Detection of monosomy 7 in interphase cells of patients with myeloid disorders

Six patients, five with acute myeloid leukemia (AML) and one with a myelodysplastic syndrome (MDS), were found to have monosomy 7 by conventional cytogenetics at diagnosis. Repetitive DNA sequences from the heterochromatic region of human chromosomes 1 and 7 were used as probes for in situ hybridization experiments on interphase cells of these patients. A double hybridization protocol was used to reveal the particular chromosomes as distinct spots or clusters of signals within interphase nuclei. The chromosome 1 sequence served as an internal control. Simultaneous detection of the sequences showed the presence of two normal number 1 chromosomes and a missing 7 chromosome from individual cells. While cytogenetic preparations showed only -7 metaphases in 3 AML and 1 MDS patients, in situ hybridization of interphase cells showed many normal cells as well as the presence of -7 in fully mature granulocytes. One AML patient studied in remission showed only normal metaphases yet had 9% interphase cells with a missing 7 and relapsed within 3 months. We conclude that examination of interphase cells by in situ hybridization provides clinically useful data since every cell including mature granulocytes can be examined, the lineage of a cell can be determined, and efficacy of differentiation therapy can be evaluated

The biology of myelodysplastic syndromes: unity despite heterogeneity

Myelodysplastic syndromes (MDS) traditionally have been grouped together as a disease entity based on clinical phenomena seen in association. Despite the similarities, there is great heterogeneity among the syndromes. Recent insights have shown, however, that there exists a biologically cohesive theme that unifies and thereby validates the conceptual interconnectedness. The first suggestion that such a relationship existed where biology could directly explain the observed cytopenias was the finding of excessive premature apoptosis of hematopoietic cells in MDS marrows. This apoptosis was mediated by paracrine as well as autocrine factors implicating both the seed and the soil in the pathology of the disease. Pro-inflammatory cytokines in the marrow microenvironment were mainly the paracrine mediators of apoptosis, but how the clonal cells committed suicide because of autocrine stimulation had remained a mystery for more than a decade. It has been shown now that deregulation of ribosome biogenesis can initiate a stress response in the cell through the p53 signaling pathway. Congenital anemias had been associated with mutations in ribosomal protein genes. The surprise came with the investigation of 5q- syndrome patients where haplo-insufficiency of the ribosomal protein gene RPS14 was found to be the cause of this MDS subtype. Similar ribosomal deregulation was shown to be present in all varieties of MDS patients, serving as another unifying characteristic. In addition to these findings, there are other DNA-related abnormalities such as uniparental disomy, mutations in the TET2 gene, and epigenetic phenomena that are associated with and occur across all types of MDS. This paper summarizes the themes unifying this heterogeneous group of diseases

Comparison of sampling adequacy between OPD based pipelle biopsy and in- patient conventional D&C, presented with abnormal uterine bleeding

Objective: To determine agreement on adequacy of sample by pipelle biopsy and conventional dilatation and curettage in patients with abnormal uterine bleeding. Study design: Cross sectional studySetting and Duration of Study: Department of Obstetrics and Gyneacology, Islamic International Medical College Trust, Railway Hospital Rawalpindi. Study was carried out over a period of six months (11-07-2012 to 14-01-2013). Patients and Methods: 84 patients presented with abnormal uterine bleeding age 45 years and older, attended Gynecology department of Railway Hospital Rawalpindi. Who qualified the inclusion criteria were enrolled in this study by non-probability consecutive sampling technique. The diagnostic intervention for endometrial sampling was by pipelle device and by conventional D&C. Both procedures were performed in the OT at the same time.First the pipelle sample was taken and was labeled as “A” then conventional D&C was performed and was labeled as “B”. Both samples were sent to the pathologist, who was blinded as to the method of sample collection for histopathology assessment. Adequacy of the sample was assessed as per operational definition. A data base was made in SPSS version 17. Kappa statistics was applied to assess the agreement. Results: Out of 84 patients, 80 (98.8%) of the patients had adequate sample with Pipelle Biopsy as compared to conventional curettage and dilatation (D & C). We therefore recommend the use of pipelle biopsy as a first line tool for endometrial assessment for our setups instead of D&C. Conclusion: Our study concluded that the Pipelle biopsy is a useful and convenient method to the patients and physicians as ompared to D&C performed in the operating theatre. It is useful in obese and high-risk patients with minimum chances of perforation of uterus due to its soft flexible tip

RADIOLOGICAL AND PATHOLOGICAL CORRELATION OF LUNG NODULES IN A BACKGROUND OF METASTATIC DISEASE

Background: Computed tomography (CT) imaging has improved the chances of detecting small indeterminate (<1 cm) lung nodules. The determination of the underlying malignant or benign nature of a lung nodule poses a great diagnostic challenge and depends on a number of factors, including the radiographic appearance of nodule, the presence of non-pulmonary metastases, characteristics of growth and histological criteria. Methods: The medical records of 89 patients admitted to our specialist cancer centre between 2008 and 2013 were reviewed. Patients of all age groups and tumour category were included in the study. Clinical data of these patients were collected and the following parameters were analysed: Radiographic diagnosis, location, size, laterality and number of nodules and histological impression. The radiological findings were then correlated with histopathological findings. The nodules were sub-classified into groups on the basis of size (A = 0–0.5 cm; B = 0.5–0.9 cm; C = 1.0–1.5 cm and D = >1.5 cm). Results: CT scan reports of 89 patients with lung nodules were reviewed. On radiology, 73/89 (82%) were reported to be malignant nodule. Histopathological review of the biopsies of these 89 nodules confirmed malignancy in 50/89 (56.2%) patients. CT scan was found to be highly sensitive (94%, 95% confidence interval [CI]: 83.43–98.68%) but with a very low specificity (33.3%, 95% CI: 19.10–50.22%). CT scan was found to have a higher negative predictive value (81.2%, 95% CI: 54.34–95.73%) and a lower positive predictive value 64.4% (95% CI: 52.31–75.25%) when correlated with histopathological findings. Pathology of these nodules included metastatic sarcoma (27/89; 30.3%) and carcinoma (18/89; 20.2%). The frequency of the biopsy-proven malignant nodules on the right side was 26/45 (57.8%) and on the left side was 24/44 (54.5%) (P = 0.832). Malignant nodules were more frequent in lower lobes (28/43, 65.1%) than in upper lobes (14/32, 43.8%). These two sites combined accounted for 84% of all malignant nodules. There was a significant correlation between nodule size and likelihood of underlying malignancy. The overall prevalence of malignancy in the larger nodules (C and D) was much higher (23/30 and 76.7%) compared to the smaller sized (A and B) nodules (27/58 and 46.8%), P < 0.05.Conclusion: CT scan is a useful tool in the initial clinical assessment of indeterminate lung nodules with high sensitivity (94%) and a high negative predictive value (81.2%).Key words: Computed tomography, fibrosis, indeterminate lung nodule, infection, lung nodule, malignancy, metastase

Oral Ezatiostat HCl (TLK199) and Myelodysplastic syndrome: A case report of sustained hematologic response following an abbreviated exposure

Treatment options for patients with lower risk non-del(5q) myelodysplastic syndromes (MDS) who fail erythroid stimulating agents are restricted to one of the hypomethylating drugs with an expected response rate of ~50%. Ezatiostat HCl, an agent with the potential for producing multi-lineage responses in this population is currently in clinical investigation phase. This case report describes a 77 year old male who received less than two cycles of therapy with ezatiostat HCl which had to be aborted due to intolerable side effects, but which produced a sustained normalization of all three blood counts. This trilineage response has now lasted for more than a year. Interestingly, the patient began with a del(5q) abnormality and responded briefly to lenalidomide. Upon relapse of the anemia, a bone marrow showed the disappearance of the del(5q) but the appearance of a new clonal abnormality t(2;3). Given that the patient had a complete cytogenetic response to a truncated exposure to lenalidomide followed by a trilineage response to an even briefer course of ezatiostat HCl suggests a potential role for ezatiostat HCl in del(5q) patients who relapse following lenalidomide

Identification of RPS14 as a 5q- syndrome gene by RNA interference screen

Somatic chromosomal deletions in cancer are thought to indicate the location of tumor suppressor genes, whereby complete loss of gene function occurs through biallelic deletion, point mutation, or epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis.1 In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q- syndrome, a subtype of myelodysplastic syndrome (MDS) characterized by a defect in erythroid differentiation.2 Here, we describe an RNA interference (RNAi)-based approach to discovery of the 5q- disease gene. We find that partial loss of function of the ribosomal protein RPS14 phenocopies the disease in normal hematopoietic progenitor cells, and moreover that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-rRNA in RPS14 deficient cells that is highly analogous to the functional defect in Diamond Blackfan Anemia, linking the molecular pathophysiology of the 5q- syndrome to a congenital bone marrow failure syndrome. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function, and suggests that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes

An Erythroid Differentiation Signature Predicts Response to Lenalidomide in Myelodysplastic Syndrome

Background: Lenalidomide is an effective new agent for the treatment of patients with myelodysplastic syndrome (MDS), an acquired hematopoietic disorder characterized by ineffective blood cell production and a predisposition to the development of leukemia. Patients with an interstitial deletion of Chromosome 5q have a high rate of response to lenalidomide, but most MDS patients lack this deletion. Approximately 25% of patients without 5q deletions also benefit from lenalidomide therapy, but response in these patients cannot be predicted by any currently available diagnostic assays. The aim of this study was to develop a method to predict lenalidomide response in order to avoid unnecessary toxicity in patients unlikely to benefit from treatment. Methods and Findings: Using gene expression profiling, we identified a molecular signature that predicts lenalidomide response. The signature was defined in a set of 16 pretreatment bone marrow aspirates from MDS patients without 5q deletions, and validated in an independent set of 26 samples. The response signature consisted of a cohesive set of erythroid-specific genes with decreased expression in responders, suggesting that a defect in erythroid differentiation underlies lenalidomide response. Consistent with this observation, treatment with lenalidomide promoted erythroid differentiation of primary hematopoietic progenitor cells grown in vitro. Conclusions: These studies indicate that lenalidomide-responsive patients have a defect in erythroid differentiation, and suggest a strategy for a clinical test to predict patients most likely to respond to the drug. The experiments further suggest that the efficacy of lenalidomide, whose mechanism of action in MDS is unknown, may be due to its ability to induce erythroid differentiation

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)

<p>Abstract</p> <p>Purpose</p> <p>In a departure from conventional strategies to improve treatment outcome for myeloid malignancies, we report the isolation of leukemia-specific peptides using a phage display library screened with freshly obtained human myeloid leukemia cells.</p> <p>Results</p> <p>A phage display library was screened by 5 rounds of biopanning with freshly isolated human AML cells. Individual colonies were randomly picked and after purification, biologic activity (growth and differentiation) on fresh AML cells was profiled. Ten peptides were synthesized for further biological studies. Multiple peptides were found to selectively bind to acute myeloid leukemia (AML) cells. The peptides bound to leukemia cells, were internalized and could induce proliferation and/or differentiation in the target patient cells. Two of the peptides, HP-A2 and HP-G7, appeared to have a novel mechanism of inducing differentiation since they did not cause G1 arrest in cycling cells even as the expression of the differentiation marker CD11b increased.</p> <p>Conclusion</p> <p>Peptide induced differentiation of leukemia cells offers a novel treatment strategy for myeloid malignancies, whereas their ability to induce proliferation could be harnessed to make cells more sensitive to chemotherapy. Conceptually, these leukemia specific peptides can also be used to refine diagnosis, document minimal residual disease, and selectively deliver toxins to malignant cells.</p