Inducible T-cell receptor expression in precursor T cells for leukemia control.

Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T-cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. As expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8(+) T-cell development was required to obtain a mature T-cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T-cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity.

Naive tumour-specific CD4+ T cells were efficiently primed in acute lymphoblastic leukaemia.

The recognition and neutralization of tumour cells is one of the big challenges in immunity. The immune system has to recognize syngeneic tumour cells and has to be primed and respond in an adequate manner. Priming of a leukaemia-specific immune response is a crucial step in tumour immunology that can mislead to tumour tolerance either by T cell ignorance, deletion or Treg induction. To resemble the situation of acute lymphoblastic leukaemia (ALL) in patients, we used the murine BALB/c model with syngeneic BM185 tumour cells. We established a tumour cell line that expresses the neo-antigen ovalbumin (BM185-OVA/GFP) to allow the application of T cell receptor transgenic, antigen-specific CD4(+) T cells. Here, we demonstrate that effective anti-ALL immunity can be established by in vivo priming of CD4(+) T cells that is sufficient to differentiate into effector cells. Yet they failed to control tumour alone, but initiated a Th1 response. An efficient tumour clearance was dependent on both antigen-specific CD4(+) T cells and CD8(+) effector T cells from the endogenous repertoire. The tolerogeneic milieu was characterized by increased Tregs numbers and elevated IL-10 level. Tregs hamper effective antitumour immune response, but their depletion did not result in reduced tumour growth. In contrast, neutralization of IL-10 improved median mouse survival. Future therapies should focus on establishing a strong CD4+ T cells response, either by adjuvant or by adoptive transfer.

Induced transgene expression for the treatment of solid tumors by hematopoietic stem cell-based gene therapy.

Tumor microenvironment is composed of different cell types including immune cells. Far from acting to eradicate cancer cells, these bone marrow-derived components could be involved in carcinogenesis and/or tumor invasion and metastasis. Here, we describe an alternative approach to treat solid tumors based on the genetic modification of hematopoietic stem and progenitor cells with lentiviral vectors. To achieve transgene expression in derivative tumor infiltrating leukocytes and to try to decrease systemic toxicity, we used the stress inducible human HSP70B promoter. Functionality of the promoter was characterized in vitro using hyperthermia. Antitumor efficacy was assessed by ex vivo genetic modification of lineage-negative cells with lentiviral vectors encoding the dominant-negative mutant of the human transforming growth factor-ß receptor II (TßRIIDN) driven by the HSP70B promoter, and reinfusion of cells into recipient mice. Subsequently, syngeneic GL261 glioma cells were subcutaneously injected into bone marrow-transplanted mice. As a result, a massive antitumor response was observed in mice harboring TßRIIDN under the HSP70B promoter, without the need of any external source of stress. In summary, this study shows that stem cell-based gene therapy in combination with spatial and temporal control of transgene expression in derivative tumor-infiltrating cells represents an alternative strategy for the development of novel antitumor therapies.

Primary malignant melanoma of the uterine cervix: case report with world literature review.

A 63-year-old patient with a malignant melanoma of the uterine cervix is described. Subtle epitheliotropism of the neoplastic cells within the endocervical columnar epithelium suggested melanoma in situ and the possibility of a primary uterine cervical melanoma, despite a negative anti-S-100 protein immunohistochemical stain. An exhaustive clinical workup, and ultimately, complete autopsy failed to reveal any other primary tumor site, and the diagnosis of melanoma was confirmed by histology and immunohistochemistry on the hysterectomy specimen. A world literature review revealed 54 previously reported cases of uterine cervical melanoma of which 43 had been reported as primary uterine cervical melanoma. A true intraepithelial melanocytic component was found in only 14 of those cases, however, and none of those reports illustrated this with the clarity with which it was seen in the endocervical glandular and surface columnar epithelium of the present case. Primary uterine cervical melanoma is usually discovered at an advanced stage and is no longer amenable to curative therapy. Even when this tumor is discovered early, however, the diagnosis may be unnecessarily delayed if the often subtle interaction of the neoplastic cells with the benign cervical squamous or glandular epithelium is not appreciated, or if the possibility of malignant melanoma is not entertained based on other histologic or immunohistologic characteristics of the tumor cells.

Pancreas allograft rejection: correlation of transduodenal core biopsy with Doppler resistive index.

PURPOSE: To determine the usefulness of sonographically obtained resistive indexes (RIs) in the diagnosis of pancreas allograft rejection. MATERIALS AND METHODS: Findings were studied from 78 transduodenal pancreas allograft biopsies that were ultrasound-guided and cystoscopically directed. The 78 biopsies included 40 that were compared directly with baseline RI data. Biopsies were categorized by result and correlated with concurrent RIs (including 26 RIs obtained within 24 hours of biopsy) with the chi2 test for categoric variables and the Student t test for continuous variables. Sensitivity, specificity, and positive and negative predictive values were calculated with standardized formulas. RESULTS: The mean RIs between the no rejection, mild acute rejection, and moderate acute rejection groups were not statistically significantly different; however, the mean RI associated with chronic rejection was statistically significantly higher (P < .05) than that in the other groups. The sensitivity, specificity, and positive and negative predictive values of either an elevated RI (> 0.70) or greater than 10% increase in the RI above the baseline value in the diagnosis of acute rejection were approximately 50%. CONCLUSION: Neither the absolute level of the RI nor the relative increase was correlated with acute rejection proved at biopsy. Changes in RIs after pancreas transplantation were a poor indicator of acute rejection, but the absolute value of the RI was elevated in cases of chronic rejection.

Immunocytochemical assay for estrogen receptor with monoclonal antibody D753P gamma in routinely processed formaldehyde-fixed breast tissue. Comparison with frozen section assay and with monoclonal antibody H222.

BACKGROUND: Estrogen receptor (ER) immunocytochemical assay (ICA) can be a useful procedure in patients with breast cancer. The authors sought to study the utility of monoclonal antibody D75P3 gamma (D75) in paraffin-embedded sections of breast cancers. METHODS: Sixty-seven cases of breast cancer were studied by ICA for ER using monoclonal antibody D75 in routinely processed formaldehyde-fixed paraffin-embedded tissue, employing ficin predigestion of sections before application of D75. The results were tabulated using a modified histochemical score (HS). In 59 cases, frozen sections of tumors were also studied with D75. In 40 cases, paraffin-embedded sections were stained with both D75 and monoclonal antibody H222 (which is commercially available). Quantitative biochemical ER assays were performed by the standard dextran-coated charcoal method. RESULTS: Statistical analysis of the data showed a highly significant correlation (P < 0.0001) between biochemical ER and paraffin-embedded section HS using D75. The results of frozen section assay and paraffin-embedded section assay using D75 were also highly correlated (P < 0.0001). In addition, paraffin-embedded section HS using D75 correlated well with paraffin-embedded section HS using H222 (P < 0.0001). However, D75 HS generally were higher than H222 HS because of the more intense staining with D75, and this difference was statistically significant (P < 0.0001). Assuming that the biochemical ER data were "true," paraffin-embedded section ICA with D75 had a diagnostic sensitivity of 100%, a diagnostic specificity of 70%, a positive predictive value of 89%, and a negative predictive value of 100%. The results of biochemical ER status and paraffin-embedded section D75 ICA ER status agreed in 61 of 67 cases (91%). In all six cases where the ER status differed, the biochemical ER status was negative, and the ER status by ICA with D75 was positive, leading the authors to suspect that some of these cases may have been biochemical false-negative findings. CONCLUSIONS: It was concluded that ICA for ER with D75 using a 2-minute ficin predigestion of formaldehyde-fixed paraffin-embedded tissue is an acceptable alternative to frozen section ICA. In addition, paraffin-embedded section ICA with D75 is easier to interpret than frozen section ICA, and the results are superior to ICA with H222 on paraffin-embedded sections.

The establishment of normal limits for serum proteins measured by the rate nephelometer. Concepts of normality revisited.

A study of the distribution of nephelometrically determined data for IgG, IgM, IgA, C3, C4, and haptoglobin was made in two geographically separate, healthy populations. Evaluation of these data by several tests of statistical normality revealed that the tests vary considerably in power; i.e., data that one rejects as gaussian may be accepted by the other. No rules exist to enable one to decide which is the best test to apply in a given situation. This study indicates that the uncritical application of X +/- 2 S.D. for the reference range of clinical laboratory tests may be fraught with error, which may not be obvious and may not provide clinically useful information in many cases. A large scale clinical investigation designed to empirically obtain limits of different laboratory tests with desired levels of specificity and sensitivity for different disease states may prove rewarding and yield knowledge more useful for clinical purposes. Until such an investigation is carried out, however, we recommend determination of reference ranges by a nonparametric percentile estimate (2.5 to 97.5 per cent), which makes no a priori assumptions about the distribution of the data in the population.

The optically clear nucleus. A reliable sign of papillary carcinoma of the thyroid?

The clear ("Orphan Annie Eye") nucleus has been accepted as one of the important microscopic features of papillary carcinoma of the thyroid. This study undertook an examination of 100 consecutive thyroid lesions exclusive of papillary, mixed, and follicular carcinomas for the presence of these nuclei. Only two lesions (2%), a follicular adenoma and diffuse hyperplasia, had such nuclear morphology but as focal changes. Thirty-seven cases of papillary, mixed, and follicular carcinoma were also studied. Clear or empty nuclei were present in 83% of papillary carcinomas. One carcinoma of follicular type had clear nuclei in a diffuse distribution. "Pseudoclear" nuclei were noted in a variety of situations ranging from normal thyroids to diffuse hyperplasia, where they were present in 65% of cases. We conclude that clear nuclei when present as a diffuse changes in a thyroid tumor are a reliable sign of papillary carcinoma but are not pathognomonic. If the character of the clear nuclei is questionable, other histologic features of papillary carcinoma should be looked for, such as papillae with overlapping nuclei, psammoma bodies and multicocality. It was also fould that frozen sections and imprints do not demonstrate the nuclei; they appear only in fixed tissues.

Mucocele of the appendix secondary to obstruction by endometriosis.

A mucocele of the appendix secondary to obstruction by endometriosis is reported and the relevant literature reviewed. The theories of the pathogenesis of appendiceal mucocele are reviewed and discussed. To our knowledge, no similar, well documented case has been reported.