Philip D. Noguchi

Prognostic significance of dna ploidy in carcinoma of prostate

Flow cytometry was used to measure the DNA content in archived paraffin-embedded human prostatic cancer tissue for 69 patients with known outcomes that presented between 1975 and 1982. Of these, 51 patients had clinically localized lesions and were surgically staged prior to radical prostatectomy, while 18 patients presented with advanced Stage D2 disease. Thirty-six of 37 (97.3%) pathologic Stage B lesions were diploid. In contrast, the majority (72.2%) of patients with metastatic disease had aneuploid tumors. The average Gleason grade for aneuploid tumors was 8.2 +/- 1.98 versus 5.5 +/- 1.89 for diploid tumors (p less than 0.01). For 51 patients with clinically localized tumors, 13.9 percent of diploid tumors with a low Gleason sum (2 to 6) had extracapsular spread of tumor or regional lymph node involvement compared with 83.3 percent of aneuploid tumors with high Gleason scores (7 to 10). The addition of DNA ploidy to degree of glandular differentiation may enhance the prognostic evaluation of prostatic tumors and eventually improve our ability to select patients who are likely to benefit from radical prostatectomy.

Augmentation of tumor antigen expression by recombinant human interferons: Enhanced targeting of monoclonal antibodies to carcinomas

The use of Mabs for the detection and treatment of human carcinoma lesions can still be regarded in its infancy. As with other new approaches to cancer therapy, several conceptual as well as real problems exist when designing clinical protocols for Mab-directed immunotherapy. From the Mab standpoint, studies using the intact IgG have shown that, in a majority of patients injected with IgG, human anti-mouse IgG antibodies develop that hamper the effectiveness of subsequent antibody administration. It is believed that the human anti-mouse antibody response is directed against the Fc region of the IgG molecule. The elimination of this region through fractionation of the Mab to obtain the minimum binding site could result in a less immunogenic molecule. Another approach aimed at reducing the immunogenicity of the Mab would be to clone the genes encoding for individual Mabs, reduce them via restriction endonuclease techniques, and insert human immunoglobulin constant regions. The resulting chimeric antibodies are believed to reduce the development of human anti-mouse antibodies. Effective Mab therapy of human tumor lesions may also be achieved through the recruitment of a portion of the hosts immunologic defense system. An example is the use of anti-idiotype Mabs that use as immunogen a Mab to a tumor antigen. The anti-idiotype antibodies are selected for binding to the antigen binding, or idiotype, region of the first Mab. The binding sites of the new anti-idiotype Mabs should reflect the internal image of the original antigen. The anti-idiotype antibodies may be used to immunize patients (i.e., vaccines) in an attempt to mount an active immune response against the antigen-positive tumor cells. Recent studies have shown a synergism between interferon-alpha and an anti-idiotype Mab for the in-vivo antitumor activity in a murine B-cell lymphoma experimental model. Whether an interferon-mediated increase in the tumor antigen or the Fc receptor was part of the synergism was not investigated. Mabs alone have also been shown to elicit cytotoxic activity in vitro and tumoricidal activity in vivo. Antibodies of the IgG2a isotype can direct macrophage-mediated cytotoxicity. These studies revealed the importance of the number of antibody sites per cell as well as the number of cells that bind the IgG2a Mab, thus suggesting a threshold requirement for the demonstration of effective tumor cell lysis in vitro and in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Interferon-γ induces altered oncogene expression and terminal differentiation in A431 cells

Abstract In tumor cell lines in which oncogene expression is abnormal, modulation of the expression of the oncogene (myc, src, or ras) by interferons (IFNs) has been observed concurrently with cell growth inhibition or phenotypic reversion. Oncogene expression has also been reported to vary during the differentiation of several neoplastic cell lines. Treatment of monolayer cultures of A431, a human epidermoid carcinoma cell line, with IFN-γ resulted in rapid morphological alterations and cell death not seen with either IFN-α or IFN-β These changes were accompanied by elevated expression of mRNAs for p21 (the c-ras gene product) and the epidermal growth factor receptor as well as increases in the biosynthetic rate of their respective proteins. These effects likewise appeared to be specific for IFN-γ. Growth inhibition by IFN-γ was also observed when A431 cells were grown in a three dimensional in vitro culture system. Immunohistochemical staining of these “tumoroids” with a differentiation specific, anti-keratin antibody indicated that IFN-γ enhanced expression of this keratin. This observation suggests that the killing by IFN-γ of A431 cells may result from an acceleraton of terminal differentiation.

Heterogenous expression of a murine B16 melanoma-associated antigen correlates with cell cycle

SummaryA murine monoclonal antibody (MM2-3C6) that reacts with a B16 murine melanoma-associated membrane antigen was used to study the relationship of antigen expression to the cell cycle. Dual-parameter flow cytometric measurements of membrane antigen and DNA revealed that antigen-positive cells were present throughout the cell cycle. Peak antigenic expression was noted during the late log phase of the cell growth curve with negligible antigen-negative population. The emergence of a distinct antigen-negative population (30%–40%) was noted in the late stationary phase. Cell cycle analysis indicated that the negative subpopulation was restricted to the G0/G1 phase, thus, demonstrating antigenic heterogeneity within the tumor cell population. Cell sorting was performed to analyze the origin of such heterogeneity. Following two sequential sortings, the antigen-negative cells became antigenic upon reculture. Again, at the late stationary phase, a distinct antigen-negative population (30%–40%) emerged. The sorted antigen-positive cells showed flow cytometric profiles identical to the sorted antigen-negative population upon reculture. Therefore, in this murine model, it appears that antigen expression is cell cycle dependent and such expression seems to be associated with proliferation.

Expression of a murine B16 melanoma-associated antigen analyzed by flow cytometry

Understanding factors regulating expression of tumor antigens is vital to the design of rational immunotherapeutic strategies. A murine monoclonal antibody (MM2-3C6) that recognizes a B16 murine melanoma-associated membrane antigen was used to study antigen expression in relation to cell cycle, clonal heterogeneity, and growth density. Dual-parameter flow cytometric measurements of DNA content and membrane antigen demonstrated that antigen-positive cells were found throughout the cell cycle. However, some antigen-negative cells were observed and were restricted to the G0/G1 phase demonstrating the antigenic heterogeneity of this tumor line. Three sublines of B16, F1, F10, and F1r with variable metastatic potential, and 50 B16 F1 clones all expressed the antigen with a mean antigen density above background of 8.84 +/- 2.52 [( mean cell fluorescence/cell size] X 10(-3] ranging from 3.68 to 16.57. Further studies of three sublines and five clones showed antigen density fluctuated over an 8-day growth period in culture with daily changes of medium. During log growth from Day 1 (1.2 +/- 0.08 X 10(4) cells/cm2) to Day 4 (20.4 +/- 3.01 X 10(4) cells/cm2), antigen density increased from 4.09 +/- 0.29 to 6.86 +/- 0.29. By Day 8 (26.5 +/- 2.86 X 10(4) cells/cm2) when the cells had been confluent for 3 days, antigen density decreased to 2.54 +/- 0.26 which was significantly lower than other days measured (P less than 0.05). It is concluded that tumor cell proliferation and cell density can modulate antigen expression in this system and, therefore, this may be a useful model to study tumor cell escape from immunotherapy.