Clinical implications of acellular mucin pools in resected rectal cancer with pathological complete response to neoadjuvant chemoradiation.

AIM: Approximately 20% of rectal cancers treated with neoadjuvant chemoradiation achieve a pathological complete response (pCR), which is associated with an improved oncological outcome. However, in a proportion of patients with a pCR, acellular pools of mucin are present in the surgical specimen. The aim of this study was to evaluate the clinical implications of acellular mucin pools in patients with rectal adenocarcinoma achieving a pCR after neoadjuvant chemoradiation followed by proctectomy. METHOD: A single-centre colorectal cancer database was searched for patients with clinical Stage II and Stage III rectal adenocarcinoma who achieved a pCR (i.e. ypT0N0M0) after neoadjuvant chemoradiation followed by proctectomy between 1997 and 2007. Patients were categorized according to the presence or absence of acellular mucin pools in the resected specimen, and groups were compared. Patient demographics, tumour and treatment characteristics, and oncological outcomes were recorded. Primary outcomes were 3-year local and distant recurrences, and disease-free and overall survivals. RESULTS: Two hundred and fifty-eight patients with clinical Stage II or Stage III rectal adenocarcinoma were treated by neoadjuvant chemoradiation. Fifty-eight of these patients had a 58 pCR. Eleven of the 58 patients with a pCR had acellular mucin pools in the surgical specimen. The median follow up was 40 months. The groups were statistically similar with respect to demographics, chemoradiation regimens, distance of tumour from the anal verge, clinical stage and surgical procedure. No patient had local recurrence. Patients with acellular mucin pools had increased distant recurrence (21%vs 5%), decreased disease-free survival (79%vs 95%) and decreased overall survival (83%vs 95%) rates, although none of these differences was statistically significant. CONCLUSION: The presence of acellular mucin pools in a proctectomy specimen with a pCR does not affect local recurrence, but may suggest a more aggressive tumour biology.

Modulation of erbB kinase activity and oncogenic potential by single point mutations in the glycine loop of the catalytic domain.

Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products which have constitutive tyrosine kinase activity and can induce erythroleukemia but not sarcomas. We have previously found that a valine-to-isoleucine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sarcomagenic potential of the oncogene and increase the kinase activity of this oncoprotein. This mutation lies at position 157 of the insertionally activated c-erbB product, affecting a highly conserved valine residue of the glycine loop involved in ATP binding and phosphate transfer. To investigate the functional importance of this residue in the catalytic activity of kinases, we have introduced at this position, by site-directed mutagenesis, codons representing the remaining 18 amino acid residues. Most of the mutants have diminished activity, with six of them completely devoid of kinase activity, indicating the sensitivity of this region to conformational changes. Some of these mutants displayed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various erbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are important exceptions such as the V157G mutant, which lacks enhanced autophosphorylation but is highly sarcomagenic. Studies of this and other autophosphorylation site mutants point to the existence of an autophosphorylation-independent pathway in sarcomagenesis. The requirement for leukemogenic potential is much less stringent and correlates with positivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full-length erbB protein, the mutation relaxed the ligand dependence and had a positive effect on the transforming potential of the full-length c-erbB.

Xenografts of primary human prostatic carcinoma.

BACKGROUND: Prostatic carcinoma is both the most common invasive cancer and the second most common cause of cancer deaths in men in the United States. Before 1991, attempts to propagate prostatic carcinoma from primary tumors for periods longer than 3 months were unsuccessful in vivo and in vitro with rare exceptions. In 1991, we reported establishment of slowly growing tumors for six of 10 human primary prostatic carcinomas approximately 2-6 months after transplantation. However, none of the tumors were larger than 5 mm or serially transplantable. PURPOSE: Our purpose in this study was to determine whether human primary prostatic carcinoma could be grown as serially transplantable xenografts. METHODS: Cells from primary prostatic carcinomas obtained from transurethral prostatic resections or total prostatectomies in 20 patients were injected subcutaneously into male nude mice on the day of surgery. Sustained-release testosterone pellets were placed subcutaneously in the mice 2-24 days before transplantation of tumors and at intervals of 10-12 weeks. Serial transplantations in subsequent generations of mice were carried out by similar methods. Chromosome analysis was performed on six tumors. RESULTS: Six of 20 primary prostatic carcinomas have grown sufficiently to permit serial transplantation into second mice; four have been documented histopathologically in the second mouse and serially transplanted into three or more successive mice. When a single primary tumor was injected into several mice by the same procedure, tumors failed to grow in some recipients but became serially transplantable in others. Growth of these tumors is slow and irregular, with frequent regressions. Short-term cultures of 10 tumors, eight of which were injected into mice in parallel, were initiated on the day of surgery; CWR31, which was successfully transplanted serially, exhibited only aberrant metaphases and showed clonal, chromosomal changes in culture. Including CWR31, three of the six tumors for which chromosomal analysis was successful contained clonal aberrations. Preliminary studies of SCID (severe combined immunodeficient) mice suggest that they are not superior to nude mice for establishment of serially transplantable prostatic carcinoma xenografts. CONCLUSIONS: A proportion of human primary prostatic carcinomas can be grown as xenografts. Four new serially transplantable xenografts (CWR21, CWR31, CWR91, and CWR22) are currently propagated in our laboratory, a resource that was not previously available. IMPLICATIONS: Our experience suggests that the most important factor in serial transplantation is the collaboration of urologists and pathologists in expediting placement of the tumor in cold saline, examination of the frozen section, and transplantation.

Eradication of neuroblastoma cells in vitro by monoclonal antibody and human complement: method for purging autologous bone marrow.

We describe an in vitro method which is useful for purging autologous bone marrow of neuroblastoma cells. The method utilizes a single murine monoclonal antibody 3G6 (an immunoglobulin MK) which we have previously developed against the ganglioside GD2; undiluted human complement; and unfractionated whole bone marrow at 1 X 10(7) nucleated cells/ml. Tumor cell clonogenic assays, Hoechst 33342 fluorescent nuclear stain, and trypan blue viability stain methods were used to assay cytotoxicity. This complement-mediated cytotoxicity technique killed 99.9-100% of neuroblastoma cell lines NMB-7, LAN-1, LAN-5, and IMR-6, while normal marrow precursor cells were not detectably damaged. The presence of normal bone marrow did not inhibit the human complement-mediated cytotoxicity. Applying the cytotoxicity method to whole unseparated bone marrow demonstrated killing of seeded neuroblastoma cells, with no gross hemolysis or cell clumping. The method did not require expensive special equipment, use of animal complement sera, or prior fractionation of the bone marrow. The average marrow nucleated cell recovery was 95%. These studies indicate that in vitro purging of autologous marrow infiltrated with neuroblastoma with monoclonal antibody 3G6 and human complement is both technically feasible and effective in eradicating residual tumor while preserving bone marrow stem cells.

Fluorimetric studies of tryptophyl exposure in concanavalin A.

Studies of the iodide ion quenching of the intrinsic fluorescence of Concanavalin A indicate that 50% of the tryptophyl fluorescence originates from exposed residues. This agrees with the X-ray crystallographic determination that two of the four tryptophan residues in a Concanavalin A monomer are on the surface. Previous studies have indicated that conformational changes induced by sugar binding alter the environment of aromatic residues. The present investigation finds that neither the specific binding of alpha-methyl-D-mannoside nor alteration of the Concanavalin A quaternary structure changes the number or accessibility of the solvent-exposed tryptophan residues. It therefore appears that the major conformational transitions in Concanavalin A do not affect steric access to the surface tryptophans and the effects previously observed may be ascribed to structurally internal tryptophan residues.

Prevention of ultraviolet radiation-induced suppression of contact and delayed hypersensitivity by Aloe barbadensis gel extract.

We investigated the ability of Aloe barbadensis gel extract to prevent suppression of contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses in mice by ultraviolet (UV) irradiation. Local immune suppression was induced in C3H mice by exposure to four daily doses of 400 J/m2 UV-B (280-320 nm) radiation from FS40 sunlamps, followed by sensitization with 0.5% fluorescein isothiocyanate (FITC) through the irradiated skin. Topical application of 0.167-1.67% Aloe gel after each irradiation significantly reduced this suppression. Aloe treatment partially preserved the number and morphology of Langerhans and Thy-1+ dendritic epidermal cells in skin, compared to those in the skin of mice given only UVR or UVR plus the vehicle. Experiments using a single (2 kJ/m2) dose of UVR followed by Aloe treatment showed that the effect of Aloe was not due to screening of the UVR. Systemic suppression of DTH to Candida albicans or CHS to FITC was induced in C3H mice exposed to 5 or 10 kJ/m2 UV-B radiation, respectively, on shaved dorsal skin and sensitized 3 d later with a subcutaneous injection of formalin-fixed Candida or FITC painted on unirradiated, ventral skin. Treatment of the UV-irradiated skin with Aloe immediately after irradiation prevented suppression of both DTH to Candida and CHS to FITC. Aloe treatment did not prevent the formation of cyclobutyl pyrimidine dimers in the DNA of UV-irradiated skin or accelerate the repair of these lesions. These studies demonstrate that topical application of Aloe barbadensis gel extract to the skin of UV-irradiated mice ameliorates UV-induced immune suppression by a mechanism that does not involve DNA damage or repair.

Aloe barbadensis extracts reduce the production of interleukin-10 after exposure to ultraviolet radiation.

Cutaneous exposure to ultraviolet radiation suppresses the induction of T cell mediated responses such as contact and delayed type hypersensitivity (DTH) by altering the function of immune cells in the skin and causing the release of immunoregulatory cytokines. Extracts of crude Aloe barbadensis gel prevent this photosuppression. Because the regulation of contact hypersensitivity and DTH responses differ, we investigated whether protection was afforded by a single or multiple agents in Aloe and the mechanism by which this material prevents suppression of DTH immunity. The ability of Aloe gel to prevent suppression of contact hypersensitivity responses to hapten decayed rapidly after manufacture. In contrast, agents that protected against systemic suppression of DTH responses to Candida albicans were stable over time. Oligosaccharides prepared from purified Aloe polysaccharide prevented suppression of DTH responses in vivo and reduced the amount of IL-10 observed in ultraviolet irradiated murine epidermis. To assess the effect of Aloe extracts on keratinocytes, Pam 212 cells were exposed in vitro to ultraviolet radiation and treated for 1 h with Aloe oligosaccharides. Culture supernatants were collected 24 h later and injected into mice. Supernatants from ultraviolet irradiated keratinocytes suppressed the induction of DTH responses, whereas Aloe oligosaccharide treatment reduced IL-10 and blocked the suppressive activity of the supernatants. These results indicate that Aloe contains multiple immunoprotective factors and that Aloe oligosaccharides may prevent ultraviolet induced suppression of DTH by reducing keratinocyte derived immunosuppressive cytokines.

Role of chemotherapy in the palliation of gastrointestinal malignancies.

In 1994, more than 200,000 patients in the United States were diagnosed with colon, gastric, or pancreatic cancer. Over half of these patients eventually will have recurrent, incurable disease and require palliative therapy. Although chemotherapy has no role in the primary treatment of most gastrointestinal cancers, and only a limited role as a form of adjuvant treatment in colon cancer, it is sometimes the only systemic treatment available for palliation. Effective chemotherapeutic agents for gastrointestinal cancers are few, and after more than three decades of research, 5-fluorouracil remains one of the few useful agents. This article reviews the role of chemotherapy in palliative therapy for the common gastrointestinal cancers. Discussion also includes recent advances in modulating 5-fluorouracil activity and the many attempts to study newer agents.

The major serological antigen (MSA1) from Schistosoma mansoni eggs is a "circumoval" precipitinogen.

A monoclonal hybridoma antibody to the major serological egg antigen of Schistosoma mansoni (anti-MSA1) reacted with schistosome eggs, forming a circumoval precipitate. This precipitate was seen when anti-MSA1 was incubated with S. mansoni, S. haematobium, and S. japonicum eggs.

Demonstration of a common antigen between Schistosoma mansoni and Fasciola hepatica.

The presence of a common antigen between Schistosoma mansoni eggs and Fasciola hepatica adult worms was demonstrated by utilizing in an anti-S. mansoni adult worm antiserum. Although not one of the three major serologic S. mansoni egg antigens, its complete cross-reactivity suggests that serologic tests done with these crude antigenic extracts will result in many false-positive cases in areas where both parasites are endemic.

Immunity and immunopathology following reinfection of mice cured of chronic schistosomiasis mansoni.

The effect of curative treatment of murine schistosomiasis mansoni on resistance to reinfection and on granulomatous hypersensitivity following reinfection was studied. The sera of mice with light chronic Schistosoma mansoni infection of 32 weeks duration were tested by immunodiffusion analysis with soluble egg antigens for a precipitin band containing an antigen specific for eggs in order to identify bisexual infections. A group of the 32 week bisexually-infected mice were treated with niridazole, as was a similar group of uninfected mice. Six weeks later (at 38 weeks) the previously infected mice were injected subcutaneously with 30 cercariae and the uninfected mice with 20 cercariae. On perfusion 12 weeks later the secondarily infected mice had a percentage worm yield following infection of 16 while that in the primarily infected mice was 60. Twelve weeks after reinfection (at 50 weeks), the previously infected mice had moderate hepatosplenic disease similar to the mice which had remained constantly infected for 50 weeks, and formed relatively small granulomas around eggs injected into their pulmonary microvasculature. In spite of much lower liver egg counts, particularly severe hepatosplenic disease was seen in the previously uninfected mice. When eggs were injected into their lungs, however, very large granulomas were observed. These experiments suggest that both immunity and modulation of immunopathology are residual after curative treatment.

The Schistosoma japonicum egg granuloma. II. Cellular composition, granuloma size, and immunologic concomitants.

Studies of granulomatous hypersensitivity to Schistosoma japonicum eggs were performed at various time periods up to 20 wk after the induction of light infections in mice. Cell populations which were determined in granulomas isolated from the livers revealed a maximum in the total number of cells at 6 wk with a decline of 36% by 20 wk. Large mononuclear cells were predominant at all time periods, with eosinophils being the second most common cell. Measurements of granuloma diameters around single viable eggs in the livers also revealed peak size at 6 wk with a decline of 51% between 16 and 24 wk. Immunodiffusion analysis demonstrated the presence of precipitating antibodies as early as 7 wk after infection. Investigations of lymphocyte blastogenesis revealed a profound depression in response to T-cell mitogens by 8 wk of infection. Studies of footpad swelling to soluble S. japonicum egg antigens revealed massive immediate reactions starting at 6 wk. but no delayed reactivity over a period from 3 to 20 wk. All of these results are related to differences in the biology of S. japonicum in comparison with S. mansoni with respect to the earlier onset of egg production, the much larger numbers of eggs produced, and the possibility of differences in the antigens emitted by the eggs.

Analysis of the immunoglobulins responsible for the circumoval precipitation reaction.

Two morphologically distinct types of circumoval precipitates (COP) have been observed in human Schistosoma japonicum infections. An elongated segmented COP occurs in chronic human infections. An unsegmented "reaction of recent infection" (RRI) occurs in serum from humans with recently acquired infections and is morphologically similar to the reaction observed in the sera of mice infected with S. japonicum. Sera from infected mice and humans were separated by G-200 chromatography to determine whether the unsegmented "RRI" was due to IgM antibody and the segmented COP reaction due to IgG. There was an elevation of the 19S fraction of sera of mice with 10 and 16 week infections. In addition, the murine 7S fraction was elevated in the 16 week infections. The COP activity was confined to the 7S fraction in the murine sera. Sera from Philippine patients which produced reactions of recent infection (acute sera), segmented COP reactions (chronic sera), and mixed reactions (believed to be from a transition stage between acute and chronic schistosomiasis) were tested. All human sera had elevation of both the 19S and 7S fractions of the acute serum. However, COP-reactive antibodies were confined to the 7S fraction of sera from the transition stage and acute stage infections. The results suggest that although IgM antibodies do in certain cases participate in the COP and produce reactions of recent infection, antibody class is not responsible for the different morphology of this reaction.

Efficacy of purified schistosoma japonicum egg antigens for ELISA serodiagnosis of human Schistosomiasis japonica: specificity and sensitivity.

At present, there is no consensus that purified schistosome egg antigens offer any advantage in the diagnosis of schistosomiasis by enzyme linked immunosorbent assay (ELISA). Previously, we demonstrated by multiple techniques that the major serologic antigens in Schistosoma japonicum soluble egg antigen (SEA) are glycoproteins, and that the glycoproteins with highest specificity and sensitivity are hydrophobes. We therefore tested these materials for their specificity, sensitivity and cost effectiveness in the ELISA. In this study we used five SEA fractions that varied in their purity and antigenicity. The order of immunologic specific activity in the ELISA, measured by titration of a standard sera pool, was: hydrophobic glycoproteins (highest), crude SEA glycoproteins, hydrophilic glycoproteins, crude SEA, and SEA proteins (lowest). Complexity (purity) of these materials were (in rank order), hydrophilic glycoproteins (purest), hydrophobic glycoproteins, crude glycoproteins, SEA proteins, and crude SEA (most complex). Epidemiologic sensitivity in the ELISA was tested on limited but well characterized populations. At high antigen coating concentration (0.5 microgram/well), the only antigen fraction with poor sensitivity was SEA proteins. There was little difference in epidemiologic sensitivity between the purer fractions with highest immunologic sensitivity (hydrophobic glycoproteins and crude SEA glycoproteins) and the crude SEA which possesses intermediate immunologic sensitivity. Differences in epidemiologic sensitivity were most pronounced when wells were coated at an antigen concentration (0.1 microgram/well) where crude SEA began to fail. Specificity for all preparations, assessed by reactivity with sera from patients with other trematode infections and with cestode and nematode infections, was excellent. The clinical sensitivity of the ELISA employing crude S. japonicum SEA is so high, and the specificity so good, that the increased immunologic sensitivity of partially purified antigens had little effect on epidemiologic sensitivity. This is not true for the S. mansoni ELISA where crude antigens had inferior sensitivity and specificity.

Humoral immune responses in human hepatosplenic schistosomiasis mansoni.

The development of hepatosplenic schistosomiasis in humans cannot always be related to the intensity of infection. A study was designed to identify different humoral immunologic responses to Schistosoma mansoni in patients with and without hepatosplenic disease. Twenty-four patients with active hepatosplenic disease were closely matched for age, sex, and fecal egg counts with twenty-four patients with only intestinal disease. A serum sample from each of these patients was tested for antibodies to the major soluble egg antigen (MSA1) by radioimmunoassay, for total and IgM antibodies to egg and worm antigenic preparations by ELISA, and for its ability to suppress antigen stimulated lymphocyte blastogenesis. No difference was found using these assays between the hepatosplenic and the intestinal schistosomiasis patients.

A phase II pharmacodynamic study of pyrazoloacridine in patients with metastatic colorectal cancer.

PURPOSE: To perform a phase II trial of pyrazoloacridine (PZA), a novel DNA intercalator, in patients with metastatic colorectal carcinoma and no previous therapy. METHODS: PZA was administered at a dose of 750 mg/m2 intravenously over 3 h every 21 days. Pharmacokinetic studies to determine PZA plasma concentrations were performed. RESULTS: No responses were seen in 14 response-evaluable patients. Patients received a median of two cycles of PZA (range 1-6). Toxicity included neutropenia and neurologic side-effects, which were > or = grade III in 73% and 14%, respectively. High plasma concentrations of PZA (Cmax) correlated with low neutrophil counts (P = 0.04). CONCLUSIONS: PZA is inactive at this dose and schedule in colorectal cancer, and produces moderately severe toxicity.

Inhibition of UV-induced immune suppression and interleukin-10 production by plant oligosaccharides and polysaccharides.

Application of Aloe barbadensis poly/oligosaccharides to UV-irradiated skin prevents photosuppression of delayed-type hypersensitivity (DTH) responses in mice. We tested the hypothesis that these carbohydrates belong to a family of biologically active, plant-derived polysaccharides that can regulate responses to injury in animal tissues. C3H mice were exposed to 5 kJ/m2 UVB from unfiltered FS40 sunlamps and treated with between 1 pg and 10 micrograms tamarind xyloglucans or control polysaccharides methylcellulose or dextran in saline. The mice were sensitized 3 days later with Candida albicans. Tamarind xyloglucans and purified Aloe poly/oligosaccharides prevented suppression of DTH responses in vivo and reduced the amount of interleukin (IL)-10 observed in UV-irradiated murine epidermis. Tamarind xyloglucans were immunoprotective at low picogram doses. In contrast, the control polysaccharides methylcellulose and dextran had no effect on immune suppression or cutaneous IL-10 at any dose. Tamarind xyloglucans and Aloe poly/oligosaccharides also prevented suppression of immune responses to alloantigen in mice exposed to 30 kJ/m2 UVB radiation. To assess the effect of the carbohydrates on keratinocytes, murine Pam212 cells were exposed to 300 J/m2 UVB radiation and treated for 1 h with tamarind xyloglucans or Aloe poly/oligosaccharides. Treatment of keratinocytes with immunoprotective carbohydrates reduced IL-10 production by approximately 50% compared with the cells treated with UV radiation alone and completely blocked suppressive activity of the culture supernatants in vivo. The tamarind xyloglucans also blocked UV-activated phosphorylation of SAPK/JNK protein but had no effect on p38 phosphorylation. These results indicate that animals, like plants, may use carbohydrates to regulate responses to environmental stimuli.

Induction of primary cutaneous melanomas in C3H mice by combined treatment with ultraviolet radiation, ethanol and aloe emodin.

The role of ultraviolet (UV) radiation in the induction of nonmelanoma skin cancer is widely accepted, although its precise contribution to the development of primary cutaneous melanoma skin cancer requires further definition. We found that painting aloe emodin, a trihydroxyanthraquinone from Aloe barbadensis, in ethyl alcohol vehicle on the skin of mice in conjunction with exposure to UVB (280-320 nm) radiation results in the development of melanin-containing skin tumors. C3H/HeN mice were treated thrice weekly with aloe emodin in a 25% ethanol in water vehicle and exposed to 15 kJ/m2 UV radiation. Neither ethanol vehicle nor aloe emodin alone induced skin tumors in the absence of UV radiation. In two separate experiments, 20-30% of the mice treated with a combination of UV radiation and ethanol vehicle and 50-67% of the UV-irradiated animals given aloe emodin in ethanol vehicle developed primary cutaneous melanin-containing tumors. The diagnosis of melanoma was established using Fontana silver stain for melanin; these tumors were negative for vimentin and keratin. Melanin-containing melanosomes were observed by transmission electron microscopy in tumors diagnosed as melanomas. Although the mechanism of carcinogenesis in these mice is currently unknown, our findings have led to the development of the first facile murine model for the induction of primary melanoma. This model has the potential to clarify the role of UV radiation in the etiology of malignant melanoma.

Treatment of colon and rectal cancer.

This article discusses multimodal treatment of noncomplicated colon and rectal cancer, considerations for specific types of colon cancer, considerations that may modify the extent and technique of surgery, the role of adjuvant chemotherapy for colon adenocarcinoma and rectal cancer, and surgical treatment of complicated colorectal cancer.

Polymyositis, prednisone, and pheomycotic cyst.

An immunosuppressed patient with polymyositis presented with an apparent ganglion of the left foot. During the operative procedure, a cystic mass inconsistent with a ganglion was excised and immediately sent to the Quantitative Bacteriology Laboratory. A rapid slide examination revealed yeastlike bodies present in the tissue. The remainder of the tissue was sent to pathology for special staining. The H&E and GMS stains revealed findings compatible with the diagnosis of a pheomycotic cyst, and the appropriate cultures confirmed this. This represents an unusual opportunistic infection in an immunosuppressed host. As more patients are managed with immunosuppressive drugs, this diagnosis will need to be considered much more frequently if treatment is to be effective.