Kaposi's sarcoma-associated herpesvirus gene sequences are detectable at low copy number in primary amyloidosis.

Primary amyloidosis (AL), like multiple myeloma (MM), results from a clonal proliferation of plasma cells. Recent detection of Kaposi's sarcoma-associated herpesvirus (KSHV) gene sequences in MM patients, although controversial, suggested that KSHV may also be present in AL. In the present study, we assayed for KSHV gene sequences in patients with primary AL independently in 2 laboratories. Nested polymerase chain reaction (PCR) was performed on DNA isolated from 21 bone marrow (BM) core biopsy samples to amplify orf26 and orf72, 2 regions of the KSHV genome. Eighteen of 21 (86%) BM core biopsy samples were KSHV PCR positive. BM aspirates from 16 of these 21 AL patients were cultured for 4-6 weeks to generate long term bone marrow stromal cells (LT-BMSCs), and 13 of 16 (81%) LT-BMSCs were also KSHV PCR positive. Results in all but 1 sample were consistent in the 2 laboratories. Sequencing of the PCR products in the 2 laboratories confirmed 94-98% and 95-98% homology to the published orf 26 and orf 72 KSHV gene sequences respectively, with interpatient base pair differences. Despite the presence of KSHV gene sequences, only 4/18 (22%) KSHV PCR positive patients demonstrated KSHV lytic antibodies by immunoblot assay. A sensitive assay performed on the BCBL-1 cell line confirmed the presence of KSHV at a very low copy number in AL. PCR using patient specific light chain gene primers also amplified DNA isolated from 2 AL BM core biopsies and 3 AL LT-BMSCs which were KSHV PCR positive, suggesting the presence of clonotypic cells. Our results therefore demonstrate KSHV gene sequences albeit at a very low copy number in the majority of BM core biopsies and LT-BMSCs from AL patients, and serological responses in only a minority of cases. Ongoing studies to identify viral transcripts and gene products will determine the biological relevance of KSHV in AL disease pathogenesis.

Clonal immunoglobulin light chain variable region germline gene use in AL amyloidosis: association with dominant amyloid-related organ involvement and survival after stem cell transplantation.

AL (primary or immunoglobulin light chain) amyloidosis (AL) differs from myeloma per se in that tissue deposits of amyloid are found, typically in association with small numbers of clonal plasma cells producing lambda light chains, and also in that AL patients typically present with a predominantly dysfunctional organ-system. This constellation of features - fibrillar deposits comprised of light chains, lambda light chain predominance, and organ-system tropism and dysfunction - remains unexplained. Select patients with AL respond to haemopoietic stem cell transplantation (SCT) with clinical improvement and extended survival, particularly those who do not have cardiac involvement. In order to ascertain whether the organ-system tropism of AL was associated with immunoglublin light chain variable region (Ig VL) germline gene utilization, we attempted to clone, sequence and assign germline donors to the clonal Ig VL genes of 62 AL patients, all of whom were treated with SCT. We succeeded in 39 cases, identifying clonal AL genes derived from donors of the lambdaI (n = 10), lambdaII (n = 5), lambdaIII (n = 6), lambdaVI (n = 11) and KI (n = 7) subtypes. The majority of the donors (IGLV6S1, DPL5, DPL2, DPL23 and LFVK431) were genes that appear in the expressed repertoire <5% of the time, suggesting an intrinsic propensity to form amyloid under certain conditions. Patients whose clones derived from the lambdaVI IGLV6S1 donor uniformly presented with dominant renal involvement while those with other Vlambda or unknown donors often had dominant cardiac or other organ involvement, particularly patients whose clones derived from the lambdaI DPL2 donor. In addition, both early (<3 months) and overall post-SCT survival were significantly better in lambdaVI IGLV6S1 patients compared to patients with other Vlambda donors. These findings indicate that there are important associations in AL amyloidosis among Ig VL gene utilization, organ-system tropism and post-SCT survival.

Identification of a novel substitution in the constant region of a gene coding for an amyloidogenic kappa1 light chain.

Current concepts regarding the association between immunoglobulin (Ig) light chain structure and AL amyloidosis (AL) emphasize Ig variable region amino acid substitutions because the majority of light chain amyloid fibrils that have been sequenced contain amino termini of the variable region with only small amounts of the constant region. In this report, we describe a patient with rapidly progressive AL whose amyloid deposits contained primarily monoclonal kappa light chain constant region fragments. We sequenced and analyzed this AL protein, determining that it was an O18-O8 kappa1 variant and that the constant region possessed an unusual Ser-->Asn substitution at position 177. Using pre-mortem bone marrow cells, we cloned and sequenced the cDNA for this AL protein (HCAK1) and, using DNA from post-mortem somatic tissue, we cloned and sequenced the patient's kappa germline O18-O8 donor and kappa constant region (Ckappa) gene segments. The cDNA that coded for HCAK1 contained a variable region that was derived from O18-O8, showing 96.1% homology to germline, and a Ckappa that had a nucleotide substitution (AGC to AAC), resulting in the 177Ser-->Asn replacement. Two Ckappa genes were cloned from somatic tissue DNA, one identical to a known Ckappa sequence and another containing this substitution which likely is a new Ckappa allotype. Our findings indicate that further investigation is warranted into the contributions genetic polymorphisms and light chain constant regions may make to amyloidogenesis.

Mobilized CD34+ cells selected as autografts in patients with primary light-chain amyloidosis: rationale and application.

BACKGROUND: Concern about tumor cell contamination in stem cell preparations has led to the use of CD34+ cell selection as a means of purging. Increasing the number of CD34+ cells per leukapheresis may help to provide an adequate dose of CD34+ cells. STUDY DESIGN AND METHODS: The reverse transcriptase polymerase chain reaction (RT-PCR) was employed to clone overexpressed clonotypic immunoglobulin light-chain variable region genes (Ig VL) from bone marrows of patients with primary light-chain amyloidosis (AL). Patient-specific primers were designed to evaluate stem cell collections for contamination. CD34+ cell selection was performed on components from AL patients who underwent mobilization with granulocyte-colony-stimulating factor (G-CSF) (filgrastim; 16 microg/kg/d for 4 days) and collection by large-volume leukapheresis (LVL;25L) on Days 4 and 5. The selected cells alone were transfused after patients received mephalan (200 mg/m2). RESULTS: Contamination was found in collections from 4 to 7 patients, which provided the rationale for a subsequent trial of CD34+ cell selection. The median number of CD34+ cells per kg collected on Days 4 and 5, and in toto, was 4.0 x 10(6)(1.1-12.7), 7.9 x 10(6)(1.8-12.7), and 10.7 x 10(6)(2.9-25.4), respectively (n = 9 patients). The median yield per selection was 38 percent, with a purity of 85 percent (45-97%), and the viability of CD34+ cells averaged 96.4 +/- 3.6 percent (n = 18 selections). The median number of CD34+ cells infused was 5.9 x 10(6) per kg (2.1-10.1). In comparison with AL patients given unselected autografts, patients receiving selected CD34+ cells experienced similar reconstitution of neutrophils and platelets but slower lymphocyte recovery. CONCLUSION: Patients with AL often have contamination with clonotypic cells in their blood autografts. G-CSF mobilization and LVL provide components that allow the selection of adequate doses of CD34+ cells. The use of CD34+ cells in patients with AL achieves rapid neutrophil and platelet recovery but delayed lymphocyte recovery. CD34+ cell selection is feasible in the treatment of AL, but its effectiveness in purging clonotypic cells remains to be ascertained.