressor activities of an attenuated p53 mutant.

Growing evidence suggests the Δ133p53α isoform may function as an oncogene. It is overexpressed in many tumors, stimulates pathways involved in tumor progression, and inhibits some activities of wild-type p53, including transactivation and apoptosis. We hypothesized that Δ133p53α would have an even more profound effect on p53 variants with weaker tumor-suppressor capability. We tested this using a mouse model heterozygous for a Δ133p53α-like isoform (Δ122p53) and a p53 mutant with weak tumor-suppressor function (mΔpro). The Δ122p53/mΔpro mice showed a unique survival curve with a wide range of survival times (92-495 days) which was much greater than mΔpro/- mice (range 120-250 days) and mice heterozygous for the Δ122p53 and p53 null alleles (Δ122p53/-, range 78-150 days), suggesting Δ122p53 increased the tumor-suppressor activity of mΔpro. Moreover, some of the mice that survived longest only developed benign tumors. In vitro analyses to investigate why some Δ122p53/mΔpro mice were protected from aggressive tumors revealed that Δ122p53 stabilized mΔpro and prolonged the response to DNA damage. Similar effects of Δ122p53 and Δ133p53α were observed on wild-type of full-length p53, but these did not result in improved biological responses. The data suggest that Δ122p53 (and Δ133p53α) could offer some protection against tumors by enhancing the p53 response to stress.

Expression of interleukin-6 and its effects on growth of HP75 human pituitary tumor cells.

The role of IL-6 in the pathogenesis of pituitary adenomas is unclear, as tumor biology is difficult to study in primary culture. We have shown here that the human pituitary cell line HP75 synthesizes IL-6 mRNA and expresses and secretes IL-6 (6167 +/- 56 pg/ml/72 h for 30,000 cells). IL-6 receptor (IL-6R) mRNA was identified by in situ hybridization and RT-PCR. Exogenous IL-6 in low dose (1 ng/ml) stimulated, whereas higher doses (100 ng/ml) inhibited, growth. This diverse effect occurs in other cell types as a result of receptor down-regulation. Cell growth was inhibited by IL-6-blocking antibody (76 +/- 6.5% inhibition; P < 0.0001). This demonstrates that IL-6 is an important growth regulator in HP75 cells, having an autocrine growth stimulatory effect under basal conditions. IL-1alpha and dibutyryl cAMP stimulated and dexamethasone inhibited IL-6 secretion; however, bacterial lipopolysaccharide, forskolin, and cholera toxin had no effect. This implies that there is a defect in the control of IL-6 secretion. Soluble IL-6R was not detected, but soluble gp130 receptor was present in the conditioned medium. Stimulation of cleavage of soluble IL-6R from the membrane-bound IL-6R could not be induced by phorbol ester or dexamethasone. Whether IL-6 has a similar effect in human pituitary adenomas requires further investigation.

Subcellular localisation of cyclin B, Cdc2 and p21(WAF1/CIP1) in breast cancer. association with prognosis.

The heterodimeric cyclin B/Cdc2 protein kinase governs entry into mitosis, and can be negatively regulated through p53-mediated transcriptional induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Ectopic expression of p21(WAF1/CIP1) in cultured cells has been shown previously to influence the subcellular distribution of the cyclin-dependent kinases (CDKs) including Cdc2. In this study, we have examined the subcellular localisation of Cdc2, cyclin B and p21(WAF1/CIP1) by immunohistochemistry in a well characterised series of primary breast cancers. Surprisingly, p21(WAF1/CIP1) was predominantly cytoplasmic in many of the tumours, where it was associated with high p53 levels; cytoplasmic p21(WAF1/CIP1) and high cyclin B levels were also significant predictors of poor prognosis. We conclude that breast tumorigenesis may be characterised by abnormalities in pathways determining not only levels of expression of key regulatory molecules, but also their subcellular localisation. Investigation of the subcellular distribution of cell cycle regulatory proteins, particularly p21(WAF1/CIP1), could provide valuable prognostic markers in breast cancer.

IL-8 mRNA expression by in situ hybridisation in human pituitary adenomas.

Several cytokines have been shown to be expressed in normal and adenomatous pituitary tissue. Recently, interleukin-8 (IL-8) mRNA was identified by reverse transcription (RT)-PCR in each of a series of 17 pituitary tumours examined. We have investigated further the presence of IL-8 mRNA, using in situ hybridisation in two normal human anterior pituitary specimens and 25 human pituitary adenomas. IL-8 mRNA was not identified in either of the two normal pituitary specimens. Only three of the 25 adenomas were positive for IL-8 mRNA. In these three tumours, which included two null cell adenomas and one gonadotrophinoma, the majority of tumour cells (>90%) were positive for IL-8 mRNA. The remaining 22 adenomas were completely negative. There was no difference in tumour size or type between the IL-8 positive and the IL-8 negative tumours, and immunocytochemistry for von Willebrandt factor showed that the two groups were also similar in their degree of vascularisation. In conclusion, IL-8 mRNA was found in 12% of pituitary adenomas studied and was histologically identified within the tumour cells. In situ hybridisation is a more appropriate technique for assessing cytokine mRNA production by human pituitary tumours because RT-PCR may be too sensitive, identifying very small, possibly pathologically insignificant, quantities of mRNA that could be produced by supporting cells such as fibroblasts, endothelial cells or macrophages.

An investigation of beta enolase as a histological marker of rhabdomyosarcoma.

Sections from 21 tumours diagnosed as primary or metastatic rhabdomyosarcoma were stained for alpha and beta enolase. The cases were subdivided into embryonal and alveolar subtypes (38% and 62%, respectively). Positive cytoplasmic staining for alpha enolase was seen in all but one case, and cytoplasmic staining for beta enolase was seen in some cells in 18 of the 21 cases (86% of the total, 88% of the alveolar subgroup, and 85% of the embryonal subgroup). No cells stained positively for beta enolase in the control series of neuroblastomas, fibrosarcomas, Wilms' sarcomas, and an osteosarcoma. The results show that beta enolase is a sensitive marker of muscular differentiation in rhabdomyosarcoma.

Comparison of beta enolase and myoglobin as histological markers of rhabdomyosarcoma.

A comparative study of beta enolase and myoglobin as markers of muscle differentiation in rhabdomyosarcoma was carried out, using an immunoperoxidase peroxidase antiperoxidase technique. Material from 26 cases of childhood rhabdomyosarcoma was studied and subdivided into embryonal and alveolar types. Positive cytoplasmic staining for beta enolase was seen in 85% of tumours studied (91% alveolar, 79% embryonal), whereas positive staining for myoglobin was detected in only 69% of tumours (82% alveolar, 64% embryonal). beta Enolase and myoglobin are useful in the histological diagnosis of rhabdomyosarcoma, and of the two, beta enolase seems to be the more sensitive.

Serum aldolase isoenzymes in benign and malignant liver disease.

Using a radio-immunoassay, aldolase A and B isoenzyme concentrations have been measured in the sera of patients in order to assess their specificity and sensitivity in a variety of hepatic disorders. Serum aldolase A has been confirmed to be elevated in some patients with malignant infiltration of the liver, but its sensitivity is not sufficient to be of clinical value. Aldolase B is a sensitive marker of liver cell damage which correlates closely with conventional biochemical markers of inflammation. It appears to distinguish successfully between hepatic and cardiac damage.

Enolase isoenzymes in uveal melanomas--a possible parameter of malignancy.

Seven uveal melanomas were stained for gamma enolase by an immunoperoxidase PAP (peroxidase-antiperoxidase) technique, and biochemical assays were carried out on tissue homogenates. A correlation between the biochemical assay and the immunoperoxidase staining was demonstrated. Two tumours with the highest biochemical assays showed positive staining for the enzyme, and 2 tumours with the lowest levels showed no appreciable staining. The highest level of enolase was present in a tumour which both clinically and histologically appeared to be benign, and the lowest level occurred in a mixed cell tumour which was large in size; the low level presumably related to its relatively fast rate of growth. Estimation of gamma enolase activity in ocular melanomas may provide an accurate quantitative method for assessing the malignant potential of these tumours.

The nature of the cardiac myxoma.

Ten archive cases of cardiac myxoma were evaluated for proliferative activity, metastatic potential and expression of oncogene/tumor suppressor gene products by means of PCNA, MIB1, nm23, p53, Bcl-2 and Rb-1 immunohistochemistry. The myxomas showed variable proliferative activity (PCNA 0-41%, average 12.6%, MIB1 0-13%, average 3.2%) contrasting with the absence of mitotic activity histologically. All the myxomas showed nm23 staining. None showed p53 reactivity. Eight cases were negative for Bcl-2 expression, with two cases giving weak cytoplasmic staining. Rb-1 reactivity showed a variable pattern (staining indices 0-86%) paralleling the cases' proliferative activity. The cardiac myxoma is interpreted as a weakly proliferative lesion with little metastatic potential and no modulation of oncogene/oncogene suppressor products. Whilst not excluding a neoplastic aetiology, the results are considered more in keeping with a reactive/hamartomatous process.

p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors.

We propose that the apoptotic function of p53 has an important role in B-cell homeostasis, which is important for the prevention of B-cell lymphomas. We created a mouse model (mDeltapro) that lacked residues 58-88 of the proline-rich domain of p53. mDeltapro is defective for apoptosis, but is able to arrest cell-cycle progression in hematopoietic tissues. mDeltapro develops late-onset B-cell lymphoma, but not the thymic T-cell tumors found in p53-null mice. Interestingly, mDeltapro lymphomas comprised incorrectly differentiated B cells. B-cell irregularities were also detected in mDeltapro before tumor onset, in which aged mice showed an increased population of inappropriately differentiated B cells in the bone marrow and spleen. We predict that by keeping B-cell populations in check, p53-dependent apoptosis prevents irregular B cells from eventuating in lymphomas.

Hypoxia and reoxygenation: a pressure for mutant p53 cell selection and tumour progression.

Recent findings indicate that in a hypoxic environment, oncogenically transformed cells with a mutant form of the tumour suppressor gene p53 may have a survival advantage over similar cells with wild-type p53. This is because the extent of hypoxia-induced apoptosis has been observed to diminish with the loss of wild-type p53 function in certain cell lines. Hypoxic conditions, common in most solid tumours, may thus provide a physiological pressure to select for cells with mutations in the p53 gene. A new model incorporating cell-specific parameters is proposed here to quantify the survival advantage of mutant or null p53 cells over their wild-type counterparts at any level of oxygen deprivation. Predictions are in good agreement with previous monolayer culture experiments comparing hypoxic survival of null and wild-type p53 cells. By extending the model we are able to investigate the effects of repeated rounds of hypoxia and reoxygenation on a mixture of wild-type and mutant or null p53 cells and determine how many rounds are required before a subpopulation of mutant or null p53 cells overtakes a given population of wild-type p53 cells.

Effect of mutation and conformation on the function of p53 in colorectal cancer.

In vitro studies have shown that immunoprecipitation with conformation-specific antibodies allows discrimination between different forms of p53; however, the significance of this has not been determined for human tumours. This study therefore examined p53 conformation in colorectal tumours and correlated this with mutational status and evidence of in vivo p53 downstream activity. Moreover, it was shown that for in vitro cell lines, DNA-damaging agents induce wild-type p53 to form a mutant conformation (PAb240+), with a concomitant rise in p21(WAF-1) expression. Induction of p53-mediated apoptosis, on the other hand, is associated with a wild-type conformation (PAb1620+). These results were confirmed for wild-type p53 in colorectal tumours. A range of p53 point mutations were found in exons 5-8 in colorectal tumours. Mutants with a wild-type conformation gave weak immunohistochemical staining, whereas mutations with flexible conformation (240+/1620+) gave intense positivity. Interestingly, this latter group of flexible mutants was also associated with features of poor prognosis. These studies show that not all p53 mutants are equal; thus, knowledge of the p53 status of a tumour may be required for a more precise prediction of prognosis and response to treatment for cancer patients.