Anderson Hospital

Based upon observations on 48 cases of retinoblastoma and published reports, the hypothesis is developed that retinoblastoma is a cancer caused by two mutational events. In the dominantly inherited form, one mutation is inherited via the germinal cells and the second occurs in somatic cells. In the nonhereditary form, both mutations occur in somatic cells. The second mutation produces an average of three retinoblastomas per individual inheriting the first mutation. Using Poisson statistics, one can calculate that this number (three) can explain the occasional gene carrier who gets no tumor, those who develop only unilateral tumors, and those who develop bilateral tumors, as well as explaining instances of multiple tumors in one eye. This value for the mean number of tumors occurring in genetic carriers may be used to estimate the mutation rate for each mutation. The germinal and somatic rates for the first, and the somatic rate for the second, mutation, are approximately equal. The germinal mutation may arise in some instances from a delayed mutation.

Carcinoma of the breast and ovary account for one-third of all cancers occurring in women and together are responsible for approximately one-quarter of cancer-related deaths in females. The HER-2/neu proto-oncogene is amplified in 25 to 30 percent of human primary breast cancers and this alteration is associated with disease behavior. In this report, several similarities were found in the biology of HER-2/neu in breast and ovarian cancer, including a similar incidence of amplification, a direct correlation between amplification and over-expression, evidence of tumors in which overexpression occurs without amplification, and the association between gene alteration and clinical outcome. A comprehensive study of the gene and its products (RNA and protein) was simultaneously performed on a large number of both tumor types. This analysis identified several potential shortcomings of the various methods used to evaluate HER-2/neu in these diseases (Southern, Northern, and Western blots, and immunohistochemistry) and provided information regarding considerations that should be addressed when studying a gene or gene product in human tissue. The data presented further support the concept that the HER-2/neu gene may be involved in the pathogenesis of some human cancers.

Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.

BACKGROUND: Children with high-risk neuroblastoma have a poor outcome. In this study, we assessed whether myeloablative therapy in conjunction with transplantation of autologous bone marrow improved event-free survival as compared with chemotherapy alone, and whether subsequent treatment with 13-cis-retinoic acid (isotretinoin) further improves event-free survival. METHODS: All patients were treated with the same initial regimen of chemotherapy, and those without disease progression were then randomly assigned to receive continued treatment with myeloablative chemotherapy, total-body irradiation, and transplantation of autologous bone marrow purged of neuroblastoma cells or to receive three cycles of intensive chemotherapy alone. All patients who completed cytotoxic therapy without disease progression were then randomly assigned to receive no further therapy or treatment with 13-cis-retinoic acid for six months. RESULTS: The mean (+/-SE) event-free survival rate three years after the first randomization was significantly better among the 189 patients who were assigned to undergo transplantation than among the 190 patients assigned to receive continuation chemotherapy (34+/-4 percent vs. 22+/-4 percent, P=0.034). The event-free survival rate three years after the second randomization was significantly better among the 130 patients who were assigned to receive 13-cis-retinoic acid than among the 128 patients assigned to receive no further therapy (46+/-6 percent vs. 29+/-5 percent, P=0.027). CONCLUSIONS: Treatment with myeloablative therapy and autologous bone marrow transplantation improved event-free survival among children with high-risk neuroblastoma. In addition, treatment with 13-cis-retinoic acid was beneficial for patients without progressive disease when it was administered after chemotherapy or transplantation.

Ameboid microglia are isolated from the cerebral tissue of neonatal rat by selective cell adhesion to plastic. Histochemical markers show that the microglial preparations are homogeneous (95 +/- 3%) and represent a 10% yield from starting cultures. Isolated ameboid microglia contain nonspecific esterase activity, the macrophage surface antigens MAC-1 and MAC-3, and acetylated low-density lipoprotein receptors. Ameboid cells have functional properties similar to those of macrophages, including the ability to engulf 5 micron latex beads, to secrete Interleukin-1 (IL-1) and to release superoxide anion. Unlike monocytes and adherent spleen cells, ameboid microglia do not show peroxidase activity by histochemical stain. Unlike resident peritoneal macrophages, ameboid microglia proliferate in vitro. Scanning electron microscopy shows that ameboid cells have short, spinous processes that can be distinguished from the ruffled surfaces of body macrophages. Our observations suggest that ameboid microglia are a distinct class of mononuclear phagocytic cells. Retinoic acid and dimethyl sulfoxide, agents known to accelerate differentiation in vitro, stimulate ameboid cells to develop thin processes several hundred microns in length. These "process-bearing" microglia eventually lose the capacity to engulf latex beads and to proliferate. They also show reductions in nonspecific esterase activity and in the binding of acetylated low-density lipoprotein. We suggest that in vitro ameboid microglia differentiate into nonphagocytic cells similar to ramified microglia found in normal adult brain. The isolation techniques described here provide the opportunity to study the composition and function of different microglial subpopulations during the development of the CNS.

Summary During 1973, 4 new epithelial tumor cell lines were isolated from pleural effusions from breast cancer patients. We describe 3 of these lines: MDA-MB-134, with a mean chromosome number of 43; MDA-MB-175, with a mean chromosome number of 49; and MDA-MB-231, with a mean chromosome number between 65 and 69. We isolated the same cell type from 4 of 10 effusions from MDA-MB-134 and from 6 of 8 effusions from MDA-MB-175. We found that pleural effusions as a source of breast tumor cells to be cultured and studied in vitro have the following advantages: 1) large amounts of material and the possibility of obtaining sequential samples from the same patient; 2) high viability of tumor cells; 3) scarcity or absence of fibroblasts; and 4) the possibility of separating the tumor cells from other “contaminating” cell types by differences in their speed or degree of attachment to the flask. All lines from different patients differed, as seen grossly and microscopically. All lines from sequential pleural effusions from the same patient were apparently alike. No viruses or mycoplasmas were detected in any line.

The records of 2,044 patients with previously untreated squamous cell carcinomas of the head and neck were reviewed in order to define the incidence and topographical distribution of lymph node metastasis on admission. The common regions of metastasis are presented for each of the seven individual head and neck sites selected for study. Knowledge of the preferred areas of spread and those that are almost never involved allows the design of more adequate plans to manage the individual lesions.

The relationship between histopathology and tumor behavior was examined in 71 cases of chondrosarcoma. The tumors were grouped into Grades I, II, and III on the basis of mitotic rate, cellularity, and nuclear size. The five-year survival rates of Grades I, II, and III are 90%, 81%, and 43%, respectively, while the corresponding 10-year survival rates are 83%, 64%, and 29%. None of the Grade I chondrosarcomas metastasized, while metastasis was observed with 10% of the Grade II tumors and 71% of the Grade III tumors. No definite relationship between tumor grade and local recurrence was found. In addition, the significance of the extent of the original operative procedure was evaluated for chondrosarcoma, Grades I and II. Local recurrence which could not be controlled by additional surgery occurred in 46% of the patients treated initially by local excision, but in only 9.5% of those who were treated by amputation or resection of all or part of the involved bone. It is concluded that chondrosarcomas range from locally aggressive non-metastasizing neoplasms to high-grade malignancies with marked metastatic potential, and that these groups may be defined and separated by the use of histopathologic criteria. The rate of local recurrence, however, is primarily dependent on the adequacy of surgical therapy rather than histologic grade. Cancer 40:818–831, 1977.

Heterochromatic regions in chromosomes of man, mainly at the centromeric areas, can be demonstrated with consistency using a special staining procedure. This procedure includes treatments of cytological preparations with HCl, RNase, NaOH (denaturing DNA), and renaturing DNA in a saline citrate solution at 65°C. The heterochromatic regions can be observed after staining with Giemsa. These regions are believed to be composed of constitutive heterochromatin. Facultative heterochromatin regions, e.g., the repressed X in female cells, do not respond to this series of treatments.

Although bladder cancers are very common, little is known about their molecular pathogenesis. In this study, invasive bladder cancers were evaluated for the presence of gene mutations in the p53 suppressor gene. Of 18 tumors evaluated, 11 (61 percent) were found to have genetic alterations of p53. The alterations included ten point mutations resulting in single amino acid substitutions, and one 24-base pair deletion. In all but one case, the mutations were associated with chromosome 17p allelic deletions, leaving the cells with only mutant forms of the p53 gene products. Through the use of the polymerase chain reaction and oligomer-specific hybridization, p53 mutations were identified in 1 to 7 percent of the cells within the urine sediment of each of three patients tested. The p53 mutations are the first genetic alterations demonstrated to occur in a high proportion of primary invasive bladder cancers. Detection of such mutations ex vivo has clinical implications for monitoring individuals whose tumor cells are shed extracorporeally.

Pseudomonas aeruginosa has emerged as an important pathogen during the past two decades. It causes between 10% and 20% of infections in most hospitals. Pseudomonas infection is especially prevalent among patients with burn wounds, cystic fibrosis, acute leukemia, organ transplants, and intravenous-drug addiction. P. aeruginosa is a common nosocomial contaminant, and epidemics have been traced to many items in the hospital environment. Patients who are hospitalized for extended periods are frequently colonized by this organism and are at increased risk of developing infection. The most serious infections include malignant external otitis, endophthalmitis, endocarditis, meningitis, pneumonia, and septicemia. The likelihood of recovery from pseudomonas infection is related to the severity of the patient's underlying disease process. The introduction of the antipseudomonal aminoglycosides and penicillins has improved substantially the prognosis of these infections. Ticarcillin and carbenicillin have been especially beneficial in neutropenic patients; however, prompt institution of therapy is mandatory for optimal benefit. Many new drugs with antipseudomonal activity, including penicillins, cephalosporins, and other beta-lactams, have been introduced in recent years and offer the potential for new approaches to therapy for these infections.

The functional and phenotypic characteristics of cells in human peripheral blood that mediate "natural killer" (NK) cytolysis have been examined with the use of multiparameter flow cytometry analysis and cell sorting. Essentially, all lymphocytes expressing NK and ADCC activity reacted with the anti-Leu-11a monoclonal antibody. The Leu-11a antigen was expressed on cytotoxic large granular lymphocytes (LGL), neutrophils, and basophils, but was not present on B cells, mitogen-activated T lymphoblasts, or Leu-1+ and Leu 4+ resting T cells. Anti-Leu-11a antibody selectively inhibited the binding of FITC heat-aggregated IgG complexes to granulocytes and LGL, and it may recognize a type of Fc receptor on these cells. Two-color FACS cell sorting indicated the existence of four lymphocyte subsets defined by the expression of Leu-11a and Leu-7 antigens. The Leu-11a+, -7- cells were highly active in 4-hr NK assays with the use of 51Cr-labeled K562 as the target. In contrast, the Leu-11a-, -7+ cells demonstrated weak activity and the Leu-11a-, -7- cells demonstrated no activity. The function of the Leu 11a+, -7+ cells varied considerably among several individuals examined. Multiparameter analysis with the use of two-color flow cytometry was used to determine the relationship between the expression of these NK-associated antigens and T and B cell-associated markers. These data indicate that considerable heterogeneity exists within human peripheral lymphocytes with regard to cell phenotype and function, but that several defined cellular subsets can be clearly revealed by using multiparameter FACS analysis and sorting.

We treated seven patients who had progressive hairy-cell leukemia with daily doses of 3 million units of partially pure alpha (leukocyte) interferon by the intramuscular route. Three patients had a complete remission, and four had a partial remission, according to strict criteria for a response. After treatment, bone-marrow aspirates showed an absence of leukemia cells in three patients and 5 per cent or fewer in three others. Normalization of subnormal peripheral-blood values occurred in six of six patients with anemia, in seven of seven with granulocytopenia, and in four of four with thrombocytopenia. Remissions have been maintained for over 6 to over 10 months. Alpha interferon appears to be highly effective in patients with hairy-cell leukemia.

Combination chemotherapy with CHOP (cyclophosphamide, Adriamycin, vincristine, and prednisone) and HOP (Adrimycin, vincristine, and prednisone, was used as treatment for patients with pathologically staged, advanced non-Hodgkin's lymphoma. Among 204 evaluable patients treated on CHOP there were 71% complete remissions with 92% overall responses. Among the 216 evaluable patients on HOP there were 61% complete remissions and 88% responses. Complete remission rates among patients with histiocytic lymphoma were comparable to those of patients with lymphocytic disease. Patients with nodular lymphoma had higher rates of complete remission than their counterparts with diffuse lymphoma. This was noted with both CHOP (78% vs. 67%) and HOP (67% vs. 60%) induction therapy. Rapid responses were common, as more than 14% of complete remissions and 66% of overall responses were achieved with the first course of treatment. Patients in complete remission have been maintained with either cyclophosphamide, vincristine, and prednisone (COP) or arabinosyl cytosine, vincristine, and prednisone (OAP). After 1 year, 86% of patients on COP and 80% on OAP are projected to be free of disease.

SummaryA microcolony technique for assaying the survival of small proportions of the clonogenic cells of the mucosa of the small intestine of mice is described. The radiosensitivity of intestinal crypt cells measured by the technique is the same as that measured by a macrocolony method. However, the microcolony technique has the special advantage of being useful for assaying cell survival after whole-body radiation.

Colorectal (CR) tumors are usually curable if detected before metastasis. Because genetic alterations are associated with the development of these tumors, mutant genes may be found in the stool of individuals with CR neoplasms. The stools of nine patients whose tumors contained mutations of K-ras were analyzed. In eight of the nine cases, the ras mutations were detectable in DNA purified from the stool. These patients included those with benign and malignant neoplasms from proximal and distal colonic epithelium. Thus, colorectal tumors can be detected by a noninvasive method based on the molecular pathogenesis of the disease.

We treated 17 patients who had Philadelphia-chromosome-positive chronic myelogenous leukemia (4 of whom had not received therapy and 13 of whom had been treated with hydroxyurea or busulfan for less than six months) with recombinant human interferon alpha A (Roferon-A). The interferon was given as 5 X 10(6) units per square meter of body-surface area per day intramuscularly during induction therapy. Fourteen patients responded to the treatment, of whom 13 had a hematologic remission and 1 had a partial hematologic remission. The median number of white cells in those patients declined from 60.9 X 10(3) to 3.4 X 10(3) per microliter, and the median number of platelets decreased from 476 X 10(3) to 231 X 10(3) per microliter. Among the five responding patients who had splenomegaly before treatment, the spleen size returned to normal in four and decreased by 75 percent in one, although it remained enlarged. Bone marrow cellularity declined from a median of 92.5 percent to a median of 57.5 percent. In six of the patients with hematologic remission, complete suppression of Philadelphia cells was observed on at least one examination. Of the 14 patients who responded, 11 have received the interferon therapy for 9 to 15 months. One patient relapsed during the treatment, and the treatment has been temporarily interrupted in two patients because of toxicity. These data are preliminary and will need further confirmation, but they suggest that recombinant human interferon alpha A is effective in inducing hematologic remission in most patients with benign-phase chronic myelogenous leukemia and in suppressing the Philadelphia chromosome in some of these patients.

Twenty-nine patients with advanced refractory multiple myeloma were treated with intermittent high-dose dexamethasone in combination with four-day infusions of vincristine and doxorubicin. Rapid and marked tumor-mass reduction (greater than 75 per cent) was noted in 14 of 20 patients whose disease was resistant to alkylating agents and in 3 of 9 patients with additional resistance to doxorubicin--a result far superior to those in previous trials with similar patients. In responsive patients, remissions were of excellent quality, and survival was significantly longer than in unresponsive patients.

Seventy-nine patients with adenoid cystic carcinoma arising in salivary glands were studied to determine whether a correlation existed between the morphologic features of the tumor and the prognosis. Three histologic grades were established: Grade I, tumors with tubular and cribriform areas but without solid components; Grade II, cribriform tumors that were either pure or mixed with less than 30% of solid areas; and Grade III, tumors with a predominantly solid pattern. Cumulative survival rates at 15 years were 39%, 26%, and 5%, for Grades I, II, and III, respectively. Grade III tumors were larger, recurred frequently, and killed the patients within 4 years. Grade I lesions were smaller, were amenable to complete surgical excision, and had a protracted clinical course. Grade II tumors lay between the other two forms both clinically and pathologically. Other important prognostic features of the adenoid cystic carcinoma were its primary site, its presence or absence at surgical margins, and the anatomic structures it involved.

The myelodysplastic syndromes are characterized by ineffective hematopoiesis and refractory cytopenias. In an attempt to improve hematopoiesis, we administered recombinant human granulocyte--macrophage colony-stimulating factor (GM-CSF) to eight patients with myelodysplastic syndrome, as part of a Phase I trial. The GM-CSF was given by continuous intravenous infusion daily for two weeks and then again after a two-week rest period. Over the entire dose range tested (30 to 500 micrograms per square meter of body-surface area), treatment was associated with marked increases in peripheral-blood leukocytes (5- to 70-fold), including granulocytes (5- to 373-fold), in all eight patients. The absolute number of monocytes, eosinophils, and lymphocytes increased in all patients. Three of eight patients also had 2- to 10-fold increases in platelet counts and improvement in erythropoiesis, with the result that two of three patients who had required red-cell and platelet transfusions no longer needed them (at 20 to 27 weeks of follow-up). Treatment was also associated with increased marrow cellularity and a decreased percentage of blasts in the bone marrow of patients with excess blasts, resulting in an increase in the ratio of differentiated myeloid cells to immature myeloid cells. We observed relatively few side effects, but bone pain was dose-limiting when it was associated with high white-cell counts. Our results showed that GM-CSF is a potent stimulator of hematopoiesis in vivo and may produce hematologic improvement in the short term (8 to 32 weeks of observation) in patients with myelodysplastic syndrome. More experience, with longer follow-up periods, will be necessary to assess the long-term safety and efficacy of this new treatment.