Evan Vosburgh

Intermediate-dose intravenous melphalan and blood stem cells mobilized with sequential GM+G-CSF or G-CSF alone to treat AL (amyloid light chain) amyloidosis.

AL amyloidosis patients ineligible for dose‐intensive melphalan (200 mg/m2) were enrolled on a phase II trial to be treated with two cycles of intermediate‐dose melphalan (IDM 100 mg/m2) and mobilized blood stem cells (BSC). For mobilization patients were randomized to either GM‐CSF 250 μg/m2 for 3 d followed by G‐CSF 10 μg/kg for 3 d (GM+G), or G‐CSF 10 μg/kg for 6 d (G‐alone), with leukaphereses on days 5, 6 and 7. To minimize morbidity, we planned to support each cycle with 3.5 × 106 CD34+ cells/kg and had a collection target of 7 × 106 CD34+ cells/kg. Those who did not achieve the target were treated with one cycle of IDM. 30 patients, a median of 62 years old and 7 months from diagnosis, were enrolled. Both mobilization regimens were generally well tolerated, and similar in terms of CD34+ cells and CFU‐GM collected, but only 6/28 patients achieved the collection target (GM+G four, G‐alone two). Despite a 19% incidence of grade 4 toxicities, IDM therapy was well tolerated. At a median follow‐up of 24 months (19–36) 57% of patients had survived, 17% with durable complete haematological responses and 40% with improved or stable amyloid organ involvement, including 3/9 patients with predominant cardiac amyloid who are alive 2–3 years after treatment. The 100 d mortality was 20%. In conclusion, no definitive differences were identified between the mobilization regimens and IDM was an active regimen in AL for selected patients.

Collection of mobilized blood progenitor cells for hematopoietic rescue by large-volume leukapheresis

BACKGROUND: Mobilized blood progenitor cells rapidly reconstitute hematopoiesis in patients after dose‐intensive chemotherapy. However, optimal timing and methods of mobilized blood progenitor cell collection have yet to be fully defined. STUDY DESIGN AND METHODS: The utility of large‐volume leukapheresis (LVL; > 15 L blood processed) in collecting target doses of mononuclear cells (7 × 10(8)/kg) for use in autologous hematopoietic rescue was investigated. LVL was begun at a standardized interval (14 days) after a course of limited chemotherapy and subsequent daily recombinant human granulocyte‐macrophage‐colony‐ stimulating factor administration to mobilize blood progenitor cells into the circulation. With each LVL procedure, mononuclear cells, colony‐forming units‐granulocyte‐macrophage (CFU‐GM), burst‐forming units‐erythroid, mixed colonies, total clonogenic progenitor cells, and CD34+ cells collected per kg of patient weight were counted. After high‐ dose chemotherapy and infusion of cryopreserved mobilized blood progenitor cells, the days needed for neutrophils to reach levels of > 0.5 × 10(9) per L and for platelets to reach levels of > 20 × 10(9) per L were recorded. RESULTS: In 14 previously treated cancer patients, an average of 28.9 +/− 4.9 L of blood was processed per LVL (n = 35) to collect medians of 2.5 × 10(8) mononuclear cells per kg (range, 1.0‐ 7.4), 14 × 10(4) CFU‐GM per kg (0‐208), 27 × 10(4) clonogenic progenitor cells per kg (0‐370), and 2.8 × 10(6) CD34+ cells per kg (0‐ 112.5). Fifty‐seven percent of patients (8/14) required one or two LVL procedures to collect adequate blood progenitor cells (range, 1–4). After dose‐intensive chemotherapy, 13 patients received medians of 6.8 × 10(8) mononuclear cells per kg (range, 5.1‐9.9), 53 × 10(4) CFU‐GM per kg (9‐208), and 12 × 10(6) CD34+ cells per kg (3.6‐112.5). Rapid hematopoietic reconstitution occurred with 10 days (range, 8–12) and 9 days (6‐15), respectively, for neutrophil and platelet recoveries. CONCLUSION: Scheduled LVL, beginning on Day 14 after the administration of granulocyte‐macrophage‐colony‐stimulating factor following chemotherapy, is a convenient and efficient method of collecting blood progenitor cells. The mononuclear cells so obtained effected consistent and rapid hematopoietic reconstitution in a highly reproducible manner in a group of heavily treated patients.

Report of a variant t(1;15;17)(p36;q22;q21.1) in a patient with acute promyelocytic leukemia

Chromosome analysis of bone marrow aspirate from a 46-year-old man with acute promyelocytic leukemia (APL) revealed a variant translocation, 46,XY,t(1:15;17)(p36;q22;q21.1). The breakpoints in chromosomes 15 and 17 appear to be the same as those in the more common translocation, t(15;17), associated with APL. The common translocation has been reported in up to 80% of cases of APL. Seventeen cases with variant translocations have been reported involving 15 alone, 17 alone, or 15, 17, and some other chromosome.

Immunologic recovery after autologous blood stem cell transplantation in patients with AL-amyloidosis

We prospectively studied absolute lymphocyte (ALC) and monocyte counts (AMC), lymphocyte subsets and proliferative in vitro responses to mitogen and antigen in 12 patients with AL-amyloidosis (AL) undergoing autologous blood stem cell transplantation (SCT) with high-dose i.v. melphalan. Myeloid and lymphoid recovery (>500 per μl) occurred in a median of 10 days post SCT. While there was a continuous decline in the number of CD4+ T cells at 3 months, ALC, AMC, B cells (CD19+), CD8+ T cells, and NK cells (CD16+/56+) returned to baseline. While T cell proliferative responses to phytohemagglutinin (PHA) remained depressed, B cell function measured by the proliferative response to staphylococcal antigen returned to baseline by 3 months. To supplement our findings, we retrospectively evaluated ALC, AMC and serum immunoglobulin levels in a separate group of patients treated with the same protocol at our institution. ALC and AMC recovery was similar to the pattern observed in the initial study group. Immunoglobulin levels remained within normal ranges at 3 and 12 months after SCT. Of 50 patients who were followed for a minimum of 1 year following SCT, seven (14%) developed shingles and one (2%) had PCP pneumonia. In conclusion, cellular immune function, reflected by absolute numbers of CD4+ T cells and PHA responsive T cell proliferation, is significantly suppressed at 3 months after SCT in patients with AL, and this post-transplant immunosuppression is associated with a low but clinically meaningful occurrence of opportunistic infections typical of T cell immunosuppression.Bone Marrow Transplantation (2001) 28, 1105–1109.

High-throughput image analysis of tumor spheroids: a user-friendly software application to measure the size of spheroids automatically and accurately.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application – SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary “Manual Initialize” and “Hand Draw” tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

P53 mutations in lymphomas: position matters

The p53 protein integrates the combination of mutations that cause a cancer with the set of environmental stresses that act on that cancer. Mutations in the p53 gene can therefore have dramatic impacts on the overall survival of cancer patients.

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100, 000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models. To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 °C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drugs effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

Sexual dimorphism of liver metastasis by murine pancreatic neuroendocrine tumors is affected by expression of complement C5

In a mouse model for neuroendocrine tumors of the pancreas (PanNETs), liver metastasis occurred at a higher frequency in males. Male mice also had higher serum and intratumoral levels of the innate immunity protein complement C5. In mice that lost the ability to express complement C5, there was a lower frequency of metastasis, and males no longer had a higher frequency of metastasis than females. Treatment with PMX53, a small molecule antagonist of C5aR1/CD88, the receptor for complement C5a, also reduced metastasis. Mice lacking a functional gene for complement C5 had smaller primary tumors, which were less invasive and lacked the CD68+ macrophages that have previously been associated with metastasis in this type of tumor. This is the first report of a gene that causes sexual dimorphism of metastasis in a mouse model. In the human disease, which also shows sexual dimorphism for metastasis, clinically advanced tumors expressed more complement C5 than less advanced tumors.

Abstract 267: Preclinical investigation of the HSP90 inhibitor, ganetespib, in combination with FDA-approved cytotoxic agents for their potential anti-tumor effects on human neuroendocrine tumors

The gastroenteropancreatic neuroendocrine tumor (GEP-NET) system is comprised of a heterogeneous group of tumors with increasing incidence. Current standard of care cytotoxic agents have limited efficacy, which necessitates the need for innovative therapeutic approaches. Heat shock protein 90 (HSP90) is overexpressed in a wide range of tumor types including human pancreatic neuroendocrine tumors (PanNETs). Ganetespib is a second-generation HSP90 inhibitor that is well tolerated in cancer patients (dosed into >1500 patients) and is currently being evaluated in several investigator sponsored clinical trials including acute myeloid leukemia (AML), ovarian cancer, breast cancer, and other tumor types. Here, we show that ganetespib inhibits the proliferation of NET cells and induces apoptosis in vitro with potency comparable to another second-generation HSP90 inhibitor (NVP-AUY922), but with potency two- to seven-fold more than first generation inhibitors (17-AAG, IPI-504) in BON-1, CM and H727 cell lines, and thirty-fold more in QGP-1 cell line. In mice bearing PanNET tumor xenografts, single agent ganetespib reduced the growth of tumors without signs of toxicity. Tumors from ganetespib treated animals demonstrated reduced phospho-AKT and phospho-ERK expression, and elevated HSP70 expression, supportive of exposures necessary for functional activity. In an effort to identify clinically meaningful agents that could further potentiate the antitumor activity of ganetespib, we performed in vitro combination screens evaluating proliferation in four NET cell lines with ganetespib and forty FDA approved anticancer agents. Ganetespib showed synergistic effects when combined with inhibitors of mTOR, topoisomerase I or II, or DNA synthesis. Results from our ongoing in vivo combination studies with ganetespib and these three classes of anticancer agents will be presented. Citation Format: Chung Wong, Evan Vosburgh, Arnold Levine, David Proia, Eugenia Xu. Preclinical investigation of the HSP90 inhibitor, ganetespib, in combination with FDA-approved cytotoxic agents for their potential anti-tumor effects on human neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 267.

Abstract 3185: Complement C5 promotes male bias of pancreatic neuroendocrine tumor metastasis

Among patients with neuroendocrine tumors, males are more likely than females to develop liver metastatic disease. We show that this male bias toward liver metastasis is also observed in a mouse model for pancreatic neuroendocrine tumorigenesis (PanNETs). Interestingly, primary tumors from mice with metastatic disease display hepatomimicry, expressing genes normally expressed in liver. Both male and female primary tumors from mice with metastatic disease show hepatomimicry, although one of the liver genes, complement C5, is only induced in males. In mice knocked out for the complement C5 gene, the frequency of liver metastasis dropped significantly; moreover, males and females lacking a gene for complement C5 developed liver metastasis at the same frequency. Liver metastasis was also prevented by PMX53, a small molecule antagonist of C5aR1/CD88, which is the receptor for complement C5a. Thus complement C5 is epistatic to metastasis and promotes gender bias of liver metastatic disease. Citation Format: Tanupriya Contractor, Chang Chan, Shinta Kobayashi, Richard Clausen, Yvonne Sun, Edaise da Silva, Evan Vosburgh, Arnold J. Levine, Laura Tang, Chris R. Harris. Complement C5 promotes male bias of pancreatic neuroendocrine tumor metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2015-3185