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Rationale: Despite direct immediate intervention and therapy, ST-segment–elevation myocardial infarction (STEMI) victims remain at risk for infarct expansion, heart failure, reinfarction, repeat revascularization, and death. Objective: To evaluate the safety and bioactivity of autologous CD34+ cell (CLBS10) intracoronary infusion in patients with left ventricular dysfunction post STEMI. Methods and Results: Patients who underwent successful stenting for STEMI and had left ventricular dysfunction (ejection fraction≤48%) ≥4 days poststent were eligible for enrollment. Subjects (N=161) underwent mini bone marrow harvest and were randomized 1:1 to receive (1) autologous CD34+ cells (minimum 10 mol/L±20% cells; N=78) or (2) diluent alone (N=83), via intracoronary infusion. The primary safety end point was adverse events, serious adverse events, and major adverse cardiac event. The primary efficacy end point was change in resting myocardial perfusion over 6 months. No differences in myocardial perfusion or adverse events were observed between the control and treatment groups, although increased perfusion was observed within each group from baseline to 6 months ( P P =0.05). At 1 year, 3.6% (N=3) and 0% deaths were observed in the control and treatment group, respectively. Conclusions: This PreSERVE-AMI (Phase 2, randomized, double-blind, placebo-controlled trial) represents the largest study of cell-based therapy for STEMI completed in the United States and provides evidence supporting safety and potential efficacy in patients with left ventricular dysfunction post STEMI who are at risk for death and major morbidity. Clinical Trial Registration: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01495364.

Background: Prior studies suggest an association between naturally high circulating levels of CD34+ cells (CD34) and better outcomes following ischemic events. Pts in the treatment arm of the PreSERVE-AMI study were infused with all CD34 yielded from their bone marrow harvest, resulting in pts with naturally higher CD34 cell counts being infused with a higher cell dose. To determine whether selection bias may have influenced results, we performed an analysis of the relationship between BM CD34 content and outcomes in PreSERVE-AMI. Methods: PreSERVE-AMI is a Phase 2, randomized, double-blind, placebo-controlled trial of autologous, BM derived CD34 (CLBS10) in pts with LV dysfunction (LVEF ≤48% by MRI) ≥4 days post-STEMI. All pts underwent mini BM harvest and were randomized 1:1 to intracoronary infusion of CD34 (minimum dose 10M±20% cells) or placebo (cell diluent). Primary safety endpoints included AEs, SAEs and MACE (CV mortality, HF hospitalization, reinfarction, revascularization). Secondary endpoints included LVEF change from baseline. Regression analyses were performed to assess the influence of BM CD34 content on outcomes. Results: 161 patients were randomized and received intracoronary infusion (Jan 2013-Dec 2014). Mean age was 57±10 and 82% were men. Mean follow-up time was 18 mo, and safety data are accruing. BM CD34 content was not associated with differences in baseline characteristics with the exception of diabetes prevalence being higher in those with higher BM CD34 count (p=0.03). In controls, regression analyses showed no association of BM CD34 content with rates of MACE (p=0.2), SAEs (p=0.4), or change in LVEF (p=0.9). However, in CD34 treated pts, there was a dose-dependent improvement in LV function (p=0.045) and dose-dependent trends in MACE and SAE reduction compared to controls. As previously reported SPECT perfusion improved to a similar degree in control and treated pts. Conclusions: These data suggest that intracoronary infusion of CD34 cells, rather than BM CD34 cell content, is responsible for the benefit observed in the CD34 treatment group. The finding of a dose-response relationship is an important advance for defining the CD34 as an active agent for tissue repair. Future studies will further define CD34 dosimetry.

The incidence and clinical relevance of tumor cells contaminating the stem cell products of patients with advanced breast cancer treated with high-dose chemotherapy is uncertain because prior studies used small sample sizes and lacked standardization of the immunocytochemistry (ICC) detection method used. We evaluated blood stem cell and bone marrow samples obtained from 535 women with metastatic breast cancer who received high-dose chemotherapy and unmanipulated mobilized blood stem cell support. Of the patients tested, 20.6% and 26.3% had blood stem cell and bone marrow contamination, respectively. Blood stem cell contamination was significantly more frequent in patients with marrow involvement than in patients without marrow involvement (35% versus 18.4%, respectively; P = .009). In fact, according to multivariate analysis results, marrow involvement was the only significant predictor for blood stem cell product contamination. Patients without marrow involvement who had fewer apheresis procedures were also observed to have a significantly lower incidence rate of blood stem cell contamination than patients who had more procedures (P < or = .008), and patients who received combined chemotherapy and cytokine mobilization therapy had less contamination than patients who received cytokine alone (P = .0001). Combined mobilization therapy appears to be associated with a lower incidence of contamination as a result of fewer apheresis procedures rather than through an antitumor effect of chemotherapy (P < or = .001). Patients with ICC-negative blood stem cell products had significantly longer progression-free survival (PFS) and overall survival (OS) than did patients with ICC-positive blood stem cell products (median PFS, 401 versus 291 days, respectively, P = .007; median OS, 1060 versus 697 days, P = .009) . However, multivariate analysis did not reveal any significant independent predictors of survival outcomes. Thus, further study is needed to determine if contaminating tumor cells in the stem cell products of breast cancer patients ever directly impact survival outcomes or are only indicative of residual in vivo disease in high-dose chemotherapy recipients.Biol Blood Marrow Transplant 2002;8(10):536-43.

Contaminating tumor cells in peripheral blood stem cell (PBSC) grafts infused for hematopoietic rescue after high-dose chemotherapy could potentially contribute to relapse in BrCa patients. To date the prevalence of PBSC contamination in BrCa patients, as determined by standard immuno-cytochemistry (ICC) assays, has generally been found to be relatively low. However, assay sensitivity may have an important impact on the ability to detect contamination. In this investigation a novel and highly sensitive BrCa cell assay using immunomagnetic enrichment with a panel of antiBrCa monoclonal antibodies (MAbs) and detection by ICC has been characterized. The immunomagnetic enrichment with ICC detection (IE-ICC) assay was directly compared with standard ICC in ability to detect BrCa contamination of PBSC specimens from patients with high-risk stage II/III and metastatic disease. The sensitivity of the IE-ICC assay was approximately 50-fold greater than that of standard ICC. As determined by standard ICC assay, BrCa cells were present in 1/14 patients (7%) and 2/26 (8%) specimens. In contrast, with IE-ICC assay the proportions of positive findings in patients (12/14, 86%) and specimens (19/26, 73%) were significantly higher (p = 0.001 and p < 0.0005, respectively). These preliminary findings suggest that the prevalence of PBSC contamination may be substantially higher than previously appreciated. Consequently, measures to reduce tumor contamination in the graft may have the potential to improve patient outcomes. Higher sensitivity assays such as the IE-ICC assay may play an important role in assessing the risks associated with tumor contamination and the effectiveness of tumor-purging approaches such as positive selection of CD34+ cells and in monitoring patient response to therapy.

With the advent of new therapeutic modalities, the treatment options for oncologists can vary greatly depending upon the aggressiveness of the patient's cancer. Patients may receive no therapy, adjuvant therapy, aggressive adjuvant therapy (taxane based), monoclonal antibody therapy (e.g. Herceptin) or bone marrow transplantation. It is now mandatory to determine accurate prognostic patient profiles at diagnosis and during therapy to determine who would benefit most from a particular therapeutic regimen or to determine who should be shifted into more aggressive therapy. We now have ultra-sensitive methods of tumor cell detection that can determine the presence of minimal residual cancer (MRC) in marrow, stem cell product (SCP) and lymph node to help create these prognostic profiles. The author has conducted a critical review of the literature regarding the type of testing used to detect MRC, the incidence of MRC in marrow, SCP, and lymph node, and the clinical significance of MRC at diagnosis and during therapy. To date it is now clear that immunohistochemistry is a very useful diagnostic tool with adequate sensitivity to detect MRC. The presence of MRC at diagnosis in marrow and/or lymph node is associated with a poor prognosis for a number of disorders including breast cancer, neuroblastoma, gastrointestinal tumors, and lung cancer. In addition, the presence of MRC during therapy in marrow and/or SCP is associated with a very poor prognosis for patients with breast cancer. The use of testing for MRC in the patient provides prognostic information that may be of use to the oncologist.

Background Despite the initial success of high-dose therapy and bone marrow transplant, the major reason for posttransplant failure is relapse of disease. Reinfusion of tumor cells may contribute to relapse in autologous stem cell transplants. We now have ultra-sensitive methods of tumor cell detection that can determine the presence of minimal residual cancer (MRC) in marrow and peripheral blood stem cells. Methods The author has conducted a critical review of the literature on this issue. Results The factors that are associated with an increase in contamination of the graft include (1) the number of cycles of induction therapy, (2) the type of mobilization regimen used, (3) the presence of tumor cells in the marrow, and (4) the number of phereses. A number of studies show that the presence of occult breast cancer in the marrow and/or stem cell product predicts for a poor posttransplant clinical outcome. The presence of clonogenic breast cancer or lymphoma cells in the graft is also associated with a very poor outcome. Published data regarding contamination in graft and outcome for patients with myeloma are limited. Conclusions Testing for minimal MRC in the oncology patient provides prognostic information that may be useful to the transplant physician.

This conference was held on June 23–25, 1996, at the Kunstlerhaus, Munich, Germany, and was sponsored by the International Society for Hematotherapy and Graft Engineering, as well as AMGEN...

There is increasing evidence that the presence of tumor cells within autologous hematopoietic cell grafts may increase the risk of relapse of disease post-transplant. The sensitive detection of minimal residual disease is therefore central to improving the outcome of autologous transplantation, irrespective of the source of the graft. This review presents a comparative evaluation of the sensitivity and specificity of the currently available methods for tumor detection.

Peripheral blood stem cells (PBSCs) are being used as an alternative to autologous marrow rescue for hematopoietic reconstitution after high- dose chemotherapy in patients with neuroblastoma and other solid malignancies. Use of PBSCs is preferred by some because of the belief that there is less risk of tumor contamination. Because tumor stem cell contamination is thought to be one contributing cause of relapse after myeloablative therapy and autologous reconstitution, we examined the potential risk of reinfusing circulating neuroblastoma cells by in vitro evaluation of their clonogenicity. Immunocytologic and tumor cell clonogenic analyses were performed on 74 blood samples obtained from 56 children with advanced-stage neuroblastoma. Concurrently drawn bone marrow specimens were evaluated in 30 instances. Circulating neoplastic cells were detected in 19 of 74 (26%) for all specimens and by immunologic techniques (26%). Using a clonogenic assay, 13 grew identifiable tumor colonies. Comparing results with the two techniques showed tumor colony growth in 10 of the 19 positive specimens by immunocytology. However, 3 of 53 samples (6%) that were negative by immunocytology were positive by the clonogenic assay. Of the 11 positive blood samples, 9 concurrent marrows contained neuroblastoma cells; of the 19 negative blood specimens, 3 concurrent marrows had metastatic disease. We conclude that circulating neuroblastoma cells are present in peripheral blood and have clonogenic properties in vitro. This supports the view that tumor cell contamination may well be one cause of relapse after autologous reconstitution. Consequently, PBSC collections should also undergo meticulous monitoring for tumor contamination before autologous reinfusion.

Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.

Background The objective of the study was to determine whether the effects of infarct-related artery (IRA) infusion of autologous bone marrow–derived CD34 + cells after ST elevation myocardial infarction (STEMI) are dependent on the dose (quantity and mobility) of the cells infused. Beneficial effects of IRA infusion of mononuclear cells after STEMI have been inconsistent, possibly because of differences in timing, cell type, quantity, and mobility of infused cells. Methods Patients were randomized to bone marrow harvest (n = 16) or control (n = 15). At a median of 8.3 days after coronary stenting for STEMI, CD34 + cells were infused via the IRA at 3 dose levels (5, 10, and 15 × 10 6 ) in cohorts of 5 patients each. Baseline and follow-up imaging and ex vivo CD34 + cell mobility were performed. Results Cell harvest and infusion were safe. Quantitative rest hypoperfusion score measured by single-photon emission computed tomography improved at 6 months in the ≥10 million cohorts compared with controls (−256 vs +14, P = .02). There was a trend toward improved ejection fraction at 6 months (+4.5%) in the ≥10 million cohorts compared with no change in the controls and 5 million cohort (+0.7%). Improved perfusion and infarct size reduction correlated with the quantity and mobility of the infused CD34 + cells. Conclusions The effects of CD34 + cell IRA infusion during the repair phase after STEMI are dose dependent and, at a threshold dose of 10 million CD34 + cells, associated with a significant improvement in perfusion that may limit deterioration in cardiac function (IRA infusion of CD34 + cells in patients with acute myocardial infarction [AMR-01] NCT00313339).

We have developed a multiassay system consisting of fluorescence microscopy, immunocytology and tumor colony assay to monitor the removal of tumor cells from bone marrow. This system was tested in preclinical purging experiments in which neuroblastoma cells were seeded into bovine marrow and purged by treatment with monoclonal antibodies and immunomagnetic beads. Eight experiments were performed on two different neuroblastoma cell lines seeded at 2% and/or 5% contamination. We consistently demonstrated greater than a 3 log removal with one cycle of antibody/bead treatment and greater than a 1 log further reduction by addition of a second cycle. We also demonstrated removal of all detectable tumor stem cells by this purging method. We feel that this system will prove valuable for monitoring ex vivo tumor removal in future clinical studies and should be considered for use in other purging trials.

Tumor surveillance tests are used to determine whether malignant cells are responsive or resistant to therapeutic regimens. For patients with neuroblastoma, conventional methods of surveillance are not sensitive enough. Because tumor cells are shed into the circulation, immunocytologic analysis of blood may function as a sensitive monitoring system. In this study, five blood samples were obtained from two patients with disseminated neuroblastoma at diagnosis and during therapy. These samples were analyzed with monoclonal antibodies and immunoperoxidase staining to determine whether circulating neuroblasts were present. In both patients, the presence or absence of circulating neuroblasts yielded information that was more sensitive than that from conventional tests. The authors conclude that immunocytologic analysis of blood should be included with conventional monitoring methods for surveillance of patients with disseminated neuroblastoma.

Peripheral blood stem cells (PBSC) are being used as one alternative to autologous marrow rescue for patients with neuroblastoma and other solid malignancies. Some physicians prefer use of PBSC because less risk of tumor contamination is believed to exist. This hypothesis was evaluated by immunocytologic analysis of blood samples and concurrently drawn bone marrow (BM) samples and of PBSC harvests obtained from 31 patients with disseminated neuroblastoma. We found circulating neoplastic cells in 75% of specimens analyzed at diagnosis, in 36% during therapy, and in 14% of PBSC harvests. Tumor cells in blood obtained during therapy did not appear until 3 months after the time of diagnosis. Clearance of circulating neuroblastoma cells was documented after two courses of induction chemotherapy. Six of 13 patients with minimal or no BM disease had positive blood specimens. We conclude that substantial risk of tumor contamination of PB harvests exists and recommend that induction chemotherapy be administered before hematopoietic progenitor cells are collected from blood.

Shedding of membrane gangliosides is characteristic of human and experimental tumors. Because some shed tumor gangliosides have potent tumor-enhancing properties, significant ganglioside shedding could influence tumor progression. We examined this possibility in a human tumor, neuroblastoma. Ganglioside shedding, measured as circulating tumor-derived GD2 ganglioside, and the outcome of 74 patients with advanced stage (III and IV) disease were studied. Progression-free survival (PFS) was inversely related to circulating GD2 levels at the time of diagnosis (P = .018). By Kaplan-Meier analysis, the quartile of patients having the highest circulating GD2 levels (greater than or equal to 568 pmol/mL) had a strikingly different outcome from the quartile of patients with the lowest (less than or equal to 103 pmol/mL) GD2 levels (P = .013): median PFS was shorter (9 v 28 months), and the long-term survival rate lower (2-year PFS of 24% v 70%). We conclude that more rapid disease progression and lower survival rate are associated with high circulating GD2 levels at diagnosis and speculate that shed neuroblastoma tumor gangliosides play a role in accelerating tumor progression.

ST segment Elevation Myocardial Infarction (STEMI) affects 160,000 annually in the US. Guidelines direct immediate revascularization and adjunctive medical therapies. Yet STEMI victims remain at risk for infarct expansion, heart failure, reinfarction, repeat revascularization and death. In pre

Objective To apply a new immunocytochemistry (ICC) assay to peripheral blood samples for micrometastatic circulating tumor cell detection in patients with head and neck squamous cell cancer (HNSCC). Design The ICC assay uses established monoclonal antibodies that bind to tumor-associated antigens combined with an enrichment system that uses positive selection with anti-human epithelial antigen (EpCAM antibody) to detect circulating tumor cells. Subjects Eighteen consecutive patients newly diagnosed as having HNSCC are described. Results Of the 18 patients, 8 (44%) demonstrated circulating tumor cells using the ICC assay. The numbers of patients positive for circulating tumor cells per stage are as follows: stage I, 1 of 1; stage II, 0 of 2; stage III, 2 of 5; stage IV, 5 of 6; and unknown stage, 0 of 4. The numbers of patients positive for circulating tumor cells per location are as follows: oral cavity, 1 of 2; oropharynx, 3 of 4; glottic area, 3 of 5; supraglottic area, 1 of 3; and unknown primary 0 of 4. Conclusions Circulating tumor cells were identified in almost half of the patients using the ICC assay. In a literature review, we were not able to identify previous reports of circulating tumor cell detection in patients with HNSCC from peripheral blood samples using ICC or identify any study that has attempted to quantify circulating tumor cell levels. Although the clinical implications of circulating tumor cells in micrometastatic tumor detection in patients with HNSCC are still unknown, they may be significant. Long-term follow-up may help elucidate the patients in whom conventional treatment may fail and, thus, those who may benefit from different treatment; it may also assist with the detection of recurrence with a simple blood collection.

Mutations within the Phosphatase and tensin homolog (PTEN) gene have been linked to the development of many cancers. PTEN is one of the most commonly lost tumor suppressors in human cancer; in fact, up to 40% of men with prostate cancer are estimated to have lost a copy of the PTEN gene at the time of diagnosis. Slides stained with CymogenDx's PTEN del-TECT FOUR COLOR probe, developed for reliably detecting PTEN deletion, are scanned using 3DHISTECH's Panoramic 250 FLASH II digital slide scanner and the resulting whole slides are subsequently analyzed using 3DHISTECH's FISHQuant software. The ability to capture fluorescent signals from 9 independent channels and up to 29 different Z layers with perfect co-localization enables the creation of multiple labelled fluorescent slides. 3DHISTECH's “Extended Focus” feature creates a single in-focus image where all the in-focus parts of each captured layer is merged into one composite image. This enables the use of automated algorithms to evaluate different FISH probes in tissue and cytology whole slide samples. The FISHQuant software segments nuclei and spots automatically and scores the resulting data. For PTEN deletion, it measures the deletion clusters for the 3 colors involved. It also uses the control to check for numerical deviation of the 10th chromosome's signals. These two measurements are run automatically and displayed on a two-dimensional “HistoPlot”. This proof of concept study, using prostate core needle biopsies, demonstrates that the PTEN deletion FISH signals captured on a digital slide and overlaid in bright field and fluorescence modes can be subsequently analyzed using automated algorithms. The ability to identify tumor heterogeneity in a prostate core biopsy (and other sample preps) thus becomes possible. “Extended focus” and the patent pending PTEN del-TECT FOUR COLOR probe limits truncations artifact and makes a huge difference as sections get thinner due to other tests needing to be performed. Having access to the whole slide for analysis provides enough cells of interest. Citation Format: Annamaria Csizmadia, Ferenc Szipocs, Janet Park, Mohammmed Harris, Peter Hartmayer, Thomas J. Moss. Automated analysis of four color PTEN deletion in prostate cancer using FISHQuant on a whole slide image. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2458. doi:10.1158/1538-7445.AM2014-2458

PURPOSE To determine whether occult tumor contamination of autologous bone marrow or peripheral-blood progenitor cells (PBPC) influences clinical outcome after high-dose chemotherapy in patients with stage IV breast cancer. PATIENTS AND METHODS We used an immunocytochemical assay capable of detecting one tumor cell in 5 x 10(5) hematopoietic cells to analyze bone marrow and/or PBPC collections obtained from 57 consecutive women with chemotherapy-sensitive metastatic breast cancer who received high-dose chemotherapy. The influence of occult tumor on time to progression, overall survival, and first site of recurrence (old or new) was studied. RESULTS Twenty-three of 57 (40%) patients received bone marrow (n=6) or peripheral-blood progenitor collections (n=17) that contained microscopic cancer. Median time to progression and overall survival were 9 and 22 months in patients who did not receive infused tumor cells, compared with 10 and 24 months, respectively, in those who received occult tumor (P=not significant [NS]). Worse survival, but not time to progression, was observed in six patients who received > or = 2/100,000 tumor cells. Regardless of whether occult tumor was infused, the majority of relapses occurred in prior, rather than new sites of disease. Three patients who received stem-cell products contaminated by microscopic breast cancer remain free from progression at 21+, 47+, and 52+ months. CONCLUSION Microscopic tumor was frequently detected by immunocytochemistry in hematopoietic stem-cell products, but did not predict for inferior treatment outcome in this cohort of patients with metastatic breast cancer. Quantitative information regarding infused tumor burden may have prognostic significance.

Patients undergoing high-dose chemotherapy (HDC) with autologous stem cell rescue (ASCR) may have tumor cells inadvertently infused if their stem cell product is tumor contaminated. We used an immunocytochemical (ICC) method to analyze 31 histologically negative bone marrow (BM) specimens taken from women with advanced-stage breast cancer at the time of BM harvest before HDC. All 31 patients were treated on one of three consecutive HDC protocols and received BM or BM and peripheral stem cells (PSC) as ASCR. Six of 26 evaluable patients had ICC-detectable contaminating tumor cells in their BM harvests. These 6 patients had a trend toward decreased overall survival compared with those patients without ICC-detectable tumor cells (17 months median versus 25+ months, p = 0.11, log rank test for those patients achieving complete response, CR, from HDC). The sites of relapse in the ICC-positive and ICC-negative groups were not notably different when analyzed for new sites versus previous sites of disease. Therefore, our retrospective analysis of a small cohort of patients suggests that the infusion of tumor cells in breast cancer patients undergoing HDC may confer a poor prognosis. Relapse patterns however suggest failure both in new sites and in sites of previous disease. Additional studies in expanded patient populations are needed to explore further the role of tumor cell infusion in ASCR and the possible clinical benefits of tumor cell removal procedures.

There is growing evidence that bone marrow transplantation is an effective means of treating solid tumors in the pediatric age group. Conventional chemoradiotherapy, in which the dose is limited by the degree of bone marrow suppression, does not increase long-term survival for most disseminated neoplasms. Bone marrow transplantation utilizes high-dose chemoradiotherapy without regard to marrow toxicity and may be the treatment of choice for these malignancies.

Peripheral blood stem cell (PBSC) reinfusions are being used with increasing frequency in lieu of, or in addition to, autologous bone marrow transplantation (ABMT) to rescue cancer patients from the myeloablative effects of high-dose chemotherapy. However, the incidence and quantity of tumor cell contamination in PBSC collections has not been widely investigated. This paper reviews the existing data and presents new information to demonstrate that tumor cells are detectable in PBSC harvests from patients with a variety of malignancies. Furthermore, their presence in peripheral blood may have prognostic and clinical significance. Areas of future research and applications for PBSC technologies are also discussed.

Background: Approximately 20% of patients suffering a ST segment elevated acute myocardial infarction (AMI) have progressive peri-infarct zone myocardial cell death causing ventricular remodeling and poor cardiac outcomes in spite of standard medical care. Neo-angiogenesis has been proposed as a natural, albeit insufficient response to mitigate ventricular remodeling resulting from an AMI. Stromal derived growth factor-1 (SDF-1), the ligand for the CXCR4 receptor, is expressed by bone marrow derived CD34+ cells and is produced in increased quantities in the peri-infarct zone. CD34+CXCR4+ cells are the cells naturally mobilized from bone marrow following an AMI to induce neo-angiogenesis. Thus we conducted a series of pre-clinical studies to develop a pharmaceutical grade bone marrow derived cell therapy product with neo-angiogenic potential for direct intra-coronary artery infusion in patients following an AMI. Methods: Bone marrow samples were obtained from healthy volunteer donors using a mini-bone marrow harvest technique (MMH). Bone marrow was stored at 4°C for up to 24 hours then processed using Isolex (Baxter, Ill) selection to acquire CD34+ cells, formulated into a delivery apparatus and stored again at 4°C for up to an additional 48 hours in a shipping container. Following storage the cell therapy product was assessed for cell recovery, viability, sterility, CXCR-4 mobility in an SDF-1 gradient, and CFU growth before and after perfusion through the internal port of an intra-coronary artery balloon dilatation catheter. Results :13 donors (11F and 2M), with a median age of 43 years (mean 43; range 21 – 61 years) yielded a median % CD34+ cells of total nucleated cells of 1.45% (mean 1.38%; range 0.92 – 1.71%) with a higher % in males vs. females (1.67 vs.1.33%) and older (≥ 45years) vs. younger (1.49 vs.1.26%) donors. CD34+ cell viability (median 96%; range 91–98%), % CD34+ mobility in an SDF-1 gradient (11.8; 4.5–34%), CFU growth (20; 14–54) and sterility were all maintained for up to 72 hours from MMH (table). Passage of the CD34+ cells through a balloon catheter did not adversely affect any of these parameters including % mobility (at 72 hours n=3; Pre% 34, 6, 18: Post 34, 18, 23). Multiple passages (n=3) through balloon dilatation catheters did not result in significant CD34+ cell loss (median recovery 99%; range 97–111%) or loss of viability (95%; range 92–98%). Conclusion: Bone marrow derived CD34+ cells selected using the Isolex device and formulated for shipping and administration maintain for up to 72 hours a high viability, mobility in an in vitro SDF-1 gradient, CFU potential and remain sterile despite multiple passages through a balloon dilatation catheter without significant cell loss. This formulation (AMR-001) provides a pharmaceutical grade cell therapy for clinical evaluation in AMI. | Hours from MMH | 36 hours | 60 hours | 72 hours | |:--------------------------------------- | ---------- | ------------- | ----------- | | Selected CD34+ cell viability % (range) | 96 (95–98) | 96 (91–97) | 97 (91–97) | | Selected CD34+ mobility % | 19 (11–20) | 6.2 (4.5–8.8) | 18 (5.7–34) | | CD34+ CFU (colonies) | 21 (13–54) | 15 (14–28) | 27 (14–37) | Median (n>3)

Background: Approximately 20% of patients suffering a ST segment elevated acute myocardial infarction (AMI) have progressive peri-infarct zone myocardial cell death causing ventricular remodeling and poor cardiac outcomes in spite of large vessel revascularization and medical management. Neo-angiogenesis occurs when VEGF levels peak and endothelial precursors are mobilized and recruited to the infarct site. Stromal cell derived factor-1 (SDF-1), the ligand for the CXCR4 receptor, is expressed by CD34+ cells and plays a role in cell homing to areas of ischemic damage. CD34+ CXCR4+ cells home to areas of ischemia, rich in SDF-1, including infarcted myocardium and are capable of inducing neo-angiogenesis. Natural neoangiogenesis is present but insufficient following AMI, suggesting that direct administration of CD34+ CXCR4+ progenitors could mitigate peri-infarct zone myocardial cell death and improve ventricular function. Methods: In this phase I study, patients with an ST segment (AMI) are enrolled in cohorts of 5 to receive one of four doses (5, 10, 15, 20 x 106 of bone marrow derived CD34+ cells. Cells are harvested using a mini-bone marrow harvest (MMH) technique, acquired by Isolex selection and administered by infusion via the infarct related artery 5 to 10 day following successful coronary artery stenting post AMI. The first 10 subjects accrued as subjects on this phase 1 study included 9 males and 1 female, with a median age of 52 years (range 36–70). Results: The first ten patients (of 20 planned) underwent a MMH under conscious sedation without incident. Adequate numbers of viable, enriched CD34+ cells were obtained following Isolex selection for treatment of subjects enrolled at the first two dose cohorts (5 x 106 and 10 x 106 CD34+ cells). The mean fraction of cells expressing CD34 in the marrow product was 0.75%, with a mean recovery of 40% following Isolex selection (Table). Conclusions: Our study demonstrates the feasibility of collecting up to 409 ml of bone marrow using a MMH technique in the immediate post AMI setting, with yields up to 86 x 106 CD34+ cells. All patient cells expressed CXCR4 and had in vitro migratory capacity. However the lower than expected percentage of TNC expressing CD34 (compared with 9 healthy age matched individuals (1.49% vs. 0.75%) and a low % recovery following Isolex selection may limit successful upper (>10 x 106) cohort treatments. VEGf-2 expression on enriched CD34+ cells was variable. | | mean (median) | range | |:---------------------------------------------------------------------- | -------------- | ------------- | | *N=7 (technical loss of 3 samples);** N=9 (technical loss of 1 sample) | | MMH marrow volume (ml) | 395 (396) | 377 – 409 | | Harvest TNC content (x 109) | 6.65 ( 6.73) | 3.85 – 8.59 | | Harvest CD34+ content (x 106) | 45.3 (50.2) | 16.9 – 86.7 | | Harvest CD34+ % of TNC | 0.75% (0.72%) | 0.54% – 1.06% | | Selected CD34+ content (x 106) | 17.8 (16.5) | 8.4 – 28.9 | | Selected % CD34+ recovery | 40.3% (41.9%) | 30.2 – 49.7 | | Selected %CD34+ viability | 97.1% (98.0 %) | 96% – 99% | | Selected % CD34+ purity | 82.5% (84.%) | 70% – 91% | | Total processing time (hours) | 14.2 (14.0) | 11 – 17 | | SDF-1 induced migration (% of CD34+ cells) | 20.2% (17.0%) | 9.5% – 35.4% | | CXCR-4 expression(% of CD34+ cells)* | 58.7% (52.0%) | 44% – 78% | | VEGF-2 expression (% of CD34+ cells)** | 0.82% (0.86%) | 0% – 2.39% | Processing and Product Results (N=10)

Patients with cystic fibrosis have fat malabsorption, providing an experimental model for evaluation ofthe hypothesis that a low-fat intake may prevent atherosclerosis. We studied the frequency and extent of aortic precursor lesions (fatty streaks, early fibromusculoelastic lesions, late fibromusculoelastic lesions) found at autopsy in this disease as well as in other patients with debilitating disorders but with no apparent impairment of fat absorption. Fatty streaks were less common in the cystic fibrosis group, as were the late fibromusculoelastic lesions. There was no significant difference in the frequency, length, or thickness of the early fibromusculoelastic lesions. The findings suggest that fat may be responsible for progression but not initiation of the fibromusculoelastic precursor lesions, and support the concept that early restriction of dietary fat may prevent, delay, or otherwise modify ahterosclerosis in the adult.

A monoclonal antibody that was prepared against human neuroblastoma cells was shown to react strongly with fetal brain and moderately with adult brain by quantitative absorption testing. Immunoperoxidase staining demonstrated expression of the antigen by neuronal precursor cells in the cerebellar external granular layer of a 24- to 26-week fetus but not by their mature derivatives in the granular and molecular layers of adult cerebellum. The antigen was also present on subventricular cells of fetal cerebral cortex, as well as adult and fetal astrocytes. The expression of this antigen by neuronal precursor cells in the external granular layer but not their mature derivatives suggests that it is a stage-specific marker for cerebellar neuronal development.

From 1983 to 1986, 21 patients with poor prognosis neuroblastoma were treated with bone marrow transplantation. This regimen included induction chemotherapy, delayed surgical resection, local irradiation, and intensive chemoradiotherapy followed by infusion of allogeneic or autologous marrow. This therapeutic approach resulted in a 57% long-term survival rate (follow-up: 14-48 months), which appears to be approximately three times superior to conventional chemotherapy in a comparable group of children. In addition, complete resection was possible in 11 of 17 patients operated on after induction therapy. Recurrence in the primary site after bone marrow transplantation occurred in only one of 18 evaluable patients. Thus, this approach almost always eradicates primary tumor in patients with neuroblastoma with advanced disease.

Current treatment of newly diagnosed widespread neuroblastoma may include chemotherapy, delayed surgical resection, marrow ablative chemoradiotherapy, and bone marrow transplantation. Diagnostic imaging (DI) with computerized tomography (CT) or magnetic resonance imaging (MRI) has been used to determine response to therapy and timing of delayed resection. We assessed the accuracy of DI in 25 patients (26 total cases) treated over 21 months. Tumor size and location were estimated prior to surgical resection by DI, and the sensitivity and specificity of these studies were determined from operative findings. DI consisted of CT (15), MRI (8), and MRI and CT (3). Discordance between DI and operative findings was found in ten patients (38%). This included three errors of sensitivity (12%), including two false-positives and one falsenegative. Seven errors of specificity were noted; they included a positive scan with no viable tumor identified (3), much more extensive disease (3), or less extensive disease (1). Viable tumor was identified in 18 cases, and in 11 patients, complete resection of macroscopic tumor at the primary site was carried out. Ten of 13 patients operated on within 5 months of beginning chemotherapy were rendered grossly free of neuroblastoma at the primary site after surgery. Eight of 12 patients operated on 6 months or longer after starting chemotherapy were rendered grossly free of tumor at the primary site. Bone marrow transplantation was performed in 21 patients, ten of whom are alive with a median follow up of 20 months. Survival was similar for patients who underwent surgical resection at ≤5 v >6 months after starting chemotherapy. We conclude that the definitive primary tumor status cannot be assessed by DI alone because of errors in sensitivity and specificity that total 38%. Surgical exploration remains the best way to evaluate primary tumor status, and it should be performed even in the face of a negative DI study. There appears to be no benefit in delaying surgical resection for >5 months after beginning chemotherapy.

bocytopenia highlighted the otherwise benign hospital courses. An exanthem has been reported in older children with RSV infection and croup, 4 but not in neonatal RSV infection. Thrombocytopenia has also not been reported to be associated with RSV infection in any age group, though it frequently has been observed in many other generalized neonatal viral infections? Disseminated intravascular coagulation, another common cause of thrombocytopenia in the septic neonate, 6 seems unlikely in our patients in light of their mild symptoms and the normal coagulation factor studies in Patient 2. Both patients had exanthems, but neither developed purpuric lesions. Respiratory syncytial virus is one of the commonest viral respiratory pathogens of children; however, neither of these RSV-infected infants had clinical symptoms of significant respiratory tract infection. By contrast, both initially had a sepsislike syndrome similar to that described for neonatal enterovirus infection. 7 The authors thank Drs. V. A. Fulginiti, J. J. Hutter, and J. J. Corrigan for their review and advice, Drs. C. DeBenedetti and G. Samoy for referral of these patients, and Ms. Nanci Buckley for invaluable assistance in preparation of the manuscript.

We analyzed the performance of two cohorts of surgical residents: one from "pass/fail" and the other from "graded" medical schools. A performance index indicates that the group from graded schools performed significantly better (P

Intensive chemotherapy, TBI, and BMT may improve the outcome for children with metastatic neuroblastoma (August, et al, 1982). We are testing a new four drug chemotherapy and TBI pretransplant regimen for its toxicity and efficacy. Five patients received cis-platinum, VM26, doxorubicin, melphalan, and TBI (VAMP-TBI); and 3 received melphalan and TBI (M-TBI) because they could not tolerate the other agents or because their tumor was judged resistent. Allogeneic (allo) marrow was given to 5 and autologous (auto) marrow to 3 patients. They were 1 1/2 to 7 yrs old when transplanted (median 5 yrs) and were transplanted 5-13 mos after diagnosis (median 10 mos). Auto marrow had no neuroblastoma cells by immunoperoxidase staining for neuron specific enolase and cell surface antigens, which detects 1 tumor cell/105 normal cells. The most consistent acute toxicity from VAMP-TBI was severe mucositis, vomiting, and diarrhea; M-TBI also caused these complications but to a lesser extent. One acute toxic death occurred with each regimen. Of the 5 patients receiving VAMP-TBI, 3 have no evidence of disease (NED) at 60 (auto), 195 (allo), and 317 (allo) days; and 1 (auto) is 7 days post transplantation. One of 3 receiving M-TBI is NED at 45 days (auto), and another has progressive disease at 189 days (allo). We conclude that VAMP-TBI is a tolerable conditioning regimen. The survival of 3 of 4 evaluable patients receiving VAMP-TBI with NED suggests that this regimen should continue to be investigated.

The purpose of this study was to evaluate the frequency and extent of aortic precursor lesions (fatty streaks, early fibro-musculoelastic lesions, late fibromusculoelastic lesions) found at autopsy in cystic fibrosis. Patients with this disease suffer from fat malabsorption and thus provide a unique experimental model for evaluation of the hypothesis that low fat intake may prevent atherosclerosis. Other patients with debilitating disorders but with no apparent impairment of fat absorption served as controls. Autopsy material from 35 patients, 9 with cystic fibrosis and 26 with leukemia and other malignancies were studied. Fatty streaks were less common in the cystic fibrosis group (p. < .001) as were the late fibromusculoelastic lesions (p. = .007). There was no significant difference in the frequency, length, or thickness of the early fibromusculoelastic lesions. The findings suggest that fat is responsible for progression but not initiation of the fibromusculoelastic precursor lesions. The results support the concept that early restriction of dietary fat may prevent, delay, or otherwise modify atherosclerosis in the adult.

Nervous system markers can provide information on maturation, differentiation,and intercellular interactions. Monoclonal anti-body 459 (ab 459),was previously found to react strongly with fetal brain,and moderately with adult brain. The objective of this study was to determine which neural cells express the antigen (ag 459) defined by ab 459. Brains obtained within 12 hours postmortem from adults and a 22 week fetus were frozen and sectioned with a cryostat. They were fixed with acetone, incubated with ab 459, followed by biotinylated anti-mouse immunoglobulin, and then avidin-biotin-peroxidase complexes. Ag 459 is expressed by fetal neurons in the external granular layer of cerebellum; these same cells were negative for glial fibrillary acidicprotein and neuron specific enolase (NSE). Ab 459 also labelled morphologically mature fetal neurons, which were NSE positive,in a cerebellar nucleus. Adult cerebellar neurons of the granular layer, which are derived from fetal external granular layer, and all adult cortical neurons did not display ag 459. Both adult and fetal astrocytes expressed ag 459.Ag 459 is the first anti-genic determinant to be defined on human fetal neuronsin the external granularlayer of the cerebellum. The expression of this determinant on these cells,but not their maturederivatives, suggests that ag 459 is a stage specific marker for cerebellarneuron development. Astrocytes assist in migration of cerebellarfetal neurons in vivo, and the presence of ag 459 on fetalastrocytes and fetal neurons raise the possibility that this marker may be involved in the migration process.