Michelle Dolan

Bone Marrow Transplantation for Recessive Dystrophic Epidermolysis Bullosa

BACKGROUND Recessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7 −/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans. METHODS Between October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography. RESULTS One patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal-epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies. CONCLUSIONS Increased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

Allogeneic Stem Cell Transplantation for Adults with Myelodysplastic Syndromes: Importance of Pretransplant Disease Burden

Allogeneic stem cell transplantation is the only known curative therapy for myelodysplastic syndromes (MDS). We present the transplant outcomes for 84 adult MDS patients, median age 50 (18-69 years), undergoing allogeneic hematopoietic stem cell transplantation (HSCT) at the University of Minnesota between 1995 and 2007. By WHO criteria 35 (42%) had refractory anemia with excess blasts (RAEB-1 or 2), 23 (27%) had refractory cytopenia with multilineage dysplasia (RCMD) or RCMD and ringed sideroblasts (RCMD-RS), and the remaining 26 (31%) had refractory anemia (RA), myelodysplastic syndrome-unclassifiable (MDS-U), chronic myelomonocytic leukemia (CMML), myelodysplastic/myeloproliferative disease (MDS/MPD), or myelodysplastic syndrome-not otherwise specified (MDS-NOS). Graft source was related in 47 (56%), unrelated donor (URD) marrow in 11 (13%), and unrelated cord blood (UCB) in 26 (31%). The conditioning regimen included total body irradiation (TBI) in 94% of transplantations; 52 (62%) myeloablative (MA) and 32 (38%) nonmyeloablative (NMA) regimens. Cumulative incidence of neutrophil engraftment by day +42, acute graft-versus-host disease (aGVHD) by day +100, and chronic GVHD (cGVHD) by 1 year were 88% (80%-96%, 95% confidence interval [CI]), 43% (36%-50%, 95% CI), and 15% (10%-20%, 95% CI), respectively. One-year treatment-related mortality (TRM), relapse, disease-free survival (DFS), and overall survival (OS) were 39% (28%-50%, 95% CI), 23% (12%-32%, 95% CI), 38% (28%-48%, 95% CI), and 48% (38%-58%, 95% CI) respectively. Cumulative incidence of relapse at 1 year in patients with pre-HCT complete remission (CR) or <5% blasts was improved at 18% (8%-28%, 95% CI) compared to 35% (16%-54%, 95% CI) in patients with 5%-20% blasts (P = .07). Additionally, with MA conditioning, the incidence of relapse at 1 year trended lower at 16% (6%-26%, 95% CI) versus 35% (18%-52%, 95% CI) in NMA (P = .06), and a statistically significant decrease in relapse was noted in patients entering HCT with CR or <5% blasts with an incidence of 9% (0%-18%, 95% CI) (MA) versus 31% (11%-51%, 95% CI) (NMA) (P = 0.04). For those patients with > or =5% blasts, MA conditioning did not significantly decrease relapse rates. One-year TRM was similar between MA and NMA conditioning. For patients entering transplant in CR or with <5% blasts, prior treatment to reach this level did not impact rates of relapse or transplant-related mortality when all patients were analyzed; however, when broken down by conditioning intensity, there was a trend toward improved DFS in those NMA patients who were pretreated. Finally, 1-year DFS was similar using related donor peripheral blood stem cell (PBSC)/marrow, URD marrow, or UCB grafts. These data suggest that (1) blast percentage <5% at HSCT is the major predictor of improved DFS and relapse and prior treatment to reach this disease status may have value in leading to improved DFS; (2) MA conditioning is associated with lower relapse risk, particularly in patients with CR or <5% blasts, but is not able to overcome increased disease burden; (3) NMA conditioning yields equivalent TRM, DFS, and OS, and is reasonable in patients unsuited for MA conditioning; (4) the donor sources tested (PBSC, bone marrow [BM], or UCB) yielded similar outcomes.

Long-term metabolic, endocrine, and neuropsychological outcome of hematopoietic cell transplantation for Wolman disease

Wolman disease is the infantile form of autosomal recessive acid lipase deficiency, typically presenting in early infancy with diarrhea, massive hepatosplenomegaly, failure to thrive, and calcification of adrenal glands. Hematopoietic cell transplantation (HCT) is the only therapy reported to prevent hepatic failure and death, which without treatment occurs within the first year of life. We report a single institutions experience with HCT treatment of four Wolman patients, two of whom are long-term survivors (the longest survival reported to date, (4 and 11 years). Survivors showed resolution of diarrhea within weeks after engraftment, normalized hepatic function, improved hepatosplenomegaly, and in one patient normal adrenal function. The older patient has normal adaptive functions but mild to moderate neurocognitive deficiencies thought to be secondary to treatment and other medical problems. The younger patient has age-appropriate neurodevelopmental and adaptive abilities. We conclude that Wolman disease can be successfully treated with HCT, and that hepatic and cognitive function can be preserved with early diagnosis and timely referral to a transplant center.

Comparison of Immunohistochemical and Fluorescence In Situ Hybridization Assessment of HER-2 Status in Routine Practice

Because HER-2 expression in invasive carcinoma of the breast has well-documented ramifications for treatment and prognosis, accurate assessment of HER-2 status is critical. Comparative studies have shown high concordance rates between immunohistochemical analysis and fluorescence in situ hybridization (FISH) in cases with immunohistochemical scores of 0 or 1+ (negative) and 3+ (strongly positive) and low concordance rates among cases with immunohistochemical scores of 2+. The present study was performed to determine concordance rates in a setting more representative of routine clinical practice, in which multiple pathologists submit specimens to a single cytogenetics referral laboratory. We found a higher rate of discordance between immunohistochemical analysis and FISH (approximately 92%) in the groups with immunohistochemical scores of 2+ than reported in other studies. These results strongly support the practice of performing FISH in all cases with immunohistochemical scores of 2+, particularly in routine practice, in which interobserver variability in immunohistochemical scoring among multiple pathologists is likely to be high.

MLL amplification in myeloid malignancies: clinical, molecular, and cytogenetic findings

Structural rearrangements involving the MLL gene at 11q23 are common recurring abnormalities in de novo and therapy-related hematologic disorders. MLL rearrangement most often results from translocation or partial tandem duplication, although recent published reports suggest a different mechanism by which MLL might participate in leukemogenesis: MLL amplification. We report two patients with myeloid disorders who showed amplification of MLL at diagnosis and who, like the majority of reported cases, had an older age at onset and on aggressive clinical course. Additionally, we summarize the salient clinical, cytogenetic and molecular findings of the 29 other cases of MLL amplification that have thus far been reported.

A novel microdeletion/microduplication syndrome of 19p13.13

Purpose: Whole genome interrogation by array-based comparative genomic hybridization has led to a rapidly increasing number of discoveries of novel microdeletion and/or microduplication syndromes. We here describe the clinical and cytogenomic correlates of a novel microdeletion/microduplication of 19p13.13.Methods: Among patients referred to the Cytogenetics laboratory for array-based comparative genomic hybridization analysis, we identified four with a deletion and one with a duplication within 19p13.13. Confirmatory fluorescence in situ hybridization and parental studies were performed. Detailed clinical findings and array profiles were reviewed and compared.Results: Patients with deletions of 19p13.13 share a unique constellation of phenotypic abnormalities. In addition to developmental disabilities, the microdeletion manifested in overgrowth, macrocephaly, and ophthalmologic and gastrointestinal findings; in contrast, the single microduplication manifested in growth delay and microcephaly.Conclusion: The consistent constellation of clinical findings associated with copy number variation of this region warrants the designation of microdeletion/microduplication syndrome of 19p13.13. An approximately 311–340 Kb smallest region of overlap encompassing 16 genes was identified. Candidate genes include MAST1, NFIX, and CALR. Identification of this syndrome has led to recommendations for diagnostic work-up and follow-up of patients with this copy number variant. Integration of detailed clinical and array data is critical for advancing both patient care and human genomic research.

The BCR/ABL Transgene Causes Abnormal NK Cell Differentiation and Can Be Found in Circulating NK Cells of Advanced Phase Chronic Myelogenous Leukemia Patients

NK cells from the blood of chronic myelogenous leukemia (CML) patients are progressively decreased in number as the disease progresses from chronic phase to blast crisis. We hypothesize that BCR/ABL may be directly responsible by interfering with NK cell differentiation. CD34+HLA-DR+ cells from CML patients were studied for their capacity to differentiate into NK cells. The NK cell cloning frequency was significantly decreased from CML CD34+HLA-DR+ cells compared with cells from normal donors, yet CD34+HLA-DR+ cells gave rise to BCR/ABL+ NK cells in some patients. This finding prompted us to further investigate circulating NK cells from the blood of CML patients. CD56+CD3− NK cells were sorted from CML patients and examined by fluorescence in situ hybridization (FISH). In contrast to chronic phase CML, significant numbers of NK cells from advanced phase CML patients were BCR/ABL+, whereas T cells were always BCR/ABL− regardless of the disease stage. To test the effects of BCR/ABL as the sole genetic abnormality, BCR/ABL was transduced into umbilical cord blood CD34+ cells, and NK development was studied. p210-enhanced green fluorescence protein-transduced cells gave rise to significantly decreased numbers of NK cells compared with enhanced green fluorescence protein transduction alone. In addition, the extrinsic addition of BCR/ABL-transduced autologous CD34+ cells suppressed the NK cell differentiation of normal umbilical cord blood CD34+CD38− cells. This study provides the first evidence that BCR/ABL is responsible for the altered differentiation of NK cells and that the NK cell lineage can be involved with the malignant clone in advanced stage CML.

Favorable outcome of allogeneic hematopoietic cell transplantation for 8p11 myeloproliferative syndrome associated with BCR-FGFR1 gene fusion

We report the case of a child who presented with nonspecific symptoms suggestive of a rheumatologic disorder, whose bone marrow had a complex translocation involving the FGFR1 locus. Hematopathologic findings were subtle and did not definitively indicate malignancy. Because he responded poorly to initial treatment with hydroxyurea, and in light of the progressive clinical course associated with the 8p11 myeloproliferative syndrome, he underwent an unrelated‐donor hematopoietic stem cell transplant. This patients atypical presentation highlights the importance of obtaining cytogenetic analysis at the time of bone marrow sampling and considering this uncommon entity in the differential diagnosis of hematologic disorders. Pediatr Blood Cancer 2012; 59: 194–196.

Correlation between HER2 determined by fluorescence in situ hybridization and reverse transcription-polymerase chain reaction of the oncotype DX test.

The human epidermal growth factor receptor 2 (HER2) gene is amplified and its protein product overexpressed in about 20% of invasive breast cancers. Despite more than a decade of efforts to standardize HER2 testing, controversy persists regarding the most optimal testing method. Recently, Oncotype DX reports have begun including HER2 results in addition to the previously reported Recurrence Score. We compared HER2 results obtained by fluorescence in situ hybridization (FISH) in our laboratories with HER2 results obtained by reverse transcription-polymerase chain reaction (RT-PCR) as documented in the Oncotype DX report. We then sought to identify potentially significant characteristics in the discrepant cases. We identified breast cancer patients with estrogen receptor-positive, lymph node–negative tumors who had Oncotype DX testing performed between September 2008 and March 2012. Patient and tumor characteristics including HER2 FISH and Oncotype DX test results were recorded. Image analysis was performed on cases with discrepancy between the HER2 FISH and Oncotype DX HER2 results to determine the relative proportion of invasive tumor. Eight of 194 (4.1%) cases showed discrepancy between HER2 FISH and Oncotype DX RT-PCR results. Although the overall percent agreement (96%) and percent negative agreement (100%) were high, percent positive agreement was only 50%. Three of 8 (38%) discrepant cases showed heterogeneous amplification by FISH. Seven of 8 (88%) discrepant cases had <50% invasive tumor in the Oncotype DX tissue block. Percent positive agreement between HER2 FISH and Oncotype DX RT-PCR is low. Multiple factors may contribute to this discrepancy including a suboptimal microdissection and possibly heterogeneous amplification of HER2 gene in some cases.

Monosomal karyotype at the time of diagnosis or transplantation predicts outcomes of allogeneic hematopoietic cell transplantation in myelodysplastic syndrome.

Various cytogenetic risk scoring systems may determine prognosis for patients with myelodysplastic syndromes (MDS). We evaluated 4 different risk scoring systems in predicting outcome after allogeneic hematopoietic cell transplantation (alloHCT). We classified 124 patients with MDS using the International Prognostic Scoring System (IPSS), the revised International Prognostic Scoring System (R-IPSS), Armands transplantation-specific cytogenetic grouping, and monosomal karyotype (MK) both at the time of diagnosis and at alloHCT. After adjusting for other important factors, MK at diagnosis (compared with no MK) was associated with poor 3-year disease-free survival (DFS) (27% [95% confidence interval, 12% to 42%] versus 39% [95% confidence interval, 28% to 50%], P = .02) and overall survival (OS) (29% [95% confidence interval, 14% to 44%] versus 47% [95% confidence interval, 36% to 59%], P = .02). OS but not DFS was affected by MK at alloHCT. MK frequency was uncommon in low-score R-IPPS and IPSS. Although IPSS and R-IPSS discriminated good/very good groups from poor/very poor groups, patients with intermediate-risk scores had the worst outcomes and, therefore, these scores did not show a progressive linear discriminating trend. Cytogenetic risk score change between diagnosis and alloHCT was uncommon and did not influence OS. MK cytogenetics in MDS are associated with poor survival, suggesting the need for alternative or intensified approaches to their treatment.