BACKGROUND. Laboratory evidence suggests that many anticancer agents exert their effect by altering the ratio between apoptosis and cellular proliferation. The objective of this clinical study was to examine in vivo changes in apoptotic index (AI), Bcl-2 expression, proliferation (Ki-67 and S-phase fraction [SPF]), and ploidy as potential indicators of chemoresponsiveness.METHODS. Twenty-eight women with primary operable breast carcinoma received 2M (mitoxantrone 11 mg/m(2), methotrexate 35 mg/m(2) every 3 weeks) for 4 cycles before surgery/radiotherapy, and an additional 2 cycles were given after surgery. Clinical response was assessed after four cycles of treatment according to World Health Organization criteria. Changes in molecular markers were assessed from biopsies obtained by fine-needle aspiration performed before treatment, repealed after 24 and/or 72 hours (T1), and on Days 7 and 21 after the first cycle of chemotherapy. Flow cytometric analysis was used to assess SPF, ploidy, and Al (in situ DNA nick end labeling assay) whereas Ki-67 and Bcl-2 were evaluated by immunocytochemical analysis.RESULTS. The overall response rate was 61% (17 of 28 patients), with a 14% (4 of 28 patients) complete response rate. Patients with diploid carcinomas (P = 0.04) with high Ki-67 (P = 0.0001) and SPF (P = 0.09) were more likely to respond to treatment. Median AI increased by 3.4% with interquartile (IQ) range of 3.2 in responders, compared with only -0.1% (IQ range, 2.2) in nonresponders at T1 (P = 0.03). Median Ki-67 decreased by -12.0% (IQ range, 22.9) in responders and increased by 18.5% (IQ range, 15.1) in nonresponders on Day 21 (P = 0.003). Median Bcl-2 scores increased by 1.0 (IQ range, 4.0) in responders and were unchanged at 0.0 (IQ range, 0.5) in nonresponders (P = 0.08). Changes in SPF or ploidy were not significantly predictive of response.CONCLUSIONS. The results of this preliminary study support evidence that chemotherapy may increase apoptosis, decrease Ki-67, and increase Bcl-2 expression in primary breast carcinomas that subsequently respond to therapy. Methodology allowing morphological confirmation of apoptosis would be advantageous. Cancer 2000;89:2145-52. (C) 2000 American Cancer Society.

The proliferation markers Ki67 and S-phase fraction (SPF) are important biological variables in determining the course of malignant disease. Changes in these variables may provide additional prognostic information. We have studied changes in Ki67 (measured by immunocytochem-istry using the Mib 1 antibody) and SPF (by flow cytometry) on samples obtained by FNA from patients with early breast cancer. In a contrai group of 25 patients repeat FNAs were taken 2 weeks apart, with no intervening treatment, in order to determine the normal variation. For Ki67 the median % + ve for the first sample was 3.0% (range 0–23%) and the second was 4.0% (range 0–23%). For SPF the median for the first sample was 7.8% (range 1.5–21.8%) and for the second 10.3% (range 0.8–22.5%). This demonstrates (i) the good re-producibility of the technique and (ii) that FNA itself does not affect subsequent measurement of proliferation in the same tumour. In 24 patients repeat FNA was performed at 10 or 21 days after chemoendocrine therapy (CET) with Mitozantrone, Methotrexate and tamoxifen. Pre-CET the median Ki67 was 12.9% (range 1–37.7%)andpost-CET 5.5% (range 0–14.7%), P < 0.05. Pre-CET the median SPF was 4.1% (range 0.9–27.7%)and the post-CET 3.2% (range 0.4–19.2%), P = NS. These changes in Ki67 may be used as an intermediate marker of response to evaluate the effectiveness of different therapeutic agents in groups of patients. For individuai patients change in relation to response to therapy needs to be evaluated with more patients. Additional quantitative measurement of apoptosis might enhance the biological and clinical significance of these measurements. The proliferation markers Ki67 and S-phase fraction (SPF) are important biological variables in determining the course of malignant disease. Changes in these variables may provide additional prognostic information. We have studied changes in Ki67 (measured by immunocytochem-istry using the Mib 1 antibody) and SPF (by flow cytometry) on samples obtained by FNA from patients with early breast cancer. In a contrai group of 25 patients repeat FNAs were taken 2 weeks apart, with no intervening treatment, in order to determine the normal variation. For Ki67 the median % + ve for the first sample was 3.0% (range 0–23%) and the second was 4.0% (range 0–23%). For SPF the median for the first sample was 7.8% (range 1.5–21.8%) and for the second 10.3% (range 0.8–22.5%). This demonstrates (i) the good re-producibility of the technique and (ii) that FNA itself does not affect subsequent measurement of proliferation in the same tumour. In 24 patients repeat FNA was performed at 10 or 21 days after chemoendocrine therapy (CET) with Mitozantrone, Methotrexate and tamoxifen. Pre-CET the median Ki67 was 12.9% (range 1–37.7%)andpost-CET 5.5% (range 0–14.7%), P < 0.05. Pre-CET the median SPF was 4.1% (range 0.9–27.7%)and the post-CET 3.2% (range 0.4–19.2%), P = NS. These changes in Ki67 may be used as an intermediate marker of response to evaluate the effectiveness of different therapeutic agents in groups of patients. For individuai patients change in relation to response to therapy needs to be evaluated with more patients. Additional quantitative measurement of apoptosis might enhance the biological and clinical significance of these measurements.

Seven populations of human leukaemic cells were implanted i.v. into sublethally irradiated severe combined immunodeficient (scid) mice. Growth of leukaemia was monitored by labelling murine peripheral blood (PB) cells with an anti-HLA monoclonal antibody and flow cytometric analysis. Two of the populations transplanted were fresh acute lymphoblastic leukaemia (ALL) bone marrow (BM) cells which both caused sustained proliferative growth in scid mice. Human cells accounted for up to a mean of 87% of the total nucleated cells (TNC) in the PB of these mice between weeks 12-15. One of these populations was passaged into fresh mice and frank leukaemia was again established. Three populations of cryopreserved acute myeloblastic leukaemia (AML) cells (2 obtained from PB and 1 from BM) and one population of cryopreserved biphenotypic acute leukaemia BM cells, only grew to a maximum of 4% within the 15 week period of the experiment. A cell population from an AML cell line (HL60), however, did engraft and proliferate resulting in a rapid deterioration of these mice between weeks 3-6 when the proportion of human cells accounted for 9% of the TNC in the PB.