Philip G. Archer

Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral-blood progenitors and growth factors on engraftment.

PURPOSE To evaluate the capacity of enriched CD34-positive (CD34+) progenitor cells to reconstitute hematopoiesis in poor-prognosis breast cancer patients following administration of a high-dose alkylating agent chemotherapy regimen. PATIENTS AND METHODS Forty-four breast cancer patients received high-dose chemotherapy followed by autologous bone marrow support (ABMS) with CD34+ hematopoietic progenitor cells in five sequentially treated cohorts. Following infusion of CD34+ marrow, cohort no. 1 received no growth factor, cohort no. 2 received granulocyte colony-stimulating factor (G-CSF), and cohort no. 3 received granulocyte-macrophage colony-stimulating factor (GM-CSF). Cohort no. 4 received the CD34+ fractions of both marrow and peripheral-blood progenitor cells (PBPCs) plus G-CSF. Cohort no. 5 received only the CD34+ PBPCs plus G-CSF. Immunohistochemical staining for breast cancer was performed on all hematopoietic cell products before and after the positive selection procedure, to assess quantitatively the level of tumor-cell contamination. RESULTS Cohorts no. 1, 2, 3, 4, and 5 achieved a granulocyte count > or = 500 x 10(9)/L in a median of 23, 10, 16, 11, and 11 days, with a platelet count greater than 20,000 x 10(9)/L documented in a median of 22, 23, 32, 12, and 10 days, respectively. The time to granulocyte reconstitution was significantly shorter for patients who received CD34+ PBPCs alone (cohort no. 5), or in combination with CD34+ marrow (cohort no. 4), when compared with those who received only the CD34+ marrow fraction (P < .01). From 1 to greater than 4 logs of breast cancer cell depletion were documented after CD34-selection, for patients in whom tumor was initially detected. CONCLUSION CD34+ marrow and/or PBPCs provide reliable and timely hematopoietic reconstitution in breast cancer patients receiving high-dose chemotherapy. Contamination of both marrow and PBPCs with breast cancer cells was reduced using this positive selection technique.

Chromosome aberrations in individuals occupationally exposed to ethylene oxide, and in a large control population☆

Chromosome aberration frequencies in 61 employees potentially exposed to ethylene oxide (ETO) were compared with those in unexposed control groups. We studied 3 worksites with differing historical ambient levels of ETO. Within worksites, groups were classified as high potential exposed, low potential exposed, or controls. Further control groups including an off-site community control group were added to give a total of 304 control individuals. Blood samples were drawn several times over a 24-month period. Aberrations were analyzed in 100 cells per sample after culture for 48-51 h. Worksites I, II and III respectively represented increasing levels of potential ETO exposure. At worksites I and II, no consistent differences in aberration frequencies were found among groups. At worksite III aberration frequencies in potentially exposed individuals were significantly increased compared with controls. The frequencies of cells with aberrations were 5.6% for the 2 individuals in the high potential exposure category and 2.6% for 23 persons in the low potential exposure group. The overall frequency of cells with aberrations in the matched control individuals was 1.4%. In the total control group of 304 individuals we found significant increases in aberrations associated with smoking and with increasing age. We have also reported previously an association between sister-chromatid exchange (SCE) frequency and ETO exposure (Stolley et al., 1984). When aberration frequencies were compared with levels of SCEs there was only a weak overall association. The correlation was found in potentially exposed but not in control groups, and for any individual, one observation could not be used to predict the other.

Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support

SummaryDetection of small numbers of breast cancer cells is important in staging the disease and can be helpful in assessing the efficacy of purging regimens prior to autologous stem cell infusion. Immunohistochemical methods are potentially useful and broadly applicable for this purpose since they are simple to perform, sensitive, and may be quite specific. We have used a combination of four monoclonal antibodies [260F9, 520C9, 317G5 (Baxter Corp); BrE-3 (Dr. R. Ceriani)] against tumor cell surface glycoproteins in a sensitive immunocytochemical assay to identify breast tumor cells in bone marrow and peripheral blood. Immunostained cytospin preparations were fixed prior to staining to preserve cytological details of immunopositive cells. After immunostaining, slides were counterstained with hematoxylin to confirm the identity of labeled cells. In cytocentrifuge experiments in which small numbers of CAMA human breast tumor cells were added to bone marrow mononuclear cells, a linear relationship between the number of tumor cells added and the number of tumor cells detected was obtained over a broad range of tumor cell concentrations. The probability of detecting tumor cells was dependent on the number of cytocentrifuge slides examined. When ten slides (5 million cells) were examined, the probability of detecting tumor at a concentration of 4 tumor cells per million bone marrow mononuclear cells was 98%. In clinical specimens, tumor cells were detected in marrow aspirates from 73 of 240 (30%) patients undergoing autologous transplantation, including 70 (37%) of 190 patients with clinical stage IV disease, 0 of 7 patients with clinical stage III disease, and 3 of 43 (7%) patients with clinical stage II disease. Seventy-three of 657 peripheral blood specimens from 26 of 155 patients (17%) contained breast cancer cells with counts ranging from 1 to 97 tumor cells per million leukocytes. Tumor cells were most frequently found in the blood of patients with stage IV disease [21 of 107 (20%)] but were also found in a substantial number [5 of 44 (11%)] of patients with stage II disease. Positive selection of CD34-positive hematopoietic progenitor cells as well as negative purging methods such as incubation with 4-hydroxyperoxy-cyclophosphamide (4-HC) were evaluated with respect to tumor cell depletion. Selection of CD34-positive progenitor cells from bone marrow or peripheral blood resulted in log reduction of 1 to > 4 tumor cells reinfused at autologous transplantation. A lesser log reduction (up to 1) was demonstrated following 4-HC purging. We conclude that properly performed and controlled immunocytochemical staining of bone marrow and peripheral blood cytospins is a sensitive and simple way to detect and quantitate breast cancer cells in hematopoietic specimens harvested for autotransplantation and that CD34-positive progenitor cell selection results in significant reduction in the number of breast cancer cells reinfused with marrow or peripheral blood stem cells.

The closest-individual method in the analysis of the distribution of capillaries ☆

Abstract An ecological survey method termed the closest-individual method was adapted for measuring the distribution of capillaries in tissue. Models were constructed in which dots represented transversely sectioned capillaries in known densities, and in ordered, random, or contagious (clustered) array. The distances from randomly selected points on a model to the nearest capillary were measured, and the frequency distributions of these distances were plotted. This distribution was compared to the mathematically predicted distribution of distances for each model. All values derived by the closest-individual method were statistically similar to predicted values, and were within approximately 5% of predictions. In addition, the kurtosis of each set of data was used to evaluate whether the data from the closest-individual method could correctly identify the nature of the array of capillaries in each model. This method is potentially of great use to studies of capillarity since it calculates median diffusion distance more accurately than estimates from capillary density alone; it calculates maximal diffusion distance, which cannot be estimated from capillary density alone; and by identifying the array of capillaries as either random, ordered, or contagious, it assesses the nature of interaction among capillaries. Other similar measurement methods are discussed and compared.

Tissue factor activity in hela cells measured with a continuous chromogenic assay and elisa reader

Tissue factor activity expressed by Hela cells cultured in 96-well plates has been quantitated in situ using a continuous spectrophotometric assay. Following the assay, cells assayed without physical disruption remained as viable as cells not subjected to the assay. Very little (or no) tissue factor was expressed in nondisrupted cells relative to that available in cells disrupted by freeze-thawing and sonication. Total tissue factor activity (that available in disrupted cells) decreased not as a simple function of time after subculturing, but was inversely related to cell density.

Evaluation of the concentric-circles method for estimating capillary-tissue diffusion distances

Abstract The concentric-circles method for estimating the complete distribution of distances from any point in the tissue of animals to the nearest capillary was evaluated. Models representing capillaries in transverse sections of muscle tissue were devised, using dots at various densities in ordered, random, or contagious (clustered) arrays. The distribution of distances from random points to the nearest capillary in each model was derived mathematically. These values were compared to values estimated by a method using sets of concentric circles placed over the models. In each set of circles, the smallest circle containing a capillary was recorded. The concentric-circles method generated estimates of median and maximal distances to the nearest capillary, and provided complete distributions of distances that were statistically similar to predictions, and deviated from predictions by an average of less than 10%.

Bone marrow involvement by lobular carcinoma of the breast cannot be identified reliably by routine histological examination alone

The aims of this study were twofold: (1) to evaluate the ability of pathologists to recognize infiltration of bone marrow core biopsy specimens by breast carcinoma, particularly lobular carcinoma, using routine hematoxylin-eosin (HE) sections; and (2) if indicated, to determine the reasons for difficulties in diagnosis. Thirty-six bone cores obtained before bone marrow harvest were involved by breast carcinoma and were confirmed by pancytokeratin immunostains. Thirty of the 36 were ductal carcinomas and six were lobular carcinomas. Fourteen negative bone core biopsy specimens (from patients with breast cancer or lymphoma) were included as controls. These 50 bone cores were reviewed by three surgical pathologists. Lobular carcinoma was correctly identified in only 39% of positive specimens as compared with 88% for ductal carcinoma. After instruction, sensitivity for the detection of lobular carcinoma improved to 61% but at the expense of an unacceptably high rate of false-positive diagnoses (18%). None of the three pathologists was able to achieve both high sensitivity and high specificity in recognizing lobular carcinoma in the bone marrow. Lobular carcinoma was difficult to detect because of tumor cell size similar to hematopoietic cells, infiltration as single cells, presence of bland cytological features, and paucity of tissue reaction to the tumor. Although the number of cases of bone marrow involved by lobular carcinoma is small, these findings suggest that pancytokeratin stains should be performed routinely in the evaluation of bone core biopsy specimens from patients with lobular carcinoma, and probably from patients with ductal carcinoma whose HE-stained bone core biopsy specimens are considered negative for tumor.

Changes in Milk Composition After Six Months of Lactation: The Effects of Duration of Lactation and Gradual Weaning

We followed lactation to one year or more in 8 women measuring both milk yield and composition. During the period from six months to one year four women showed milk yields which declined significantly while four women had yields which remained above 450 g per day. In order to determine whether changes in composition were due to changes in duration of lactation, declining milk yield, or both, the time-dependance of the concentration of several milk components was compared with the time-dependance of the milk yield. The following conclusions were drawn: (i) Within the accuracy of the analysis lactose, potassium and copper concentrations depended neither on milk yield nor duration of lactation, (ii) Lipid increased with duration of lactation and citrate and zinc decreased with duration of lactation. Changes in milk yield did not affect these components, (ii) Sodium, chloride, total protein, and magnesium increased and glucose decreased with decreasing milk yield. Duration of lactation did not affect these components, (iv) Calcium decreased with duration of lactation and increased with declining milk yield. These conclusions are compared with conclusions derived from multiple regression analysis of the same data. It is concluded that both types of analysis used in conjunction with inspection of graphical data are useful in the analysis of longitudinal data.