Molecular Quantitation of Minimal Residual Disease in Multiple Myeloma Patients Achieving Complete Remission after Treatment with Revlimid or Velcade.

In multiple myeloma (MM), new therapeutic strategies utilize the proteasome inhibitor bortezomib (Velcade) and the thalidomide immunomodulatory analog lenalidomide (Revlimid). While promising, MM invariably recurs, necessitating a better understanding of the malignant cells that persist during periods of complete remission (CR). In MM, minimal residual disease (MRD) is a valuable prognostic indicator for predicting time to relapse. There are no published studies to quantify the MRD or malignant cells that resist Velcade or Revlimid. In MM, the uniquely rearranged IgH VDJ gene provides a molecular signature. PCR strategies incorporating amplification of genomic DNA using patient specific primers provide a means to quantify MRD in MM patients. To date, the patient cohort tested included three MM patients achieving CR as part of a randomized trial for Revlimid plus dexamethasone, one MM patient with CR in response to dexamethasone (dex) alone, and two MM patients achieving CR after Velcade. All of the CR patients had clonotypic VDJ mRNA transcripts in BM by RT-PCR. Analysis of genomic DNA (gDNA) quantifies the number of malignant cells, as each cell has only one copy of the MM IgH VDJ. Three strategies were employed to quantify the extent of minimal disease during CR to these agents: semi-quantitative PCR analysis of purified gDNA from BM aspirates, quantitative realtime PCR using SYBR Green, and PCR analysis of cells captured from BM aspirate slides (laser pressure catapulting). 1) The semi-quantitative method measures the amount of gDNA template in the PCR reaction required to attain a positive signal, independent of cell type or morphology. 2) The quantitative PCR confirms method 1 by plotting rate of amplification of gDNA for a control sequence as compared to the clonotypic sequence, to calculate the frequency of all clonotypic MM cells in BM. 3) Laser pressure catapulting of BM cells from slide preparations provides a measure that most closely approximates the in vivo situation because it does not introduce potential artifacts arising from purification of cells and DNA that could confound interpretation of the results, and enables morphological identification of the cell types harboring clonotypic IgH VDJ genes. BM aspirate slides were viewed and numerous small patches of cells (8–15 cells) were captured into individual tubes for PCR analysis. Overall, we found that CR patients treated with Revlimid+ Dex have a 100 fold lower frequency of clonotypic MM cells (requiring 1.59e5 cells to detect a positive signal) than did the MM patient in CR from dex (detectable in 1.01e3 cells), indicating that Revlimid substantially reduced, but did not eradicate the malignant clone. Furthermore, for one patient, for whom sequential BM samples were available, the clonal frequency continued to decrease over time. Patients in CR after Velcade treatment have MM cells detectable in 1.32e4 cells. Both therapies exert more depletion as compared to dex alone. Larger patient cohorts are being analyzed to further quantify levels of MRD achieved in response to these agents.

Spore morphotypes of Thelohania solenopsae (microsporidia) described microscopically and confirmed by PCR of individual spores microdissected from smears by position ablative laser microbeam microscopy

Development of Thelohania solenopsae, a parasite of the red imported fire ant (Solenopsis invicta), until recently was thought to include formation of two types of spores: unicellular meiospores, maturing inside sporophorous vesicles in sets of eight (octospores); and Nosema-like binuclear free spores. Megaspores, discovered in 2001, develop primarily in alates and are morphologically distinct from the two previously known types of spores. The role of megaspores in the T. solenopsae life cycle, as well as their existence, has been questioned. The current research includes light and electron microscopic descriptions of the three major spore morphotypes characteristic of T. solenopsae development. In addition, individual octospores and megaspores were isolated into groups of 8–20 from methanol-fixed and Calcofluor-stained smears of the infected ants for subsequent PCR analysis by the laser pressure catapulting function of a position ablative laser microbeam microscope, a technique applied for the first time to research of microsporidia. The PCR-amplified SSU rDNA nucleotide sequences from octospores and megaspores were identical. This, along with the consistency with which megaspores are detected in infected ants, demonstrates that megaspores are integral to the life cycle of T. solenopsae.