Ig VH gene mutational patterns indicate different tumor cell status in human myeloma and monoclonal gammopathy of undetermined significance

Plasma cell tumors display a wide spectrum of clinical progression, ranging from aggressive multiple myeloma to a benign form known as monoclonal gammopathy of undetermined significance (MGUS), which requires no treatment. Because both diseases involve mature Ig- secreting plasma cells, the reason for this variation in malignant behavior is unclear. However, assessment of malignant potential is desirable for choice of treatment protocols. Ig variable (VH) gene sequences analysis has previously shown the tumor cell of multiple myeloma to be postfollicular, with mutated homogeneous clonal sequences indicating no continuing exposure to the somatic hypermutation mechanism, and this was confirmed in 7 of 7 patients. Comparison of the VH gene sequences in the monoclonal cells in MGUS yielded a different result, with 3 of 7 patients demonstrating mutated heterogeneous sequences consistent with the tumor cells remaining under the influence of the mutator. In 1 of 3 of these patients, an IgM-positive precursor cell was identified that expressed heterogeneous VH sequences similar to those of the isotype-switched plasma cell. These results indicate that the clonal cells in MGUS differ from those in myeloma and suggest that the difference may reflect malignant potential.     

Favored use of immunoglobulin V(H)4 Genes in AIDS-associated B-cell lymphoma

We examined the lg heavy chain variable region genes (Ig V(H) genes) expressed in biopsy specimens of 10 patients with acquired immunodeficiency syndrome (AIDS)-associated lymphoma. Eight expressed Ig V(H) genes of the V(H)4 group, indicating a bias toward expression of Ig V(H) genes of this subgroup. Sequence analyses of Ig V(H) genes isolated from any one lymphoma did not reveal evidence for intraclonal diversity. However, some lymphomas express Ig V(H) genes that apparently have undergone somatic diversification and selection. In addition, we found that the sequence encoding each examined third complementarity determining region most likely resulted from D-D fusion, a process that ordinarily contributes to the generation of a relatively small proportion of the Ig heavy chain genes expressed by normal adult B cells. The noted restriction in the use of Ig V(H) genes by AIDS-associated B-cell lymphomas suggests that antigenic stimulation contributes to lymphomagenesis in patients with AIDS.     

Differentiation state and responses to hematopoietic growth factors of murine myeloid cells transformed by myb

Murine hematopoietic cells can be transformed in vitro by recombinant retroviruses that express the myb oncogene, and hematopoietic growth factor (HGF)-dependent myeloid cell lines can be derived from these transformed primary cells. In this study, the differentiation state and responses of myb-transformed hematopoietic cells (MTHCs) have been investigated. We find that MTHCs exhibit properties of early myeloid progenitors including synergistic responses to combinations of HGFs and expression of certain surface markers. As reported previously, MTHCs respond well to granulocyte-macrophage colony-stimulating factor (GM- CSF) but can also respond to interleukin-3 (IL-3); the response to the latter factor depends on the mouse strain from which the cells are derived. Although these single factors stimulate MTHCs, combinations of these factors with colony-stimulating factor-1 (CSF-1 or M-CSF) or Steel factor (SLF or SCF) act synergistically to promote colony formation. The surface markers expressed by MTHCs include both granulocyte-macrophage lineage specific antigens Gr-1, 7/4, F4/80, and Mac-1, as well as two antigens found on early progenitors and stem cells--Thy-1 and Sca-1 (Ly6E). Expression of the latter markers is often heterogeneous and can be modulated by the growth factors to which the cells are exposed. Finally, we show that monocytic differentiation of MTHCs can be induced by exposure to tumor necrosis factor (TNF alpha). Taken together, these results suggest that MTHCs will be a useful model for studying HGF/cytokine responses in both proliferation and differentiation.     

Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine- SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.     

Serological investigation of pneumonia as it presents to the physician��s office

Purpose:

To define the etiology of pneumonia, using a battery of serological tests, among patients presenting to physicians’ offices in Cumberland County, Nova Scotia from July 2, 1989 to July 1, 1990.

Methods:

Patients presenting to their physician’s office with symptoms suggestive of pneumonia were invited to participate in the study by completing a questionnaire, having a chest radiograph and providing acute and convalescent phase serum samples. These serum samples were tested for antibodies to Mycoplasma pneumoniae, Coxiella burnetii, Legionella pneumophila, adenovirus, and influenza viruses A and B. Some of the samples were tested for antibodies to Chlamydia pneumoniae.

Results:

Seventy-five of the inception cohort of 203 patients had a chest radiograph compatible with pneumonia, a completed questionnaire and acute and convalescent phase serum samples. There were 39 females and 36 males with a mean age of 41.7 years. Twenty-six (35%) were admitted to hospital. The mortality rate was 3%. Forty-five per cent had a diagnosis made by serology: M pneumoniae, 22 (29%); influenza A virus, five (7%); C burnetii, L pneumophila, adenovirus, two (3%) each.

Conclusions:

While it is not possible to generalize about these findings because of ascertainment bias, the data suggest that M pneumoniae is a common cause of pneumonia presenting to a physician’s office and that mortality is low in this group of patients.     

Eosinophilic sialodochitis: redefinition of ‘allergic parotitis’ and ‘sialodochitis fibrinosa’

Sialodochitis fibrinosa and allergic parotitis have described rare patients with recurrent salivary gland swelling and mucus plugs, often with atopy. We have evaluated three patients with atopic disease, recurrent salivary gland swelling, and an eosinophilic sialodochitis. Two had eosinophil‐rich mucus plugs. Fifty‐six additional cases were identified in a medical literature database search, each defined by recurrent salivary gland swelling associated with eosinophil‐rich mucus plugs or sialodochitis with periductal eosinophilic infiltration. The majority (78%) were reported from Japan. Females were predominantly affected (F:M = 2.3) with a median age of 47 years at evaluation. The parotid and submandibular glands were involved, respectively, in 71% and 46%. Allergic symptoms were present in 66%, atopic disease in 63% of those with reported allergy testing, and blood eosinophilia in 71%. Contrast sialography and other imaging modalities documented ductal dilatation in 82%. Treatments included anti‐allergic medications (58%), systemic glucocorticoids (25%), duct cannulation with irrigation, steroid injection, and/or duct dilatation (36%), and glandular resection (19%). We recommend the diagnosis ‘eosinophilic sialodochitis’ be applied to patients who meet this case definition. The disease is a unique cause of chronic recurrent salivary gland swelling. Its likely allergic etiology may be amenable to current or future biologic therapies.     

Semi-automatic cone beam CT segmentation of in vivo pre-clinical subcutaneous tumours provides an efficient non-invasive alternative for tumour volume measurements

Objective:

To evaluate the feasibility and accuracy of using cone beam CT (CBCT) scans obtained in radiation studies using the small-animal radiation research platform to perform semi-automatic tumour segmentation of pre-clinical tumour volumes.

Methods:

Volume measurements were evaluated for different anatomical tumour sites, the flank, thigh and dorsum of the hind foot, for a variety of tumour cell lines. The estimated tumour volumes from CBCT and manual calliper measurements using different volume equations were compared with the “gold standard”, measured by weighing the tumours following euthanasia and tumour resection. The correlation between tumour volumes estimated with the different methods, compared with the gold standard, was estimated by the Spearman''s rank correlation coefficient, root-mean-square deviation and the coefficient of determination.

Results:

The semi-automatic CBCT volume segmentation performed favourably compared with manual calliper measures for flank tumours ≤2 cm³ and thigh tumours ≤1 cm³. For tumours >2 cm³ or foot tumours, the CBCT method was not able to accurately segment the tumour volumes and manual calliper measures were superior.

Conclusion:

We demonstrated that tumour volumes of flank and thigh tumours, obtained as a part of radiation studies using image-guided small-animal irradiators, can be estimated more efficiently and accurately using semi-automatic segmentation from CBCT scans.

Advances in knowledge:

This is the first study evaluating tumour volume assessment of pre-clinical subcutaneous tumours in different anatomical sites using on-board CBCT imaging. We also compared the accuracy of the CBCT method to manual calliper measures, using various volume calculation equations.Accurate methods for assessing subcutaneous tumour volumes are vital components of pre-clinical cancer research. Longitudinal studies comparing different cancer treatment regimens in research animals (usually mice or rats) often use tumour volume assays as the main end point for evaluating treatment efficacy.¹ The current standard for tumour volume measurements for pre-clinical subcutaneous tumours consists of using manual callipers to determine the length, width and, in some cases, also depth of the tumour. Tumour volumes are then calculated based on a chosen mathematical formula, where a formula based on a modified ellipsoid has previously been shown to perform quite well.^1,2 Calliper measures, although fast and convenient, are subject to several sources of uncertainty such as interobserver variability, differences in tumour shape, and amount of fatty tissue and fur surrounding the tumour.Non-invasive imaging methods have become the standard for clinical tumour response assessment, and CT has been the main component for more than a decade.^3,4 Previous studies have shown that small-animal ultrasound imaging or sequential micro-CT scans can be used to measure subcutaneous tumours in mice and rats more accurately than manual calliper measures.^5–7 Improving the accuracy of tumour volume measurements will not only improve the quality of data in treatment efficacy studies, but it will also reduce the variability and thus reduce the number of animals required for tumour studies.Taking micro-CT scans or ultrasound images of animals may, however, be quite a time consuming and potentially costly procedure, also requiring further anesthetizing of the animals. Here, we present a method for semi-automatic tumour volume determination based on cone beam CT (CBCT) scans taken using the on-board imager of the small-animal radiation research platform (SARRP; XStrahl®, England, UK).^8,9 With robotic-image-guided small-animal irradiators becoming increasingly available,¹⁰ this method provides a promising alternative for fast and less user-dependent tumour volume measurement using CBCT scans already obtained in the process of radiation therapy target localization. We compare the performance of CBCT volume segmentation to that of manual calliper measurements for different tumour sites and provide recommendations for pre-clinical tumour volume assessment based on these results.