Mouse spleen lymphoblasts generated in vitro: Their replication and differentiation in vitro*

[No abstract available

Mouse spleen lymphoblasts generated in vitro. Their replication and differentiation in vitro

Mouse spleen lymphoblasts induced with lipopolysaccharide and fetal calf serum were obtained in high yield and purity in their first proliferative cell cycle by floatation in dense bovine plasma albumin columns (3). The blasts were maintained in vitro for 3 more days. The cultures were examined in bulk on each day, and in addition, those cells in S phase initially were tagged with [(3)H]thymidine and followed continuously in vitro. Grain count dilution data indicated that most blasts divided but twice over a 2- to 3-day interval in vitro. [(3)H]Thymidine pulse radiolabeling and flow microfluorometry suggested that at least 50-70 percent of the proliferating blasts withdrew from proliferative activity after 2-3 days of culture. Morphologic studies demonstrated that lymphoblasts persisted as such for 1-2 days in vitro and then matured into typical plasma cells. Many of the blastprogeny had small nuclei and considerable basophilic cytoplasm on Giemsa-stained cell smears; abundant rough endoplasmic reticulum by electron microscopy; and readily detectable cytoplasmic Ig by immunocytochemistry. Reversion of blasts to small lymphocytes could not be detected; however, some blasts persisted even after 3 days of culture. The viability of the cultured lymphoblast was followed by initially tagging the cells with [(3)H]thymidine as well as several other techniques. Little cell death was documented during the first day of culture. The number of labeled progeny increased twofold whereas the grain count halved. But 40- 50 percent of the cell-associated label was lost during each of the second and third days, and fewer labeled progeny than predicted by grain count dilution were identified. The culture medium could not be implicated in this loss of lymphoblast progeny, and we suggest that the maturation of the lymphoblast to a short-lived plasma cell was responsible. Therefore mitogen-stimulated B blasts seem to mature into typical plasma cells after just two cycles of cell division. The plasma cells resemble those produced in situ during an immune response in their cytologic features, withdrawal from active proliferative activity, and short life-span

Sentinel node staging for breast cancer: Intraoperative molecular pathology overcomes conventional histologic sampling errors

BACKGROUND: When sentinel node dissection reveals breast cancer metastasis, completion axillary lymph node dissection is ideally performed during the same operation. Intraoperative histologic techniques have low and variable sensitivity. A new intraoperative molecular assay (GeneSearch BLN Assay; Veridex, LLC, Warren, NJ) was evaluated to determine its efficiency in identifying significant sentinel lymph node metastases (\u3e.2 mm). METHODS: Positive or negative BLN Assay results generated from fresh 2-mm node slabs were compared with results from conventional histologic evaluation of adjacent fixed tissue slabs. RESULTS: In a prospective study of 416 patients at 11 clinical sites, the assay detected 98% of metastases \u3e2 mm and 88% of metastasis greater \u3e.2 mm, results superior to frozen section. Micrometastases were less frequently detected (57%) and assay positive results in nodes found negative by histology were rare (4%). CONCLUSIONS: The BLN Assay is properly calibrated for use as a stand alone intraoperative molecular test

Effect of Axillary Dissection vs No Axillary Dissection on 10-Year Overall Survival Among Women With Invasive Breast Cancer and Sentinel Node Metastasis: The ACOSOG Z0011 (Alliance) Randomized Clinical Trial

The results of the American College of Surgeons Oncology Group Z0011 (ACOSOG Z0011) trial were first reported in 2005 with a median follow-up of 6.3 years. Longer follow-up was necessary because the majority of the patients had estrogen receptor–positive tumors that may recur later in the disease course (the ACOSOG is now part of the Alliance for Clinical Trials in Oncology)

Metasin — An Intra-Operative RT-qPCR Assay to Detect Metastatic Breast Cancer in Sentinel Lymph Nodes

Nodal status is one of the most important prognostic factors in breast cancer. Established tests such as touch imprint cytology and frozen sections currently used in the intra-operative setting show variations in sensitivity and specificity. This limitation has led to the development of molecular alternatives, such as GeneSearch, a commercial intra-operative real-time quantitative Polymerase Chain Reaction (RT-qPCR) assay that allows the surgeon to carry out axillary clearance as a one-step process. Since GeneSearch has been discontinued, we have developed the replacement Metasin assay, which targets the breast epithelial cell markers CK19 and mammaglobin mRNA and identifies metastatic disease in sentinel lymph nodes. The optimised assay can be completed within 32 min (6 min for RNA preparation and 26 min instrument run time), making its use feasible in the intraoperative setting. An analysis by Metasin of 154 archived lymph node homogenates previously analysed by both parallel histology and GeneSearch showed concordance for 148 cases. The sensitivity and specificity of Metasin compared with GeneSearch were 95% (CI 83%-99%) and 97% (CI 91%-99%) respectively; compared with histology they were 95% (CI 83%-99%) and 97% (CI 91%-99%), respectively. The sensitivity and specificity of GeneSearch compared with histology were 90% (CI 77%-96%) and 97% (CI 93%-99%) respectively. The positive predictive value of Metasin was 90% and negative predictive value was 98% for both histology and GeneSearch. The positive predictive value of GeneSearch was 92% and the negative predictive value was 97% compared to histology. The discordance rates of Metasin with both GeneSearch and histology were 3.89%. In comparison, the discordance rate of GeneSearch with histology was 4.5%. Metasin's robustness was independently evaluated on 193 samples previously analysed by GeneSearch from the Jules Bordet Institute, where Metasin yielded comparable results

Utilization of Genomics for Risk Assessment and Molecular Subtyping to Improve Treatment Strategy in Breast Cancer

Death from breast cancer began a decline in the 1990’s. One widely accepted reason for this reduction was the discovery that breast cancer can be uniquely defined at the molecular level by oncogenes and tumor suppressor genes including, but not limited to, EGFR, AKT, ErbB2, PI3K, TP53, BRCA1/2, and PTEN. These genes and potential mutations within, can drive constitutive activation of aberrant signaling that can induce and sustain tumorigenesis. More importantly, these genes represent new avenues for possible markers and therapies. In the wake of this molecular biology discovery, there was an influx of new breast cancer treatments to the marketplace, most notably the launch of trastuzumab (Herceptin) in 1996. Subsequently, the incidence of death continued to decrease peaking from 2002 to 2003 when it dropped by 7% in that one year alone. However, the current challenge in oncology is how to translate the wealth of information contained in the molecular biology of cancer and translate it into patient care. While progress has been made in developing treatments, less progress has been made identifying new markers for breast cancer to get that treatment to the right patients or selecting them for treatment trials. Currently, the majority of providers and healthcare systems do not utilize molecular biology to determine risk of breast cancer recurrence and treatment decision making for breast cancer. Rather, they determine which patients require what adjuvant treatment based on some form of the breast cancer tumor–node–metastases (TNM) staging system and clinical-pathological criteria, such as lymphovascular invasion (LVI), nodal status, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu) status and more. As a result, potential differences exist worldwide in the selection of patients who require adjuvant chemotherapy based on their risk of breast cancer recurrence. All of these tests and criteria are based on the anatomical extent of the tumor, with little if any insight into the patient’s breast cancer biology. A significant percentage (30 to 50%) of all early stage breast cancer patients (50,000 to 75,000 patients annually in the US alone) have clinically ambiguous or confounding clinical-pathological characteristics, making it difficult for physicians to formulate clinical risk and supports the argument for development and utilization of molecular diagnostic testing. By looking at the molecular biology of cancer, companies, such as Agendia, seek to create technologies beyond clinical screening, imaging or cell surface staining. Technologies like MammaPrint and BluePrint, reveal who is truly at risk by differentiating responders from non-responders based on genetics. By interrogating single genes or overall patterns of gene expression, it is possible to better understand the various sequences of biological events that give rise to breast cancer and in turn, better develop and guide treatments