Plasma Cell Myeloma Residual Disease Quantitation Using a Next-Generation Sequencing-Based IGH Clonal Rearrangement Assay with the Aid of a "Spike-in" Clonal Sequence

Introduction: Next-generation sequencing (NGS)-based IGH clonal rearrangement assays can characterize and subsequently track disease-associated clonal sequences for lymphoid and plasma cell neoplasms, even at very low levels. As IGH PCR primers are used, the detected clonal sequences are usually reported as % of sequencing reads, roughly corresponding to % of B and plasma cells (PC) in samples, rather than % of total cellularity, hampering accurate disease burden assessment. In this study, we evaluated a method for calculating residual disease burden as % of total cellularity, with the aid of adding a known quantity of "spike-in" clonal sequence to the samples, and compared to concurrent 10-color flow cytometry (FC) quantitation of abnormal PC. Methods: DNA was extracted from 40 plasma cell myeloma patient marrow biopsies sent for disease monitoring purposes at Memorial Sloan Kettering Cancer Center (MSKCC), with previously-characterized clonal sequences specific to the patients' myelomas. All samples had concurrent FC analyses and aspirate differential counts performed. 100 cell equivalent of DNA with a known clonal sequence (LymphoQuant®, LQ) was added to 700ng of patient DNA (~100,000 cell equivalent), and testing was performed using LymphotrackTM, a NGS-based assay. Following PCR amplification using IGH FR1 primers, sequencing was performed on the Illumina MiSeqTM instruments at the molecular laboratory of MSKCC. Reproducibility studies were conducted on a subset of samples at the laboratory of Invivoscribe, Inc. using identical methodology. LymphoTrack MRD data analysis tool (MRDDAT) v.1.0.3 was used to search for both the myeloma-specific and LQ clonal sequences. Disease as # of cell equivalent was calculated as: (% reads for myeloma clonal sequence/% reads for LQ) X 100 cells. Disease as % of total cellularity was calculated as: (# of cell equivalent/100,000 cells) X 100%. Results: Disease as % of total cellularity calculated by LQ showed a median of 0.7576% cells (range: 0.000614% to 39.89%), compared to abnormal PC as % of total WBC by FC with a median of 0.355% cells (range: 0.00061% to 44.70%). Overall, a good correlation between disease quantitation by LQ and FC could be observed for cases with ≤10% total PC by aspirate count (r=0.79), while the correlation is lower for cases with >10% total PC (r=0.51). 12/40 samples were tested in two different laboratories, and showed excellent correlation in disease quantitation by LQ (r=0.94). As expected, detectable clonal sequences as % of sequencing reads (rather than as % of total cellularity) showed poor correlation with FC quantitation (r=0.32), due to variability of total B and plasma cell content in different samples. Conclusions: Disease as % of total cellularity calculated with the aid of a known "spike-in" sequence in the NGS-based assay showed good correlation with the quantitation of abnormal PC by FC, when total PC was ≤10% by aspirate count. The correlation between the two declines when total PC was >10%. When patient samples contain a high number of B and/or plasma cells, the PCR amplification efficiency of the very small amount of the admixed "spike-in" clonal sequence may be hampered, affecting accurate quantitation. Furthermore, FR1 primers may not anneal optimally to some patients' clonal sequences due to somatic hypermutations in binding sites, underestimating the % of disease clone. Utilization of a second "spike-in" sequence and other primer sets (FR2, FR3) may improve disease % calculations in some cases. Disclosures Ho: Invivoscribe, Inc.: Honoraria. Roshal:Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Huang:Invivoscribe, Inc.: Employment. Hutt:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment. Landgren:Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Merck: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria.

A proposal for cellularity assessment for EGFR mutational analysis with a correlation with DNA yield and evaluation of the number of sections obtained from cell blocks for immunohistochemistry in non-small cell lung carcinoma

AimsDifferent approaches have been described for reporting specimen adequacy for epidermal growth factor receptor (EGFR) mutation analysis. We aimed: (1) to conduct cellularity assessment and to investigate its association with DNA yield, (2) to compare the H&E slides taken before and after the thick sections (curls) obtained for EGFR testing and (3) to evaluate the number of ancillary studies performed.MethodsCell block (CB) slides of 110 non-small cell lung carcinoma cases submitted to EGFR analysis from 2010 to 2012 were reviewed for total cellularity (ranges 1–100, 100–250, 250–500, 500–750, 750–1000 and >1000 cells), tumour cellularity (ranges 1–50, 50–100, 100–300 and >300 cells) and the percentage of tumour cells. Precurl and postcurl H&E slides were compared using the three criteria. The number of immunohistochemistry (IHC) markers and special stains and DNA yield were recorded.ResultsDNA yield was significantly associated with the total cellularity, number and percentage of tumour cells. For 46 cases with precurl and postcurl slides, only three (6.5%) were classified as being different and in two of them the postcurl slide had greater cellularity than the precurl. IHC was performed in 83 cases, with a minimum of 1 and a maximum of 11 markers (median of 3) per case.ConclusionsAn association between the total cellularity and the tumour cellularity with the DNA yield was demonstrated using the ranges described. Evaluation of a postcurl slide is an unnecessary practice. The majority of the CB had sufficient material for ancillary studies (up to 11 markers) and mutation testing.

Perturbed Hematopoiesis In Mice Lacking ATMIN (an ATM co-Factor)

ATM (Ataxia telangiectasia mutated) kinase plays important roles in hematopoiesis. Two co-factors participate in ATM activation in a signal-dependent manner: NBS1 during ionising radiation induced DNA damage and ATMIN under hypotonic stress or chloroquine treatment, presumably due to changes in chromatin structure. Such artificial treatments might not have a genuine biological significance. However, we have recently shown a potential role for ATMIN in B cells. CD19-Cre mediated deletion of Atmin led to impaired class switch recombination, greatly increased genomic instability and consequently the development of B cell lymphomas. Considerable (but not total) overlap in phenotypes was found in ATM and ATMIN deficient B cells suggesting that ablation of ATMIN alters ATM function. As Atm-/- HSCs have impaired quiescence, maintenance and reconstitution capacity due to elevated reactive oxygen species, we sought to investigate if the absence of ATMIN would affect the primitive hematopoietic compartment. We used a Vav-Cre approach to delete Atmin to test ATMIN’s functions in the hematopoietic system (simply referred as AtminΔ/Δ). At steady state, AtminΔ/Δ mice have ∼75% reduction in total splenocytes as compared to control mice. This is mainly due to reduced B cell, but also T cell, numbers. However, AtminΔ/Δ mice do not develop B cell lymphomas but they present differentiation block at the pre-B cell and at the immature/mature re-circulating B (IgM+/hi) cell stages with more than a 2-fold increase in apoptosis in these cell fractions compared to controls. The lack of lymphoma development in these mice suggests that the lack of ATMIN would also affect the more primitive compartment (e.g. CLP), not allowing tumorigenic clones to accumulate. Indeed, in the bone marrow (BM), a ∼40% reduction in total cellularity was observed in ATMIN deficient mice compared to littermate controls at 8-12 wks of age. This reduction in total cellularity is reflected across the board, from LT-HSCs (LSK/CD34-/lo/Flt3-) to the more mature cells and, more evidently, B cells. Apart from a similar B cell maturation block seen in the spleen, the frequency of CMPs (LS-K/IL7R-/CD34+/FcγR−) and GMPs (LS-K/IL7R-/CD34+/FcγR+) were reduced by 3- and 2-fold, respectively, in ATMIN deficient mice. The frequencies of other primitive compartments were not different in ATMIN deficient mice vs controls, including the CLP (LS-Klo/IL7R+) fraction. This reduction in CMP and GMP fractions translated into a ∼50% reduction in CFU capacity. Interestingly, only LT-HSCs, ST-HSCs and MPPs were found to be more cycling in the absence of ATMIN, suggesting a compensatory mechanism due to the reduction in some of the compartments downstream of LSK. Unlike in B cells, no increase in apoptosis was found in any other ATMIN deficient BM cell types, implying that the function of ATMIN is dependent on the context of the cell. When transplanting similar numbers of ATMIN or control LT-HSCs, the regenerative capacity of AtminΔ/Δ-Vav-Cre cells was reduced by ∼16-fold in peripheral blood at 16 wks post-transplantation, with an early pronounced ∼10-fold reduction in B220+ cell production that was maintained over time. We found a steady decline in the production of myeloid cells over time, reaching a ∼13-fold difference between AtminΔ/Δ-Vav-Cre and control transplanted cells. However, in secondary recipients transplanted with a similar number of LSK cells, the regenerative capacity of AtminΔ/Δ-Vav-Cre cells was only further reduced to ∼24-fold as compared to control cells at 16 wks post-transplant. Interestingly, when transplanting ST-HSCs, the regenerative capacity of this fraction was even more drastically reduced, reaching ∼51-fold difference between AtminΔ/Δ-Vav-Cre and control cells. Contrary to our predictions, oxidative stress does not seem to play any evident role in reducing CMP and GMP fractions. Interestingly, however, we observed a mild but significant phosphorylation of H2AX and a specific ATM substrate, KAP1, in only these myeloid progenitors suggesting activation of ATM signaling. Further studies are on going to test whether this to due to replicative stress. Overall, our studies highlight that ATMIN appears to have different roles in different cells and potential novel ATM-independent functions. Certainly, in the absence of ATMIN, hematopoiesis is severely altered, directly and/or indirectly affecting the function of LT-HSCs. Disclosures: No relevant conflicts of interest to declare.

Complete Hematologic and Cytologic Remission in M6A Erythroleukemia Using Decitabine Hypomethylation Monotherapy

Hypomethylating drugs (e.g. Azacitadine, Decitabine) are becoming increasingly attractive choices for the treatment of acute myelogenous leukemia (AML), particularly in populations deemed inappropriate for standard anthracycline-based induction chemotherapy (i.e., 7+3), such as the elderly. Acute erythroleukemia (French-American- British M6) is a rare, heterogeneous disorder involving increased red cell precursors and myeloblasts, that accounts for 3–5% of de novo AML and 20–30% of secondary AML. Three subsets have been described: M6A (myeloblast-rich erythroleukemia); M6B (proerythroblast-rich erythroleukemia); and M6C (myeloblast- and proerythroblast-rich mixed variant). Little data exists on the efficacy of hypomethylation therapy in this uncommon subtype of AML. We report the rapid induction of a complete hematologic and cytologic remission in a patient with the M6A variant erythroleukemia using decitabine monotherapy. A 75 year-old white male with a history of coronary artery disease, was admitted for elective cardiac catheterization because of recurrent exertional chest pain. A pre-catheterization complete blood count (CBC) revealed pancytopenia with a white blood cell (WBC) count of 1400/mm3, hemoglobin of 6.2 g/dL (mean cell corpuscular volume of 113 fL), and a platelet count of 51,000/mm3. The differential showed neutropenia, but was otherwise normal with no circulating blasts. Blood transfusion completely relieved his chest pain, and his catheterization was uneventful. A bone marrow aspirate and biopsy demonstrated a hypocellular marrow (15–20%) with erythroid hyperplasia (70% of total cellularity) and increased blasts (10% of total cellularity, 30% of non-erythroid cells), most compatible with acute M6A erythroid/myeloid leukemia. Cytogenetic studies revealed a normal male karyotype, but a single cell with a 5q- deletion. Subsequent fluorescent in situ hybridization (FISH) analysis using an LSI DNA probe (Vysis Inc.) for detection of the EGR1 gene on chromosome 5q, failed to confirm the 5q deletion, consistent with normal cytogenetics. Because of the patient’s advanced age and co-morbidities, induction with hypomethylation monotherapy was begun with intravenous decitabine (Dacogen®) 20 mg/m2 daily for five consecutive days, repeated every four weeks. Growth factor support (Neupogen® 375 mcg daily) and antibiotic prophylaxis (ciprofloxaxin, acyclovir, fluconazole) were only required after the first cycle of decitabine because neutropenia did not occur with subsequent cycles. WBC and platelet counts normalized shortly after cycle one, while the hemoglobin normalized by cycle five. An interim bone marrow aspirate/biopsy was repeated after cycle three, showing normocellularity (30%), and no evidence of residual leukemia. Other than a localized herpetic reactivation following cycle one, the patient has tolerated hypomethylation therapy extremely well. He has now completed eight cycles of therapy, and remains in complete remission. Epigenetic alterations (such as DNA methylation) may play a role in AML leukemogenesis by inducing the inhibition of tumor suppressor genes. Recent studies have also shown that high estrogen receptor-α and p15INK4B methylation levels in AML patients in clinical remission, were associated with a high risk for leukemia relapse and poor relapse-free survival. However, little data exist for epigenetic dysregulation in the etiology of human erythroleukemia. Cell culture studies using human erythroleukemia cell lines indicate that DNA methylation may have functional consequences for gene activity, including globin gene expression and cellular differentiation. The current case indicates a potential role for DNA methylation in the pathogenesis of human erythroleukemia, and argues for further investigation into this potential mechanism of leukemogenesis. Future studies will include the effect of hypomethylating compounds on the growth and differentiation of human erythroleukemia cells, and the evaluation of DNA methylation levels in bone marrow biopsy samples from M6 patients.

Cul4A Is Required for Cell Viability and Its Deficiency in Hematopoietic Cells Causes Apoptosis and Is Fatal.

Critical regulators of hematopoiesis are controlled by ubiquitin-mediated proteolysis. Cul4A encodes a core subunit of one ubiquitin ligase, and previous results with hematopoietic cell lines and with Cul4A haploinsufficient mice indicate that Cul4A is required for hematopoietic stem cell function and to maintain the homeostasis of progenitors, precursors, and mature hematopoietic cells. Because Cul4A-deficiency is embryonic lethal, we generated Cul4A conditional knockout mice to examine the requirement of Cul4A for hematopoiesis in adult mice. A mutant Cul4A allele (Cul4Aflox) was constructed where its first coding exon was flanked by loxP sites. Transgenic mice with this mutant allele and the interferon-inducible Cre transgene, Mx1-Cre, were derived. When deletion of Cul4A was induced in Cul4Aflox/flox Mx1-Cre mice, the animals died within 3–10 days of the beginning of induction. Necropsies performed four days after the beginning of induction showed that all of the tissues where Mx1-Cre was reported to be expressed appeared normal, except the bone marrow, spleen, and small intestine. The red pulp in the spleen was diminished, there were many fewer nucleated cells in the bone marrow, and the microvilli of the small intestine (duodenum) were dramatically shortened. The mass and total cellularity of mutant spleens were half of controls (Cul4Aflox/flox mice without Mx1-Cre), and bone marrow total cellularity was one-tenth of controls. The frequency of mutant hematopoietic progenitors was reduced 3800-fold in the bone marrow and 80-fold in the spleen. Peripheral blood counts of mature myeloid and lymphoid cells were also dramatically reduced. To separate the in vivo effects of Cul4A-deficiency in hematopoietic cells from those in other cell types, conditional mutant bone marrow was transplanted into wild type recipients, these cells were allowed to engraft for 2 months, and then Cul4A deletion was induced. Mutant animals died within 9–11 days of the beginning of induction with bone marrow nearly empty of cells, spleens only 29% the mass of controls, myeloid and lymphoid counts in the peripheral blood reduced to nearly zero, hematocrits at only 21% of controls, and platelet counts at only 10% of controls. The small intestine, however appeared normal, indicating that Cul4A-deficiency in hematopoietic cells is sufficient to cause death. To examine the fate of Cul4A-deficient hematopoietic cells, deletion was induced in vivo in Cul4Aflox/flox Mx1-Cre and control mice, and then bone marrow was harvested and cultured in vitro. Apoptotic cells were detected (either Annexin V positive, 7-AAD negative or TUNEL positive cells) 2–5 days after induction. At 4 and 5 days after induction, the frequency of apoptotic mutant cells was significantly greater than controls (P=0.01 and 0.03, respectively), and at 5 days the frequency of TUNEL positive cells was 4.5-fold greater in the mutant cells. Together, these results indicate that Cul4A-deficiency in hematopoietic cells results in apoptosis, a failure of the hematopoietic system, and death. Analyses of how the expression levels of Cul4A target proteins are altered by Cul4A-deficiency will be presented.

Use of spleen organ cultures to monitor hemopoietic progenitor cell regeneration following irradiation and marrow transplantation

After lethal irradiation of C57BL mice followed by the injection of 10(7) marrow cells, total cellularity and progenitor cell levels exceeded pretreatment levels within 12 days in the spleen, but regeneration remained incomplete in the marrow. The exceptional regenerative capacity of progenitor populations in the spleen was observed in organ cultures of spleen slices prepared 24 hr after irradiation and transplantation, excluding continuous repopulation from the marrow as a significant factor in splenic regeneration.

Use of spleen organ cultures to monitor hemopoietic progenitor cell regeneration following irradiation and marrow transplantation

After lethal irradiation of C57BL mice followed by the injection of 10(7) marrow cells, total cellularity and progenitor cell levels exceeded pretreatment levels within 12 days in the spleen, but regeneration remained incomplete in the marrow. The exceptional regenerative capacity of progenitor populations in the spleen was observed in organ cultures of spleen slices prepared 24 hr after irradiation and transplantation, excluding continuous repopulation from the marrow as a significant factor in splenic regeneration.

Normal Bone Marrow Total Cell and Differential Values by Quantitative Analysis of Particle Smears

1. A method is described which permits absolute, area-related bone marrow counts for the total nucleated cells and differential count. The technic is easy, the accuracy satisfactory. 2. Normal values for the marrow of young adults (18-45 years) are given. 3. The method permits comparison of any marrow cells independent from changes of the total cellularity, and it makes serial comparative marrow studies in diseases useful, especially in cases where the cellularity is greatly altered (aplasia, leukemia) and percentage-differential counts are almost impossible to judge properly. 4. Experiments in animals may be followed by absolute marrow counts, without alteration of the cells such as occurs in pipet- and shaking-methods. Experiments with ionizing radiation5 showed clearly the practical value of this technic. 5. A survey is given on some total cell counts in disease, showing the range of pathologic changes to be determined with the technic described. The range is from about 80,000 (in marrow atrophy) to 1.5 million (in small-cell leukemia).

The Effect of Cortisone on Cell Proliferation and Migration in Peripheral Nerves undergoing Wallerian degeneration

The effect of cortisone on the proliferation of tissues other than mesenchymal has received little attention. Bullough (1952) showed that cortisone had a marked effect on the mitotic activity of the epidermis in mice, and Leroy (1952) showed a similar effect on immature testes of rats, but no effect on mature ones. McColl & Weston (1953) have studied the influence of cortisone on the process of Wallerian degeneration in peripheral nerve and noted that there was less total cellularity in degenerated nerves from cortisone-treated animals. It was thought useful to undertake a quantitative study of proliferation during Wallerian degeneration in peripheral nerves from cortisone-treated animals because the normal changes in nuclear population during this process have been intensively investigated (Abercrombie & Johnson, 1946; Thomas, 1948), and it is known that different types of cells multiply at different rates.