Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences

Human T-cell receptor (TCR) delta-chain diversity mainly originates from high junctional variability, since only a limited number of germline elements is available. This extraordinary diversity at the V.J junction, due to the use of two D delta elements and extensive incorporation of N nucleotides, constitutes a specific clonal marker for cell populations exhibiting rearranged TCR delta genes. To this end we amplified in vitro by polymerase chain reaction (PCR) the TCR delta junctional region of five acute lymphoblastic leukemias (ALL), isolated respective DNA fragments, and used them directly as clonospecific probes. The combination of PCR technology and hybridization to clonospecific probes permitted the detection of leukemia DNA at dilution of 1:100,000 in all five cases. Moreover, we were able to investigate one of the ALL patients 11 months after achieving continuous complete remission. Conventional Southern blot analysis failed to detect rearranged TCR genes at this stage. However, residual leukemic cells could readily be detected by PCR technique. We conclude that the strategy proposed here is a very sensitive tool to detect minimal residual disease in a significant proportion of human lymphoid neoplasias.     

A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa)

Agonist-induced inside-out signaling results in an increased affinity of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) for soluble ligands (fibrinogen [Fg] and PAC1). Ligand binding to integrins initiates outside-in signaling that leads to cellular responses such as cell spreading and focal adhesion formation. A point mutation in the beta 3 cytoplasmic domain (S752-->P) is associated with blocked inside- out alpha IIb beta 3 signaling in a variant Glanzmann's thrombasthenia. This mutation was introduced into beta 3 and cotransfected into Chinese hamster ovary cells with a chimeric alpha subunit consisting of the alpha IIb extracellular and transmembrane domains and the alpha 6B cytoplasmic domain. The substitution of the alpha IIb cytoplasmic domain with that of alpha 6 led to activation of alpha IIb beta 3 to bind PAC1, mimicking inside-out signaling. This effect was reversed by the S752-->P mutation, indicating a disruption of inside-out signaling by the mutation. In addition, transfectants expressing this beta 3 variant showed reduced alpha IIb beta 3-mediated cell spreading on immobilized Fg, focal adhesion, and fibrin clot retraction, suggesting an impairment in outside-in alpha IIb beta 3 signaling. Therefore, a single point mutation in the beta 3 cytoplasmic domain impaired bidirectional signaling through alpha IIb beta 3.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

Circulating plasma cells detected by flow cytometry as a predictor of survival in 302 patients with newly diagnosed multiple myeloma

We detected circulating plasma cells (PCs) by flow cytometry in 302 patients with newly diagnosed multiple myeloma (MM) by gating on CD38+CD45- cells. The number of circulating PCs per 50 000 mononuclear cells was reported. In 80 (27%) patients, no circulating PC were seen; 106 (35%) patients had 1 to 10 and 115 (38%) patients had more than 10 circulating PCs. Median overall survival for the 302 patients was 47 months. Patients with 10 or fewer circulating PCs had a median survival of 58.7 months, whereas patients with more than 10 circulating PCs had a median survival of 37.3 months (P = .001). On multivariate analysis, the prognostic value of circulating PCs was independent of beta2-microglobulin, albumin, and C-reactive protein. There was only a weak correlation between tumor mass and circulating PCs, suggesting that the appearance of circulating PCs may be a reflection of tumor biology. We conclude that the number of circulating PCs measured by flow cytometry in patients with newly diagnosed MM is an independent predictor of survival.     

Panniculitis–an unusual cutaneous manifestation of systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune systemic disease characterized by small vessel involvement that leads to tissue ischemia and fibroblast stimulation resulting in accumulation of collagen (fibrosis) in the skin and internal organs. Lipomembranous panniculitis is a peculiar type of fat necrosis and has been reported with clinical conditions, commonly with peripheral vascular diseases. We describe a case of a 43‐year‐old woman with SSc manifestations, who presented with black scaly skin plaques, associated with thickening of the subcutaneous fat tissue, on the lateral surface of her thighs, her calves, gluteal area and lower abdomen. Biopsy revealed lipomembranous panniculitis. Lipomembranous changes have been seen in connective tissue disorders such as lupus profundus, morphea, systemic sclerosis and panniculitis associated with dermatomyositis, but rarely in thighs, calves, gluteal area and lower abdomen. Almeida MSTM, Lima SCB, Carvalho LL, Almeida JVM, Santos LG, Rolim JRA, Rocha TE. Panniculitis–An unusual cutaneous manifestation of systemic sclerosis.     

Outcomes and treatments of patients with immunoglobulin light chain amyloidosis who progress or relapse postautologous stem cell transplant

Immunoglobulin light chain (AL) Amyloidosis is a condition whereby misfolded proteins generated by plasma cells deposit in tissues causing organ dysfunction. Chemotherapy and autologous stem cell transplant when eligible are standard treatment options. Several studies report long‐term outcomes of patients post‐transplant. However, there is a paucity of literature describing outcomes of relapsed patients post‐transplant. We performed a retrospective study to assess outcomes and therapies employed upon relapse after transplant. Between 1996 and 2009, 410 patients received transplant at the Mayo Clinic as first‐line therapy. Of those patients, 42 (10%) died within 3 months of transplant, 64 (16%) died without documented relapse, 158 (38%) were alive without documented progression, and 146 (36%) had documented progression. Those 146 patients are the subject of our study, and their median time to hematologic relapse/progression was 23.6 months (95%CI 18.3, 26.3 months). Their median overall survival and 5‐yrs overall survival from post‐transplant relapse/progression was 51.7 months (95%CI 34.1–62.3) and 39%, respectively. The most common first regimen for treatment after relapse was lenalidomide or thalidomide. In conclusion, our study indicates that patients with AL amyloidosis fare well post‐transplant relapse/progression. Additionally, it provides a yardstick to design clinical trials to determine best treatment options.