Dennis A. Johnston

A comparative analysis of sextant and an extended 11-core multisite directed biopsy strategy.

PURPOSE The 3 tumor locations unsampled by conventional sextant biopsies that have been identified on composite 3-dimensional reconstruction of 180 radical prostatectomy specimens are the anterior transition zone, midline peripheral zone and inferior portions of the anterior horn in the peripheral zone. We evaluated an 11-core multisite directed biopsy scheme incorporating these alternate areas and conventional sextant biopsies in 362 patients from 2 institutions. MATERIALS AND METHODS Patients without a prior diagnosis of cancer underwent ultrasound guided 11-core biopsies which included conventional sextant and 3 alternate sites. All specimens were separated for specific location identification. Biopsy was performed in 183 patients at MD Anderson Cancer Center (group 1) and in 179 at Toronto General Hospital (group 2). All group 2 and 54% of group 1 patients (98 of 183) had a prior biopsy negative for cancer. RESULTS Median prostate specific antigen was higher in group 2 than in group 1 patients (11.5 versus 9.5 ng./ml., p = 0.016). Overall a 33% increase (36 of 110 patients) in cancer detection was observed when biopsy technique included the alternate areas (p = 0.0021). The anterior horn was the most frequently positive biopsy site followed by the transition zone and midline sites. The 11-core technique had significantly better cancer detection rates when digital rectal examination and transrectal ultrasound were normal, and in men with serum prostate specific antigen between 4.1 and 10 ng./ml. CONCLUSIONS Biopsies of the alternate sites suggested by our simulation studies are feasible and reproducible. This new strategy significantly enhanced (p = 0.0075) prostate cancer detection compared to conventional sextant biopsies in men undergoing a repeat procedure.

Cellular DNA content as a marker of neoplasia in man

Abstract Cellular DNA content was determined by means of flow cytometry with the use of DNA specific fluorochromes (ethidium bromide and mithramycin) in 516 human tissue samples from 440 subjects. Compared to human granulocytes as diploid reference standard, there was a 91 percent incidence of DNA content abnormality difference in DNA content of tumor G10 cells indicating aneuploidy in 118 patients with neoplastic disease (including nine patients who lacked histopathologic evidence of malignancy at the time of study). Ninety-four percent of aneuploid tumors were hyperdiploid. Except for six solid tumors with biclonal abnormalities in DNA content, the remainder of neoplasms were characterized by uniform DNA content with little dispersion (small coefficient of variation of tumor G10 populations). For the entire group of patients with malignant disease, three modal values of DNA content were recognized at low-degree hyperdiploidy, near triploidy and tetraploidy. Except for the prevalence of high-degree hyperdiploidy in melanomas and low-degree hyper- and hypodiploid abnormalities in malignant lymphomas, significant disease-specific patterns of abnormal DNA content were not apparent. The magnitude of ploidy abnormality was further influenced by patient age and proliferative activity of the tumor. Female patients displayed a preponderance of small-degree hyperdiploid and tetraploid tumors, whereas near-triploid abnormalities prevailed among male patients, who also harbored five of six biclonal tumors. Tumor cell ploidy did not vary among different sites of disease and upon sequential long-term follow-up examination. All 121 benign tumors had a diploid DNA content. Among the group of 209 patients with normal histology or reactive changes were seven patients with a previously established diagnosis of cancer with ploidy abnormality. This discrepancy indicates that monodispersal of the entire tissue aliquot for DNA flow cytometry is superior to histologic examination of focal neoplasia. There were two patients, one with recurrent benign pleural effusions and one with reactive lymphadenopathy, with ploidy abnormality by DNA content in whom malignant lymphoma developed. We conclude that flow cytometry of cellular DNA content is a rapid, objective, quantitative and sensitive method to determine a highly specific and stable tumor cell marker.

The incidence of occult nipple-areola complex involvement in breast cancer patients receiving a skin-sparing mastectomy

Background: Surgical treatment of breast cancer traditionally has included resection of the nipple-areola complex (NAC), in the belief that this area had a significant probability of containing occult tumors. The purpose of this study was to investigate the true incidence of NAC involvement in patients who underwent a skin-sparing mastectomy (SSM) and to determine associated risk factors.Methods: A retrospective chart review was conducted of 326 patients who had a SSM at our institution from 1990 to 1993. NAC involvement was reviewed in 286 mastectomy specimens. The charts were analyzed for tumor size, site, histology, grade, nodal status, recurrence, survival, and NAC involvement.Results: Occult tumor involvement in the NAC was found in 5.6% of mastectomy specimens (16 patients). Four patients would have had NAC involvement identified on frozen section if they had been undergoing a skin-sparing mastectomy with preservation of the NAC. There were no significant differences between NAC-positive (NAC+) and NAC-negative (NAC-) patients in median tumor size, nuclear grade, histologic subtype of the primary tumor, or receptor status. There were significant differences in location of the primary tumor (subareolar or multicentric vs. peripheral) and positive axillary lymph node status. NAC involvement was not a marker for increased recurrence or decreased survival.Conclusions: Occult NAC involvement occurred in only a small percentage of patients undergoing skin-sparing mastectomies. NAC preservation would be appropriate in axillary node-negative patients with small, solitary tumors located on the periphery of the breast.

Five‐year survival after pulmonary metastasectomy for adult soft tissue sarcoma

Determinants of 5‐year survival were evaluated after complete resection of pulmonary metastases from adult soft‐tissue sarcomas. Fifty‐eight patients had complete resection (median survival 25 months, P = 0.0002), with a 25.8% absolute 5‐year survival (15 of 58 patients); six patients had unresectable disease (median survival 6 months) and were excluded from additional analysis. Eleven patients remain disease free, with a median follow‐up of 76 months. Significant independent prognostic indicators associated with improved survival (P < 0.05) included metastasis doubling time of 40 days or greater (median survival 37 months versus 15 months if less than 40 days); unilateral disease on preoperative radiography (33 months versus 15 months if bilateral disease); three or fewer nodules on preoperative computed tomography (40 months versus 14 months if 4 or more nodules); two nodules or fewer resected (40 months versus 17 months if 3 or more nodules resected), and tumor histology (33 months for malignant fibrous histiocytoma versus 17 months for all others). Multivariate analysis identified the number of nodules detected by computed tomography preoperatively as having significant prognostic value.

Effect of Radiation-Induced Xerostomia on Human Oral Microflora

A longitudinal study was performed to assess the effects of radiation-induced xerostomia on the human oral microflora. Pronounced microbial population shifts were found in each of five intraoral sites tested. Cariogenic microorganisms gained prominence at the expense of noncariogenic microorganisms in concert with the saliva shutdown. These changes occurred before the onset of clinical caries irrespective of whether or not a topical fluoride gel was used as a caries preventive.

OPTIMIZATION OF PROSTATE BIOPSY STRATEGY USING COMPUTER BASED ANALYSIS

PURPOSE We evaluated and optimized the detection of cancer by prostate biopsies. We developed a stochastic computer simulation model of ultrasound guided biopsies using mathematically reconstructed radical prostatectomy specimens. Use of this technique allows rapid evaluation of a variety of factors for their effect on prostate biopsy results. We used this model to analyze the effectiveness of sextant biopsies, which have been widely adopted in clinical practice. We also analyzed other biopsy schemes. MATERIALS AND METHODS A total of 607 tumor foci from 180 serially sectioned whole mount radical prostatectomy specimens was mapped and digitized. The cancers had been clinically diagnosed by a variety of biopsy strategies. Simulated parasagittal sextant biopsies were performed for each case. Forty simulation runs (each consisting of a set of 6 biopsies) were performed for each prostate, with realistic random variations in sextant biopsy localization programmed in each run. Cancer detection by biopsy was considered reliable if 90% of the simulation runs for each prostate were positive for cancer. A summary algorithm was used to map the tumor foci. RESULTS Simulation of sextant biopsies demonstrated reliably detected cancer in only 107 of 147 patients (73%) in whom total tumor volume was greater than 0.5 cc. There was little correlation between total length of cancer in biopsy cores and tumor volume. Change of biopsy angle from 30 to 45 degrees did not result in significantly increased detection rates. Similarly, placing all biopsies more laterally did not increase overall detection rates. When we mapped tumor foci from the 40 cases in which sextant biopsies did not reliably detect tumor, we found that the foci were distributed in areas not biopsied by the sextant method, that is the transition zone, midline peripheral zone and inferior portion of the anterior horn of the peripheral zone. A 10-core biopsy scheme incorporating these areas as well as the posterolateral prostate reliably detected cancer in 141 of 147 patients (96%) with total tumor volumes greater than 0.5 cc. CONCLUSIONS Prostate cancer of significant volume can be present in areas not sampled by standard sextant biopsies. Biopsies of the transition zone, midline peripheral zone and inferior portion of the anterior horn of the peripheral zone should be considered for re-biopsy strategy after negative sextant biopsies. Sampling of these additional areas also can be incorporated in an initial biopsy scheme to increase overall initial rates of detection of prostate cancer.

Stimulation of Megakaryocyte and Platelet Production by a Single Dose of Recombinant Human Thrombopoietin in Patients with Cancer

Thrombocytopenia is an important clinical problem in the management of patients in hematology and oncology practices. In the United States, the use of platelet transfusions to manage severe thrombocytopenia has steadily increased: Approximately 4 million units were transfused in 1982, and more than 8 million units were transfused in 1992 [1, 2]. This marked increase in the need for platelets has paralleled advances in organ transplantation, bone marrow transplantation, cardiac surgery, and the use of dose-intensive therapy in the treatment of chemosensitive malignant conditions. Although platelet transfusions may decrease the risk for fatal bleeding complications, repeated transfusions increase the risk for transmission of bacterial and viral pathogens, transfusion reactions, and transfusion-associated graft-versus-host disease. These transfusions also contribute to increasing health care costs and inconvenience to patients [3]. Thus, an agent that can increase platelet production and prevent or attenuate thrombocytopenia would be an important advance. Thrombopoietin, the ligand for the c-Mpl receptor (found on platelets and megakaryocyte progenitors), was recently cloned by several investigators and was shown to be a primary regulator of platelet production in vivo [4-8]. Thrombopoietin promotes both the proliferation of megakaryocyte progenitors and their maturation into platelet-producing megakaryocytes. In preclinical studies done in normal mice and nonhuman primates, thrombopoietin increased platelet counts to a level higher than those previously achieved with other thrombopoietic cytokines [9, 10]. Moreover, in a murine model for myelosuppression, recombinant thrombopoietin given as a single dose decreased the nadir and accelerated platelet recovery in mice that had been rendered pancytopenic by sublethal radiation and chemotherapy [11]. In these studies, more prolonged treatment (for as long as 8 days) provided no additional benefit and was associated with marked thrombocytosis during the recovery phase. On the basis of these observations, we initiated a phase I and II clinical and laboratory investigation of recombinant human thrombopoietin in patients with cancer who were at high risk for severe chemotherapy-induced thrombocytopenia. This trial was divided into two parts: Part I studied thrombopoietin given before chemotherapy, and part II studied thrombopoietin given after chemotherapy. The objective of part I, the results of which are reported here, was to assess the hematopoietic effects, pharmacodynamics, and clinical tolerance of this novel agent in patients who had normal hematopoietic function before chemotherapy. Methods Patients Patients with sarcoma who had never had chemotherapy, were suitable candidates for subsequent chemotherapy, and did not have rapidly progressive disease were eligible for this trial. Patients were required to have a Karnofsky performance status score of 80 or more, adequate bone marrow (absolute neutrophil count 1.5 109/L; platelet count 150 109/L and 450 109/L), adequate renal function (serum creatinine level 120 mol/L), and adequate hepatic function (alanine aminotransferase level < 3 times normal; bilirubin level < 1.5 times normal). Patients with a history of thromboembolic or bleeding disorders, significant cardiac disease, or previous pelvic radiation were excluded. Written informed consent was obtained from all patients before study entry in accordance with institutional guidelines. Design During the phase I dose-ranging portion of this clinical cohort study, thrombopoietin was administered as a single intravenous dose 3 weeks before chemotherapy. At study entry, three patients were assigned to each of four dose levels (0.3, 0.6, 1.2, and 2.4 g/kg of body weight). Patients who had no dose-limiting toxicity and did not develop neutralizing antibodies to thrombopoietin were eligible to receive thrombopoietin at the same doses after chemotherapy. Recombinant Human Thrombopoietin The thrombopoietin used in this study was provided by Genentech, Inc. (South San Francisco, California). Thrombopoietin is a full-length glycosylated molecule produced in a genetically modified mammalian cell line and purified by standard techniques. It was mixed with preservative-free normal saline as a diluent for injections. Clinical and Laboratory Monitoring Before and during the clinical trial, patients were monitored by complete histories; physical examinations; and laboratory tests, including a complete blood cell count with differential counts, serum chemistry, coagulation profile, urinalysis, assessment of thrombopoietin antibody formation, chest radiography, and electrocardiography. Blood counts were obtained daily for the first 5 days and then at least three times per week. Peripheral smears were examined serially for platelet morphology. Platelet counts and the average size of platelets (mean platelet volume) were derived from 64-channel platelet histograms. Bone marrow aspiration and biopsy were done before and 1 week after thrombopoietin treatment. The bone marrow specimens were initially fixed in 10% neutral formalin, embedded in paraffin, cut into sections 5 m thick, and stained with hematoxylin-eosin for morphologic analysis and with Masson trichrome for analysis of collagen fiber content. Fresh, air-dried smears of bone marrow were stained with Wright-Giemsa. Bone marrow samples were examined for overall cellularity and morphology in a blinded manner. Megakaryocyte counts were measured by choosing 10 high-power (40x) fields in areas without artifactual zones or trabecula. The relative size of the megakaryocyte was assessed by examining bone marrow aspirate smears using the Magiscan Image Analysis System (Compix, Cranberry, Pennsylvania). Bone marrow aspirates were also assayed for hematopoietic progenitor cell number and cycle status, for content of CD34+ and CD41+ cell subsets (by flow cytometry), and for megakaryocyte ploidy (by flow cytometry). Blood samples were assayed for hematopoietic progenitor cell number and for platelet function. Pharmacokinetics Profiles Serum samples were collected before and at 2, 5, 10, 60, and 90 minutes and 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 hours after thrombopoietin administration. Concentration-time profile at each dose level was evaluated by using standard pharmacokinetics methods. Serum thrombopoietin levels were quantitated by enzyme-linked immunosorbent assay for thrombopoietin [12]. Hematopoietic Progenitor Cell Assays Assays for colony-forming unit-granulocyte-macrophage (CFU-GM); burst-forming unit-erythroid (BFU-E); and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) using low-density bone marrow [13] and peripheral blood cells [14] were done with methyl cellulose assays. The percentage of bone marrow CFU-GM and BFU-E in DNA synthesis (S-phase of cell cycle) was measured by a high-specific-activity tritiated thymidine suicide technique [15]. Assays for colony-forming unit-megakaryocyte (CFU-MK) and burst-forming unit-megakaryocyte (BFU-MK) were done using a fibrin clot assay [16]. Ploidy Analysis Megakaryocyte-enriched cell fractions were prepared from bone marrow cell suspensions by using a Percoll gradient technique. Ploidy was determined by flow cytometric measurement of the relative DNA content after staining with propidium iodide in hypotonic citrate solution [17]. Cells were also stained with anti-CD41b (8D9)-FITC (SyStemix, Palo Alto, California) to allow gating on CD41b+ megakaryocytes. At least 3000 CD41+ events were collected for each sample. The percentage of CD41+ cells in ploidy class was determined from the fluorescence-activated cell-sorting dot plots. Platelet Function Platelet aggregation was measured in response to three agonists: adenosine diphosphate (final concentration, 20 g/mL), collagen (6 g/mL), and thrombin (5 g/mL). Standard methods were used [18]. The concentrations of agonists were chosen on the basis of previous in vitro studies done on blood from normal controls. The instruments used for the assays were the Bio/Data Pap 4A (Horsham, Pennsylvania) and the Crono-log 560CA (Havertown, Pennsylvania). Immunophenotypic Analysis Immunophenotypic analysis was done using anti-CD34 (Becton Dickinson, San Jose, California) and anti-CD41 monoclonal antibodies (Immunotech, Westbrook, Maine) by a standard dual-color flow cytometry technique [19]. Statistical Analysis Continuous variables were compared by using the Wilcoxon matched-pairs signed-rank test. Trends for possible dose-response relation were evaluated using the Spearman rank correlation coefficients (rS) between dose and outcome. Industry Role Thrombopoietin and partial funding for the study were provided by Genentech, Inc. The study was a collaborative effort between the principal investigator and the industrial sponsor. Data collection, data analysis, the writing of the manuscript, and the decision to publish the manuscript were under the control of the principal investigator. The manuscript was reviewed by the industrial sponsor before submission. Results Twelve chemotherapy-naive patients (7 men and 5 women) with sarcoma of diverse histologic sub-types were entered into the dose-ranging portion of this phase I trial, which studied thrombopoietin before chemotherapy. All patients were considered evaluable for clinical tolerance and response to thrombopoietin. The median age of these patients was 42 years (range, 16 to 63 years), and the median Karnofsky performance status score was 90 (range, 80 to 100). Four patients had previously received radiation therapy, and eight had previously had surgery. Peripheral Blood Counts Treatment with a single dose of thrombopoietin was associated with increases (1.3-fold to 3.6-fold) in platelet counts (baseline mean, 264 109/L; maximal mean, 592 109/L) (P = 0.002). The increase in platelet count was seen at all dose levels (Figure 1) in all patients. The peak response i

Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity.

Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8+ T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-γ or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8+ T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.

Measurement of Residual Leukemia during Remission in Childhood Acute Lymphoblastic Leukemia

BACKGROUND Complete remission of B-precursor acute lymphoblastic leukemia (ALL) has traditionally been defined as the near absence of lymphoblasts in a light-microscopical examination of stained bone marrow smears, but a patient in remission may still harbor up to 10(10) leukemia cells. We investigated whether there is a relation between the outcome of treatment and submicroscopic evidence of residual disease. METHODS We conducted a prospective study of patients during a first clinical remission using a quantitative polymerase-chain-reaction (PCR) assay capable of detecting 1 viable leukemia cell among 200,000 normal marrow mononuclear cells and a clonogenic blast-colony assay. Bone marrow specimens from 24 children were sequentially evaluated during a five-year period, and the results were compared with the clinical outcome. RESULTS Seven patients relapsed and 17 remained in remission 2 to 35 months after the completion of treatment. The levels of residual leukemia-cell DNA in the two groups were significantly different (P<0.001; 95 percent confidence interval for the difference in the mean log-transformed ratio of leukemia-cell DNA to normal bone marrow-cell DNA, 0.38 to 1.28). Autoregression analyses identified trends for individual patients that were associated with relapse. Despite continued remission in 17 patients, evidence of residual leukemia was detected by PCR in 15 and by both PCR and blast-colony assays in 7. CONCLUSIONS Molecular signs of residual leukemia can persist up to 35 months after the cessation of chemotherapy in children with ALL in remission. This suggests that eradication of all leukemia cells may not be a prerequisite for cure.

Genetic epidemiology study of idiopathic talipes equinovarus.

Previous genetic studies of idiopathic talipes equinovarus (ITEV) suggest an environmental and genetic component to the etiology of ITEV. The present study was undertaken to assess the role of causal factors in the development of ITEV. A total of 285 propositi were ascertained, with detailed family history information available in 173 cases and medical records on the remaining 112 propositi. Information was collected on specific prenatal, parental, and demographic factors. No racial heterogeneity was noted among any of the factors. The overall ratio of affected males to females was 2.5:1. The incidence of twinning among all propositi was significantly increased (P=0.006) above the expected population frequency. A family history of ITEV was noted in 24.4% of all propositi studied. These findings, in addition to the detailed analysis of 53 pedigrees with ITEV history, suggest the potential role of a gene or genes operating in high-risk families to produce this foot deformity.