ICCS Portland Plenary Session IV:  Hematopathology.
Chair: Joe DiGiuseppe

Plenary session IV, chaired by the new president of ICCS, Dr. Joe DiGiuseppe,
introduced several topics in the overlapping areas of flow cytometry and hematopathology. The session provided 4 exciting presentations that all pushed forward the frontier at which these disciplines intersect.  With their presentations, Dr. Goolsby and Dr. Irish highlighted the utility of new strategies for thinking about neoplasms including AML and lymphoma by using response to a stimulus to identify neoplastic cells or predict disease course. Dr. Campana described the discovery process involved in identification of novel markers to improve MRD detection in ALL and Dr. Head challenged our traditional view of MDS.

The first speaker, Dr. Dario Campana of the National University of Singapore, discussed minimal residual disease (MRD) testing in acute lymphoblastic leukemia (ALL) with an emphasis on the utility and discovery of newly identified immunophenotypic markers for detection of MRD in ALL.  Dr. Campana began his presentation by noting that MRD is the most powerful prognostic factor in childhood ALL and reviewing principles for MRD detection in both B cell and T cell ALL at various time points post therapy.  For T cell ALL, detection of immature T cells in the blood and marrow is considered abnormal, however, the situation is more complex for B cell ALL.  Detection of MRD in B cell ALL early after a steroid containing chemotherapeutic regimen may be performed using a limited panel 1.  However, because B lymphoid blasts may share some overlapping immunophenotypic features with early B cell precursors (hematogones), MRD detection for B cell ALL at time points post induction chemotherapy, when hematogones are normally present in the marrow, can be difficult.    To this end, Dr Campana described work conducted in his laboratory to detect new potential markers for MRD detection in ALL using gene expression array analysis.  Candidate genes noted to be highly differentially expressed in ALL by gene expression arrays were validated for differential protein expression using flow cytometry.  The new markers discovered in this study allowed for more precise detection of MRD in cases with either no, or only one, marker aberrantly expressed using current methodology.  The findings of Dr. Campana and colleagues, based primarily on work conducted at St. Jude Children’s Research Hospital, are elegantly outlined in a recent publication 2.  

The second speaker of the session was Dr. Charles Goolsby of Northwestern University who discussed signaling in normal marrow and acute myeloid leukemia (AML).  Dr. Goolsby’s presentation built on his exciting discussion of cell signaling in normal marrow given on Sunday evening when he accepted the Wallace H. Coulter Foundation award.  Dr. Goolsby and colleagues studied the response of several phospho proteins to stimulation by a variety of cytokines or growth factors and established patterns in normal progenitor and myelomonocytic populations.  Similar studies were conducted on blast populations in samples from patients with AML, and differences in responses to stimuli were characterized.    Patients with AML showed abnormal responses to stimuli which fell into categories including 1) altered constitutive activation, 2) enhanced or reduced response to stimulation by a cytokine or growth factor, and 3) altered kinetics of response.  Interestingly, such alterations in cytokine/growth factor response appear to be stable, suggesting that this approach may enhance strategies for MRD detection.  Additionally, such responses appear to identify abnormal cells in populations that appear relatively homogeneous using traditional immunophenotypic markers.  Readers interested in exploring Dr. Goolsby's work further are referred to the following recent publications 3, 4.

Speaker three, Dr. David Head of Vanderbilt University Medical Center presented a new perspective on the myelodysplastic syndromes (MDS) with an emphasis on work his laboratory has conducted in quantitative assessment of myeloid nuclear differentiation antigen (MNDA) expression and flow cytometric assessment of expression of cell cycle antigens, DNA damage, and apoptosis that challenge the conventional model of MDS pathogenesis.  MNDA is a nuclear protein expressed in hematopoietic cells that is thought to be involved in regulation of programmed cell death (PCD).  Dr. Head began by demonstrating by flow cytometry that MNDA shows decreased expression in patients with MDS5, thereby confirming prior gene expression array data suggesting decreased expression of MNDA in MDS.   As this protein is thought to be involved in PCD, demonstration of decreased expression led to a discussion of the traditional paradigm of MDS.  MDS is often characterized as a disease, which, at least in early stages, is associated with both increased proliferation and increased PCD.  Dr. Head noted several problems with this traditional pathogenic paradigm and spent some time presenting data that challenges this model 6.  Specifically, Dr, Head described studies conducted in his laboratory demonstrating that hematopoietic cells from patients with MDS demonstrated no increase in mitoses or expression of Annexin V, a marker of PCD.  Patients with MDS did however have an increase in cells in G2, suggesting a delay in passage through G2, which was attributed to increased DNA damage.  These findings argue against the traditional paradigm of MDS and strengthen Dr. Head’s proposition that rather than increased apoptosis, MDS is characterized by increased DNA damage resulting in delayed passage of marrow cells though the cell cycle.  

The final presentation of the session was given by Dr. Jonathan Irish and reviewed data conducted by his group at Stanford University demonstrating how B cell signaling networks reveal a negative prognostic human lymphoma subset that emerges during tumor progression.  Similar to Dr. Goolsby, Dr. Irish’s work was based on measuring response of various phospho proteins to a stimulus.  In this case, cells from patients with follicular lymphoma were stimulated with agents including some that elicit a response via the B cell receptor.  Interestingly, sequential studies demonstrated that over time, an expanded population of cells that lacked a response to stimuli was detected.  These cells were designated lymphoma negative prognostic (LNP) cells.   Increases in LNP cells led to an increased risk of death in patients with follicular lymphoma.  The experiments described during this presentation appear to paint a clear picture with respect to prognosis in follicular lymphoma; however, the procedure and data analysis is rather complex.  To this end, Dr. Irish discussed several potential ways to simplify the assay, for instance combining readouts for response to stimuli into a single channel and making use of automated modeling for data analysis.  Dr. Irish’s works is elegantly reported in a recent publication7.

The presenters in this year’s outstanding session on hematopathology have succeeded in challenging and broadening the way we approach hematopoietic disease in the clinical flow cytometry laboratory.
 
References:
1. Coustan-Smith E, Ribeiro RC, Stow P, Zhou Y, Pui CH, Rivera GK, et al. A simplified flow cytometric assay identifies children with acute lymphoblastic leukemia who have a superior clinical outcome. Blood. 2006 Jul 1;108(1):97-102.
2. Coustan-Smith E, Song G, Clark C, Key L, Liu P, Mehrpooya M, et al. New markers for minimal residual disease detection in acute lymphoblastic leukemia. Blood. 2011 Jun 9;117(23):6267-76.
3. Marvin J, Swaminathan S, Kraker G, Chadburn A, Jacobberger J, Goolsby C. Normal bone marrow signal-transduction profiles: a requisite for enhanced detection of signaling dysregulations in AML. Blood. 2011 Apr 14;117(15):e120-30.
4. Woost PG, Solchaga LA, Meyerson HJ, Shankey TV, Goolsby CL, Jacobberger JW. High-resolution kinetics of cytokine signaling in human CD34/CD117-positive cells in unfractionated bone marrow. Blood. 2011 Apr 14;117(15):e131-41.
5. McClintock-Treep SA, Briggs RC, Shults KE, Flye-Blakemore LA, Mosse CA, Jagasia MH, et al. Quantitative assessment of myeloid nuclear differentiation antigen distinguishes myelodysplastic syndrome from normal bone marrow. Am J Clin Pathol. 2011 Mar;135(3):380-5.
6. Head DR, Jacobberger JW, Mosse C, Jagasia M, Dupont W, Goodman S, et al. Innovative Analyses Support a Role for DNA Damage and an Aberrant Cell Cycle in Myelodysplastic Syndrome Pathogenesis. Bone Marrow Research. 2011;2011.
7. Irish JM, Myklebust JH, Alizadeh AA, Houot R, Sharman JP, Czerwinski DK, et al. B-cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression. Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):12747-54.

Sindhu Cherian, MD
University of Washington, Seattle, WA