Identification of Blood-Based Biomarkers for the Prediction of the Response to Neoadjuvant Chemoradiation in Rectal Cancer.

The current standard of care for patients with locally advanced rectal cancer (LARC) is neoadjuvant chemoradiation (nCRT) followed by total mesorectal excision surgery. However, the response to nCRT varies among patients and only about 20% of LARC patients achieve a pathologic complete response (pCR) at the time of surgery. Therefore, there is an unmet need for biomarkers that could predict the response to nCRT at an early time point, allowing for the selection of LARC patients who would or would not benefit from nCRT. To identify blood-based biomarkers for prediction of nCRT response, we performed in-depth quantitative proteomic analysis of pretreatment plasma from mice bearing rectal tumors treated with concurrent chemoradiation, resulting in the quantification of 567 proteins. Among the plasma proteins that increased in mice with residual rectal tumor after chemoradiation compared to mice that achieved regression, we selected three proteins (Vascular endothelial growth factor receptor 3 [VEGFR3], Insulin like growth factor binding protein 4 [IGFBP4], and Cathepsin B [CTSB]) for validation in human plasma samples. In addition, we explored whether four tissue protein biomarkers previously shown to predict response to nCRT (Epidermal growth factor receptor [EGFR], Ki-67, E-cadherin, and Prostaglandin G/H synthase 2 [COX2]) also act as potential blood biomarkers. Using immunoassays for these seven biomarker candidates as well as Carcinoembryonic antigen [CEA] levels on plasma collected before nCRT from 34 patients with LARC (6 pCR and 28 non-pCR), we observed that levels of VEGFR3 (p = 0.0451, AUC = 0.720), EGFR (p = 0.0128, AUC = 0.679), and COX2 (p = 0.0397, AUC = 0.679) were significantly increased in the plasma of non-pCR LARC patients compared to those of pCR LARC patients. The performance of the logistic regression model combining VEGFR3, EGFR, and COX2 was significantly improved compared with the performance of each biomarker, yielding an AUC of 0.869 (sensitivity 43% at 95% specificity). Levels of VEGFR3 and EGFR were significantly decreased 5 to 7 months after tumor resection in plasma from 18 surgically resected rectal cancer patients, suggesting that VEGFR3 and EGFR may emanate from tumors. These findings suggest that circulating VEGFR3 can contribute to the prediction of the nCRT response in LARC patients together with circulating EGFR and COX2.

Genomic and Transcriptomic Characterisation of Response to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer.

Standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (NACRT), followed by surgical resection. However, >70% of patients do not achieve a complete pathological response and have higher rates of relapse and death. There are no validated pre- or on-treatment factors that predict response to NACRT besides tumour stage and size. We characterised the response of 33 LARC patients to NACRT, collected tumour samples from patients prior to, during and after NACRT, and performed whole exome, transcriptome and high-depth targeted sequencing. The pre-treatment LARC genome was not predictive of response to NACRT. However, in line with the increasing recognition of microbial influence in cancer, RNA analysis of pre-treatment tumours suggested a greater abundance of Fusobacteria in intermediate and poor responders. In addition, we investigated tumour heterogeneity and evolution in response to NACRT. While matched pre-treatment, on-treatment and post-treatment tumours revealed minimal genome evolution overall, we identified cases in which microsatellite instability developed or was selected for during NACRT. Recent research has suggested a role for adaptive mutability to targeted therapy in colorectal cancer cells. We provide preliminary evidence of selection for mismatch repair deficiency in response to NACRT. Furthermore, pre-NACRT genomic landscapes do not predict treatment response but pre-NACRT microbiome characteristics may be informative.

Preoperative radiation dose escalation for rectal cancer using a concomitant boost strategy improves tumor downstaging without increasing toxicity: A matched-pair analysis.

PURPOSE: Pathologic complete response to neoadjuvant chemoradiation therapy (CRT) is associated with improved outcomes for patients with locally advanced rectal cancer (LARC). Increased response rates have been reported with higher radiation doses, but these studies often lack long-term outcome and/or toxicity data. We conducted a case-control analysis of patients with LARC who underwent definitive CRT to determine the efficacy and safety of intensified treatment with a concomitant boost (CB) approach. METHODS AND MATERIALS: From 1995 to 2003, a phase 2 protocol examined CRT with 5-fluorouracil and CB radiation therapy (52.5 Gy in 5 weeks) for patients with LARC. Seventy-six protocol patients were matched (case-control approach) for surgery type, tumor (T) stage, and clinical nodal (N) stage with patients who received standard dose (SD) CRT (5-fluorouracil, 45 Gy). A chart review was performed. McNemar's test and Kaplan-Meier analyses were used for statistical analysis. RESULTS: The SD and CB groups did not differ in tumor circumferential involvement and length, but the tumors of CB patients were closer to the anal verge (4.7 vs 5.7 cm; P = .02). Although tumor downstaging was higher in the CB cohort (76% vs 51%; P < .01), pathologic complete response rates did not differ (CB, 17.1% vs SD, 15.8%, P = 1.00). The incidence of grade ≥3 radiation-related toxicities was low and similar in both groups (CB, 10% vs SD, 3%, P = .22). Postoperative (anastomotic leak, wound complications/abscess, bleeding) and late (small bowel obstruction, stricture) complication rates did not differ between the groups (P > .05). The median follow-up was 11.9 years. The 5-year local control rates were higher for CB (100.0%) compared with SD (90.0%) patients (P = .01). CB patients had higher rates of 10-year progression-free survival (71.9% vs 57.6%, P < .01) and overall survival (71.6% vs 62.4%, P = .01) compared with SD patients. CONCLUSIONS: CRT dose escalation for patients with LARC is safe and effective. The improved T-downstaging and local control observed in CB patients should encourage further dose escalation studies.

Prospective validation of treatment accuracy using implanted fiducial markers for spinal stereotactic body radiation therapy.

In order to accurately assess positioning errors in spinal SBRT, many institutions employ bony-fusion based imaging techniques, such as the ExacTrac™ (Brain Lab) system, in conjunction with 3D verification (performed via CT-on-rails in our practice). We hypothesized that the use of implanted gold fiducial markers could improve the accuracy of patient positioning over bony fusion alone. We addressed this question prospectively, enrolling patients on an IRB-approved protocol. Gold seeds were implanted in the vertebral pedicles flanking the target level. At treatment, setup error was calculated using two methods-standard kV image fusion, and geometric fiducial-based projection, with independent CT-on-rails verification. Analyses of residual set-up error showed that fiducial-based setup agreed with fusion-based determination, but did not significantly reduce error. Offline 6D fusion of the treatment and planning CT illustrated residual rotational error using standard or fiducial based setup. We conclude that the ExacTrac and CT-on-rails platform yields highly accurate results for spinal SBRT setup, with reduced residual error than previously reported. While the addition of fiducials did not further reduce error, the bony fusion approach is now prospectively validated in comparison to implanted fiducials. Both bony fusion and fiducial marker methods are associated with residual rotational error, thus 3D verification remains an important component of spinal SBRT treatment.

Prospective evaluation of target and spinal cord motion and dosimetric changes with respiration in spinal stereotactic body radiation therapy utilizing 4-D CT.

PURPOSE: To assess the dosimetric effects of respiratory motion on the target and spinal cord in spinal stereotactic body radiation therapy (SBRT). METHODS AND MATERIALS: Thirty patients with 33 lesions were enrolled on a prospective clinical protocol and simulated with both free-breathing and four-dimensional (4-D) computed tomography (CT). We studied the target motion using 4-D data (10 phases) by registering a secondary image dataset (phase 1 to 9) to a primary image dataset (phase 0) and analyzing the displacement in both translational and rotational directions. The study of dosimetric impacts from respiration includes both the effect of potential target and spinal cord motion and anatomic changes in the beam path. A clinical step-and-shoot IMRT plan generated on the free-breathing CT was copied to the 4-D datasets to evaluate the difference in the dose-volume histogram of target and normal tissues in each phase of a breathing cycle. RESULTS: Twenty three lesions had no motion in a breathing cycle; four lesions had anterior-posterior motion ≤ 0.2 mm; two lesions had lateral motion ≤ 0.2 mm; and eight lesions had superior-inferior motion, most ≤ 0.2 mm with the worst at 0.6 mm. The difference of maximum dose to 0.01 cm3 of spinal cord in different phases of a breathing cycle was within 20 cGy in worst case. Target volumes that received the prescription dose (V100) varied little, with deviations of V100 of each phase from the average CT < 1% in most cases. Only when lesions were close to the diaphragm (e.g., at T11) did the V100 deviate by about 7% in the worst case scenario. However, this was caused by a small dose difference of 20 cGy to part of the target volume. CONCLUSIONS: Breathing induced target and spinal cord motion is negligible compared with other setup uncertainties. Dose calculation using averaged or free-breathing CT is reliable when posterior beams are used.

Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

Outcomes and predictive factors for salvage therapy after local--regional recurrence following neoadjuvant chemotherapy and breast conserving therapy.

BACKGROUND: There are few data addressing local-regional recurrence (LRR) and salvage therapies in patients treated with neoadjuvant chemotherapy (NCT) compared to those treated with surgery first. We characterize the clinical course and predictive features of salvage treatment for LRR after breast conserving therapy (BCT) analyzed by initial treatment. METHODS: We identified 1,589 patients who underwent BCT; 1,141 (72 %) patients underwent initial surgery, and 448 (28 %) received NCT. Kaplan-Meier and Cox regression analyses were performed to analyze factors associated with overall survival (OS), local control (LC) of recurrence, and distant metastasis-free survival (DMFS) following LRR. RESULTS: 56 patients had a LRR, for a crude recurrence rate of 3 %. For patients with potentially curable recurrence (excluding distant metastases within 3 months of LRR), the 5-year OS, LC, and DMFS rates were 52, 77, and 69 %. On multivariate analysis, initial pathologically negative node status and use of surgery for salvage were significant factors associated with higher OS. Additionally, older age was associated with higher LC rates after salvage. Estrogen receptor-positive disease and surgery for LRR were associated with reduced risk of distant metastases; regional recurrence and use of initial adjuvant chemotherapy were associated with increased risk of distant metastases. For each of these endpoints, the addition of NCT to the multivariate model did not approach significance. CONCLUSIONS: LRR is an uncommon event after BCT and many patients with LRR remain curable (5-year OS >50 %). Our data indicate that NCT does not compromise salvage after LRR, providing further assurance that this strategy is safe for appropriately selected breast cancer patients.

Use of a deformable atlas to identify cryptic critical structures in the treatment of glioblastoma multiforme.

Dose constraints for traditional neural critical structures (e.g. optic chiasm, brain stem) are a standard component of planning radiation therapy to the central nervous system. Increasingly, investigators are becoming interested in accounting for the dose delivered to other non-target neural structures (e.g. hippocampi), which are not easily identified on axial imaging. In this pilot study, a commercially available digital atlas was used to identify cryptic neural structures (hippocampus, optic radiations, and visual cortices) in 6 patients who received intensity modulated radiation therapy (IMRT) as part of multimodal management of glioblastoma multiforme (GBM). The patient's original IMRT plans were re-optimized, with avoidance parameters for the newly identified critical structures. Re-optimization was able to reduce both mean and maximum dose to the volumes of interest, with a more pronounced effect for contralateral structures. Mean dose was reduced by 11% and 3% to contralateral and ipsilateral structures, respectively, with comparable reduction in maximum dose of 10% and 2%, respectively. Importantly, target coverage was not compromised, with an average change in coverage of 0.2%. Overall, our results demonstrate the feasibility of incorporating tools for cryptic critical structure identification into the treatment planning process for GBM.

Generalizable class solutions for treatment planning of spinal stereotactic body radiation therapy.

PURPOSE: Spinal stereotactic body radiation therapy (SBRT) continues to emerge as an effective therapeutic approach to spinal metastases; however, treatment planning and delivery remain resource intensive at many centers, which may hamper efficient implementation in clinical practice. We sought to develop a generalizable class solution approach for spinal SBRT treatment planning that would allow confidence that a given plan provides optimal target coverage, reduce integral dose, and maximize planning efficiency. METHODS AND MATERIALS: We examined 91 patients treated with spinal SBRT at our institution. Treatment plans were categorized by lesion location, clinical target volume (CTV) configuration, and dose fractionation scheme, and then analyzed to determine the technically achievable dose gradient. A radial cord expansion was subtracted from the CTV to yield a planning CTV (pCTV) construct for plan evaluation. We reviewed the treatment plans with respect to target coverage, dose gradient, integral dose, conformality, and maximum cord dose to select the best plans and develop a set of class solutions. RESULTS: The class solution technique generated plans that maintained target coverage and improved conformality (1.2-fold increase in the 95% van't Riet Conformation Number describing the conformality of a reference dose to the target) while reducing normal tissue integral dose (1.3-fold decrease in the volume receiving 4 Gy (V(4Gy)) and machine output (19% monitor unit (MU) reduction). In trials of planning efficiency, the class solution technique reduced treatment planning time by 30% to 60% and MUs required by ∼20%: an effect independent of prior planning experience. CONCLUSIONS: We have developed a set of class solutions for spinal SBRT that incorporate a pCTV metric for plan evaluation while yielding dosimetrically superior treatment plans with increased planning efficiency. Our technique thus allows for efficient, reproducible, and high-quality spinal SBRT treatment planning.

Stereotactic body radiation therapy for management of spinal metastases in patients without spinal cord compression: a phase 1-2 trial.

BACKGROUND: Spinal stereotactic body radiation therapy (SBRT) is increasingly used to manage spinal metastases, yet the technique's effectiveness in controlling the symptom burden of spinal metastases has not been well described. We investigated the clinical benefit of SBRT for managing spinal metastases and reducing cancer-related symptoms. METHODS: 149 patients with mechanically stable, non-cord-compressing spinal metastases (166 lesions) were given SBRT in a phase 1-2 study. Patients received a total dose of 27-30 Gy, typically in three fractions. Symptoms were measured before SBRT and at several time points up to 6 months after treatment, by the Brief Pain Inventory (BPI) and the M D Anderson Symptom Inventory (MDASI). The primary endpoint was frequency and duration of complete pain relief. The study is completed and is registered with ClinicalTrials.gov, number NCT00508443. FINDINGS: Median follow-up was 15·9 months (IQR 9·5-30·3). The number of patients reporting no pain from bone metastases, as measured by the BPI, increased from 39 of 149 (26%) before SBRT to 55 of 102 (54%) 6 months after SBRT (p<0·0001). BPI-reported pain reduction from baseline to 4 weeks after SBRT was clinically meaningful (mean 3·4 [SD 2·9] on the BPI pain-at-its-worst item at baseline, 2·1 [2·4] at 4 weeks; effect size 0·47, p=0·00076). These improvements were accompanied by significant reduction in opioid use during the first 6 months after SBRT (43 [28·9%] of 149 patients with strong opioid use at baseline vs 20 [20·0%] of 100 at 6 months; p=0·011). Ordinal regression modelling showed that patients reported significant pain reduction according to the MDASI during the first 6 months after SBRT (p=0·00003), and significant reductions in a composite score of the six MDASI symptom interference with daily life items (p=0·0066). Only a few instances of non-neurological grade 3 toxicities occurred: nausea (one event), vomiting (one), diarrhoea (one), fatigue (one), dysphagia (one), neck pain (one), and diaphoresis (one); pain associated with severe tongue oedema and trismus occurred twice; and non-cardiac chest pain was reported three times. No grade 4 toxicities occurred. Progression-free survival after SBRT was 80·5% (95% CI 72·9-86·1) at 1 year and 72·4% (63·1-79·7) at 2 years. INTERPRETATION: SBRT is an effective primary or salvage treatment for mechanically stable spinal metastasis. Significant reductions in patient-reported pain and other symptoms were evident 6 months after SBRT, along with satisfactory progression-free survival and no late spinal cord toxicities. FUNDING: National Cancer Institute of the US National Institutes of Health.

Bifocal intracranial germinoma: a retrospective analysis of treatment outcomes in 20 patients and review of the literature.

PURPOSE: Bifocal germinoma (BFG) is a rare intracranial neoplasm for which the choice of radiation therapy (RT) field is controversial. Some believe that BFG represents disseminated disease requiring craniospinal irradiation (CSI), whereas others believe that BFG represents localized disease and advocate for more limited fields. METHODS AND MATERIALS: We analyzed 20 BFG patients at our institutions with classic bifocal lesions (pineal gland and suprasellar region). In addition, we identified 60 BFG patients from the literature. The RT fields, use of chemotherapy and extent of disease were recorded and analyzed for each patient. RESULTS: There were 55 patients with bifocal lesions only (Group I), and 25 with bifocal lesions plus ventricular and/or CSF positive disease (Group II). The 5-year progression-free survival was 95% for Group I and 80% for Group II. In Group I, there were no failures in patients receiving CSI (n = 11), two spinal failures in those treated with more limited RT fields without chemotherapy (n = 17), and one spinal failure with chemotherapy (n = 23). In Group II, there were no failures in patients receiving CSI (n = 11), but four spinal failures were observed in patients receiving more limited RT fields with chemotherapy (n = 13); 1 patient who received whole-brain RT without chemotherapy experienced failure in the spine and brain. CONCLUSIONS: CSI is associated with excellent PFS in BFG. In Group I BFG patients, omission of spinal irradiation appears to be a reasonable approach, especially when chemotherapy is used. Patients with Group II BFG are best treated with CSI.

Imprinted genes that regulate early mammalian growth are coexpressed in somatic stem cells.

Lifelong, many somatic tissues are replenished by specialized adult stem cells. These stem cells are generally rare, infrequently dividing, occupy a unique niche, and can rapidly respond to injury to maintain a steady tissue size. Despite these commonalities, few shared regulatory mechanisms have been identified. Here, we scrutinized data comparing genes expressed in murine long-term hematopoietic stem cells with their differentiated counterparts and observed that a disproportionate number were members of the developmentally-important, monoallelically expressed imprinted genes. Studying a subset, which are members of a purported imprinted gene network (IGN), we found their expression in HSCs rapidly altered upon hematopoietic perturbations. These imprinted genes were also predominantly expressed in stem/progenitor cells of the adult epidermis and skeletal muscle in mice, relative to their differentiated counterparts. The parallel down-regulation of these genes postnatally in response to proliferation and differentiation suggests that the IGN could play a mechanistic role in both cell growth and tissue homeostasis.

Irgm1 protects hematopoietic stem cells by negative regulation of IFN signaling.

The IFN-inducible immunity-related p47 GTPase Irgm1 has been linked to Crohn disease as well as susceptibility to tuberculosis. Previously we demonstrated that HSC quiescence and function are aberrant in mice lacking Irgm1. To investigate the molecular basis for these defects, we conducted microarray expression profiling of Irgm1-deficient HSCs. Cell-cycle and IFN-response genes are up-regulated in Irgm1(-/-) HSCs, consistent with dysregulated IFN signaling. To test the hypothesis that Irgm1 normally down-regulates IFN signaling in HSCs, we generated Irgm1(-/-)Ifngr1(-/-) and Irgm1(-/-)Stat1(-/-) double-knockout animals. Strikingly, hyperproliferation, self-renewal, and autophagy defects in Irgm1(-/-) HSCs were normalized in double-knockout animals. These defects were also abolished in Irgm1(-/-)Irgm3(-/-) double-knockout animals, indicating that Irgm1 may regulate Irgm3 activity. Furthermore, the number of HSCs was reduced in aged Irgm1(-/-) animals, suggesting that negative feedback inhibition of IFN signaling by Irgm1 is necessary to prevent hyperproliferation and depletion of the stem cell compartment. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFN-dependent stimulation in HSCs, with an essential role in preserving HSC number and function. The deleterious effects of excessive IFN signaling may explain how hematologic abnormalities arise in patients with inflammatory conditions.

Quiescent haematopoietic stem cells are activated by IFN-gamma in response to chronic infection.

Lymphocytes and neutrophils are rapidly depleted by systemic infection. Progenitor cells of the haematopoietic system, such as common myeloid progenitors and common lymphoid progenitors, increase the production of immune cells to restore and maintain homeostasis during chronic infection, but the contribution of haematopoietic stem cells (HSCs) to this process is largely unknown. Here we show, using an in vivo mouse model of Mycobacterium avium infection, that an increased proportion of long-term repopulating HSCs proliferate during M. avium infection, and that this response requires interferon-gamma (IFN-gamma) but not interferon-alpha (IFN-alpha) signalling. Thus, the haematopoietic response to chronic bacterial infection involves the activation not only of intermediate blood progenitors but of long-term repopulating HSCs as well. IFN-gamma is sufficient to promote long-term repopulating HSC proliferation in vivo; furthermore, HSCs from IFN-gamma-deficient mice have a lower proliferative rate, indicating that baseline IFN-gamma tone regulates HSC activity. These findings implicate IFN-gamma both as a regulator of HSCs during homeostasis and under conditions of infectious stress. Our studies contribute to a deeper understanding of haematological responses in patients with chronic infections such as HIV/AIDS or tuberculosis.

The p47 GTPase Lrg-47 (Irgm1) links host defense and hematopoietic stem cell proliferation.

Hematopoietic stem cells (HSCs) are self-renewing bone marrow cells that give rise to all blood lineages and retain a remarkable capacity to proliferate in response to insult. Although some controls on HSC activation are known, little is understood about how this process is linked to natural signals. We report that the interferon-inducible GTPase Lrg-47 (Irgm1), previously shown to play a critical role in host defense, inhibits baseline HSC proliferation and is required for a normal HSC response to chemical and infectious stimuli. Overproliferating Lrg-47(-/-) HSCs are severely impaired in functional repopulation assays, and when challenged with hematopoietic ablation by 5-fluorouracil or infection with Mycobacterium avium, Lrg-47(-/-) mice fail to achieve the expected expansion response in stem and progenitor cell populations. Our results establish a link between the response to infection and HSC activation and demonstrate a novel function for a member of the p47 GTPase family.

CD150- side population cells represent a functionally distinct population of long-term hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that replenish the cellular elements of blood throughout life. HSCs represent a paradigm for the study of stem-cell biology, because robust methods for prospective isolation of HSCs have facilitated rigorous characterization of these cells. Recently, a new isolation method was reported, using the SLAM family of cell-surface markers, including CD150 (SlamF1), to offer potential advantages over established protocols. We examined the overlap between SLAM family member expression with an established isolation scheme based on Hoechst dye efflux (side population; SP) in conjunction with canonical HSC cell-surface markers (Sca-1, c-Kit, and lineage markers). Importantly, we find that stringent gating of SLAM markers is essential to achieving purity in HSC isolation and that the inclusion of canonical HSC markers in the SLAM scheme can greatly augment HSC purity. Furthermore, we observe that both CD150(+) and CD150(-) cells can be found within the SP population and that both populations can contribute to long-term multilineage reconstitution. Thus, using SLAM family markers to isolate HSCs excludes a substantial fraction of the marrow HSC compartment. Interestingly, these 2 subpopulations are functionally distinct, with respect to lineage output as well as proliferative status.

Hematopoietic fingerprints: an expression database of stem cells and their progeny.

Hematopoietic stem cells (HSCs) continuously regenerate the hematologic system, yet few genes regulating this process have been defined. To identify candidate factors involved in differentiation and self-renewal, we have generated an expression database of hematopoietic stem cells and their differentiated progeny, including erythrocytes, granulocytes, monocytes, NK cells, activated and naive T cells, and B cells. Bioinformatic analysis revealed HSCs were more transcriptionally active than their progeny and shared a common activation mechanism with T cells. Each cell type also displayed unique biases in the regulation of particular genetic pathways, with Wnt signaling particularly enhanced in HSCs. We identified approximately 100-400 genes uniquely expressed in each cell type, termed lineage "fingerprints." In overexpression studies, two of these genes, Zfp 105 from the NK cell lineage, and Ets2 from the monocyte lineage, were able to significantly influence differentiation toward their respective lineages, demonstrating the utility of the fingerprints for identifying genes that regulate differentiation.