Evaluation of the addition of rituximab to CODOX-M/IVAC for Burkitt's lymphoma: a retrospective analysis.

BACKGROUND: Burkitt's lymphoma (BL) is a highly aggressive B-cell non-Hodgkin's lymphoma (NHL) that may be cured with intensive chemotherapy. The addition of the CD20-directed monoclonal antibody rituximab to CODOX-M/IVAC (cyclophosphamide, vincristine, doxorubicin, and high-dose methotrexate, alternating with ifosfamide, etoposide, and cytarabine) has not been studied despite efficacy in other aggressive CD20-positive NHLs. PATIENTS AND METHODS: Eighty adult BL patients treated with or without rituximab were identified at our institutions. Response rate, overall survival (OS), and progression-free survival (PFS) are calculated. RESULTS: There were fewer relapses in rituximab-treated patients (3 of 40 versus 13 of 40, P = 0.01). There was a trend for improvement in outcome favoring rituximab-containing therapy, with 3-year PFS (74% versus 61%) and 3-year OS (77% versus 66%), although these did not reach statistical significance. Advanced age and central nervous system involvement were associated with poorer OS on multivariable Cox regression analysis, adjusting for treatment, human immunodeficiency virus (HIV) involvement, and risk group. CONCLUSIONS: CODOX-M/IVAC, with or without rituximab, is a highly effective regimen for the treatment of adult BL. Rituximab decreased the recurrence rate and showed a trend in favor of improvement in PFS and OS. HIV-infected patients achieved outcomes comparable with those of their non-HIV-infected counterparts.

Development and validation of a model predictive of occult nipple involvement in women undergoing mastectomy.

BACKGROUND: This prospective study aimed to build a predictive model using preoperative information to aid selection for nipple-sparing mastectomy. METHODS: Two hundred consecutive skin-sparing mastectomy specimens without overt nipple involvement were evaluated. Demographic, preoperative pathology and imaging information was collected. Nipple specimens (2 x 2 x 2 cm) were sectioned at 3-mm intervals. Haematoxylin and eosin-stained slides were examined by a breast pathologist for involvement by tumour. Logistic regression analyses of 65 therapeutic procedures identified factors associated with occult involvement and created a predictive model. This was tested on specimens from a further 65 therapeutic procedures. RESULTS: Occult nipple involvement was noted in 32 (24.6 per cent) of 130 mastectomy specimens. In the training set, imaging diameter of the lesion and its distance from the nipple predicted nipple involvement on univariable analysis (P = 0.011 and P = 0.014 respectively). The multivariable logistic regression model was validated in the test set. The areas under the receiver-operating characteristic curve were 0.824 and 0.709 for the training and test sets respectively. CONCLUSION: Three-quarters of women undergoing mastectomy did not have occult nipple involvement. A clinical tool including tumour size and distance from the nipple has been developed to improve patient selection for nipple-sparing mastectomy.

Matched pair analyses of stage IV breast cancer with or without resection of primary breast site.

BACKGROUND: Reports demonstrate improved survival of stage IV breast cancer patients with primary cancer resection. This may result from selection for surgery, rather than biological processes. METHODS: We performed matched-pair analysis that minimized potential bias in selecting surgery for primary cancer. Chart review was also performed of 5-year survivors to assess selection bias affecting breast surgery. RESULTS: 19,464 breast cancer patients were identified; 808 (4.2%) were stage IV: 622 were analyzed after eliminating wrong diagnoses or staging, and limiting patients to Massachusetts residents. Matched-pair analysis narrowed or eliminated apparent survival benefit associated with primary site surgery in several comparisons. When the impact of the sequence of systemic and surgical treatments was studied in stage IV patients, 90% 2-year survival occurred in patients receiving chemotherapy first, in contrast to receiving chemotherapy simultaneously with or after surgery, suggesting selection for delayed surgery after excellent response to initial chemotherapy. In bone metastases, the 2-year survival advantage occurred with chemotherapy before surgery; no difference in survival with or without surgery occurred when these treatments were simultaneous. Among 5-year survivors, frequency of primary site surgery after excellent response to systemic therapy, breast surgery in stage III patients incorrectly classified as stage IV, and frequency of oligo metastases all indicated selection bias. CONCLUSIONS: Case selection bias in primary breast cancer resection in state IV patients may explain most, if not all, the apparent survival advantage of such surgery.

Age-dependent effects of TWEAK/Fn14 receptor activation on neural progenitor cells.

TWEAK/Fn14 signaling regulates progenitor cell proliferation, differentiation, and survival in multiple organ systems. This study examined the effects of TWEAK (tumor necrosis factor-like weak inducer of apoptosis) treatment on cultured mouse neural progenitor cells. The receptor for TWEAK is expressed by neural progenitor cells from the early embryonic stages through postnatal development. Although embryonic day 12 (E12) and postnatal day 1 (PN1) neural progenitor cells both express the receptor for TWEAK, TWEAK treatment of cultured E12 and PN1 progenitor cells resulted in age-dependent effects on proliferation and on neurite extension by neuronal progeny. TWEAK treatment did not alter proliferation of E12 neural progenitor cells but shifted PN1 progenitor cells toward cell-cycle phases G0 and G1 and reduced the rate at which they incorporated CldU. Conversely, the effects of TWEAK on axon elongation were more prominent in the earlier developmental stage. TWEAK induced extensive neurite outgrowth by the neuronal progeny of E12 but not PN1 progenitors. Treatment of the E12 progenitor cells with a TWEAK-neutralizing antibody repressed neurite extension, indicating that endogenous activation of this pathway may be required for neurite extension by the embryonic neuronal progeny. These studies indicate that TWEAK/Fn14 receptor activation exerts different effects on neural progenitor cells and their progeny depending on the developmental stage of the cells.

Spread of human cancer cells occurs with probabilities indicative of a nongenetic mechanism.

There has been much uncertainty as to whether metastasis requires mutation at the time of spread. Here, we use clinical data to calculate the probability of the spread of melanoma and breast cancer cells. These calculations reveal that the probability of the spread of cancer cells is relatively high for small tumours (approximately 1 event of spread for every 500 cells for melanomas of 0.1 mm) and declines as tumours increase in size (approximately 1 event of spread for every 10(8) cells for melanomas of 12 mm). The probability of spread of breast cancer cells from the lymph nodes to the periphery is approximately 1 event of spread for every 10(8) cells in the nodal masses, which have a mean diameter of 5 mm, while the probability of spread of cancer cells from the breast to the periphery when the primary masses are 5 mm is also approximately 1 event of spread for every 10(8) cells. Thus, the occurrence of an event of spread from the breast to the lymph nodes appears not to increase the propensity of the progeny of those cells to spread from the lymph nodes to the periphery. These values indicate that the spread of human breast cancer and melanoma cells is unlikely to occur by a mechanism requiring mutation at the time of spread.

Analysis of benefit:risk ratio and mortality reduction for the UK Breast Screening Programme.

A quantitative analysis has been performed to predict the benefit:risk ratio and associated mortality reduction for the UK National Health Service Breast Screening Programme. The analysis is based on the results of an established biological simulation method coupled with dosimetric information and population statistics applicable to the UK breast screening programme. As well as the general breast screening population, the benefit:risk ratios for specific subgroups of women thought to be at higher risk are estimated. The effects of alterations in screening strategy are also investigated. The results indicate favourable benefit:risk ratios and mortality reductions for all women in the programme, with a breast cancer mortality reduction of approximately 9% over the whole UK female population, equivalent to a breast cancer mortality reduction in the region of 25% for the age range 55-69 years.

beta-catenin is a downstream effector of Wnt-mediated tumorigenesis in the mammary gland.

The Wnt signal transduction pathway has been implicated in mammary tumorigenesis in the mouse. beta-catenin, a key downstream effector of this pathway interacts with and thus activates the Tcf/Lef family of transcription factors. Elevated levels of beta-catenin have been found in many human tumors, notably colon carcinomas. Recently, elevated levels of beta-catenin have been associated with poor prognosis in human adenocarcinoma of the breast. In order to assess the possible role of beta-catenin in mammary carcinoma, we have created transgenic mice bearing the MMTV-LTR driving an activated form of beta-catenin. These mice develop mammary gland hyperplasia and mammary adenocarcinoma, a phenotype very similar to that of transgenic mice expressing an MMTV-driven Wnt gene. Indeed, the histopathology of the mammary tumors in Wnt-mediated adenocarcinoma is identical to that observed in our beta-catenin-mediated disease model. Furthermore, putative beta-catenin transcriptional targets, cyclin D1 and c-myc, are elevated in beta-catenin-mediated mammary tumors and cell lines. These observations support the notion that the oncogenic Wnt pathway operates via beta-catenin and its targets in the context of mammary hyperplasia and carcinoma.

The Daxx enigma.

Several reports describing Daxx and its putative role have emerged without a unifying theme. While Daxx has been implicated in apoptosis, it remains unclear whether Daxx is pro- or anti-apoptotic, and whether its role in apoptosis is direct or indirect. Moreover, whether Daxx plays alternative or additional roles in regulating transcription, centromere binding or any number of other activities within the cell, is uncertain. The ability of Daxx to interact with a wide variety of molecules in yeast-interaction trap systems (Table 1) has allowed for this range of speculation. The fact that Daxx contains no significant homology to other known proteins has rendered its study all the more challenging.

Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development.

The mammalian Daxx gene has been identified in a diverse set of yeast interaction trap experiments. Although a facilitating role for Daxx in Fas-induced apoptosis has been suggested, Daxx's physiologic function remains unknown. To elucidate the in vivo role of Daxx, we have generated Daxx-deficient mice. Surprisingly, rather than a hyperproliferative disorder expected from the loss of a pro-apoptotic gene, mutation of Daxx results in extensive apoptosis and embryonic lethality. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo.

Breast cancer: computer simulation method for estimating optimal intervals for screening.

PURPOSE: To develop and evaluate a mathematic method that can be used to determine the optimal screening interval for detection of breast cancer prior to distant metastatic spread. MATERIALS AND METHODS: A computer simulation was developed with the use of biologically based data from the literature on the rates of tumor growth and spread, which can be used to calculate the course of breast cancer growth and metastasis. RESULTS: On the basis of the data available at this time, the results of the simulations suggested that a screening interval of 2 years would result in a 22% reduction in the rate of distant metastatic disease, an interval of 1 year would result in a 51% reduction, and an interval of 6 months would result in an 80% reduction. CONCLUSION: These findings suggest that more frequent screening could dramatically reduce the death rate from breast cancer.

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.

Dyad symmetry within the mouse 3' IgH regulatory region includes two virtually identical enhancers (C alpha3'E and hs3).

The transcription of the murine Ig heavy chain locus is regulated not only by the intronic enhancer, E mu, but also by a 3' regulatory region located downstream of the C alpha membrane exon. Several DNase I-hypersensitive sites (hs1-4) and enhancer elements (e.g., C alpha3'E) have been identified in this 3' regulatory region, and some of these were suggested to comprise a locus control region. However, little is known about the coordinate regulation or function of these individual elements. Here we provide evidence that C alpha3'E and hs3 are virtually mirror images of each other and demarcate the edges of an approximately 25-kb region of quasi-dyad symmetry with 3'alphaE(hs1,2) at its center. Flanking 3'alphaE(hs1,2) are inverted repeats and families of repetitive sequences uniquely located in this region. We have observed that, like 3'alphaE(hs1,2) and hs3, C alpha3'E is DNase I hypersensitive in plasma cell lines, but not in a pre-B cell line. Additionally, we found that C alpha3'E and hs3 show significant transcriptional synergy in transfection assays only in a plasma cell line. The DNA topology of the 3' regulatory region coupled with new and existing data on the activity of its individual enhancers during B cell differentiation lead us to propose a biphasic model for the activity of this region. According to our model, one unit, consisting of the 3'-most enhancer, hs4, is active early and throughout B cell development. The second unit, which comprises C alpha3'E, 3'alphaE(hs1,2), and hs3, becomes active later in development, when it contributes to such processes as class switching and increased levels of Ig heavy chain gene transcription in plasma cells.

B cell lineage-specific activator protein (BSAP). A player at multiple stages of B cell development.

B cell lineage-specific activator protein (BSAP), encoded by the Pax 5 gene, is a critical modulator of early B cell differentiation. Binding sites for BSAP have been identified in the promoters of multiple genes as well as at multiple sites within the IgH locus. While in vivo target sites critical for early B cell differentiation have not yet been identified, candidate sites include a 3 IgH enhancer that is active early in B cell development. BSAP may play an additional role later in development, such as in the regulation of isotype class switching.

Regulation of 3' IgH enhancers by a common set of factors, including kappa B-binding proteins.

Targeted disruption of the p50 subunit of NF-kappa B resulted in isotype class switch defects resembling those observed in mice in which the downstream IgH enhancer 3'alpha E(hs1,2) was deleted. We postulated that kappa B binding proteins may regulate class switching by interacting with 3'alpha E(hs1,2) or with other IgH 3' enhancers with which 3'alpha E(hs1,2) synergizes. kappa B binding sites were identified in 3'alpha E(hs1,2) and 3' alpha-hs4, the distal 3' IgH enhancer. A kappa B binding site within 3'alpha E(hs1,2) contributes to at least half the activity of the enhancer in plasma cells, while the same kappa B binding site participates in the complex repression of the enhancer in B cells. In the case of 3'alpha-hs4, a kappa B binding complex activates the enhancer in pre-B, B cells and plasma cells. Additional binding sites within 3'alpha-hs4 for factors known to regulate 3'alpha E(hs1,2), including Oct-1 and BSAP, were identified, and their contribution to 3'alpha-hs4 regulation during B cell development was assessed. Oct-1 positively regulates the enhancer in pre-B and B cells, while BSAP is a repressor in pre-B cells and an activator at the B cell stage. These studies identify kappa B binding proteins as key modulators of 3'alpha E(hs1,2) and 3'alpha-hs4, and suggest coregulation of the two enhancers by a common set of factors.

Identification of 3' alpha-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation.

In addition to E mu, several elements downstream of the IgH cluster, i.e. 3' of the C alpha gene, are involved in regulating IgH gene rearrangement and expression. This entire downstream regulatory region was shown to be deleted in the mutant myeloma cell line, LP1.2. The deletion encompasses approximately 34 kb and is presumably responsible for the reduced levels of IgH expression in this cell line. An additional regulatory element, included in the LP1.2 deletion, was identified by investigation of a DNase I hypersensitivity site located approximately 33 kb downstream of the alpha gene and present in pre-B and plasma cells. This novel IgH gene enhancer element, termed 3' alpha-hs4, is capable of activity throughout B cell development. Transient transfection of 3' alpha-hs4 in a CAT reporter gene construct shows transcriptional enhancement activity approximating that of E mu in S194 plasmacytoma and M12.4.1 and A-20 B cell lines; while in a pre-B cell line, 18-81, the average activity is 25% that of E mu. Enhancer activity was localized to an 800 bp fragment. The activity of 3' alpha-hs4 is orientation independent and appears to be B cell specific. Tight regulation of 3' alpha-hs4 is inferred from its variable activity in different plasmacytoma cell lines and within the pre B cell line, 18-81.

Trophoblast/leukocyte-common antigen is expressed by human testicular germ cells and appears on the surface of acrosome-reacted sperm.

The murine monoclonal antibody H316 reacts with a cell-surface antigen of human trophoblast, leukocytes, certain epithelia, and several malignant cell types. We have found that the H316 antibody also recognizes an antigen synthesized by pre- and post-meiotic human testicular germ cells and is expressed in the acrosomal region of methanol-fixed testicular, epididymal, and ejaculated sperm. The antigen is poorly expressed on the surface of fresh ejaculated motile sperm, but is detectable on most viable sperm after a 6-h incubation in medium containing human serum albumin (HSA), or 60-min incubation with the calcium ionophore A23187 (both treatments induce sperm acrosomal changes termed capacitation and acrosome reaction). We found that antigen recognized by H316 is immunoprecipitated as a single, broad 50 kDa band from radiolabeled ionophore-treated sperm extracts and that preincubation of HSA-capacitated sperm with this antibody causes a moderate, but significant, inhibition of hamster egg penetration. These data indicate that the antigen recognized by the H316 monoclonal antibody is synthesized by testicular germ cells and is surface-expressed on capacitated/acrosome-reacted sperm populations. Its potential as a human sperm acrosome reaction marker, and possible biological role in sperm-egg or sperm-lymphocyte interactions, warrants further investigation.