Lurdes Zamora

Activating Mutations in the Tyrosine Kinase Domain of the Epidermal Growth Factor Receptor Are Associated with Improved Survival in Gefitinib-Treated Chemorefractory Lung Adenocarcinomas

Purpose: Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) confer a strong sensitivity to gefitinib, a selective tyrosine kinase inhibitor of EGFR. Experimental Design: We examined EGFR mutations at exons 18, 19, and 21 in tumor tissue from 68 gefitinib-treated, chemorefractory, advanced non–small cell lung cancer patients from the United States, Europe, and Asia and in a highly gefitinib-sensitive non–small cell lung cancer cell line and correlated their presence with response and survival. In addition, in a subgroup of 28 patients for whom the remaining tumor tissue was available, we examined the relationship among EGFR mutations, CA repeats in intron 1 of EGFR, EGFR and caveolin-1 mRNA levels, and increased EGFR gene copy numbers. Results: Seventeen patients had EGFR mutations, all of which were in lung adenocarcinomas. Radiographic response was observed in 16 of 17 (94.1%) patients harboring EGFR mutations, in contrast with 6 of 51 (12.6%) with wild-type EGFR (P < 0.0001). Probability of response increased significantly in never smokers, patients receiving a greater number of prior chemotherapy regimens, Asians, and younger patients. Median survival was not reached for patients with EGFR mutations and was 9.9 months for those with wild-type EGFR (P = 0.001). EGFR mutations tended to be associated with increased numbers of CA repeats and increased EGFR gene copy numbers but not with EGFR and caveolin-1 mRNA overexpression (P = not significant). Conclusions: The presence of EGFR mutations is a major determinant of gefitinib response, and targeting EGFR should be considered in preference to chemotherapy as first-line treatment in lung adenocarcinomas that have demonstrable EGFR mutations.

Prognostic significance of copy number alterations in adolescent and adult patients with precursor B acute lymphoblastic leukemia enrolled in PETHEMA protocols

Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL).

Cytogenetic and fluorescence in situ hybridization studies in 60 patients with multiple myeloma and plasma cell leukemia

We report cytogenetic results in a series of 60 patients affected with multiple myeloma (MM) and plasma cell leukemia (PCL) and compare the results with those previously reported. In our series, a total of 41% of MM patients and 71% of PCL patients displayed chromosome abnormalities. To evaluate the clinical value of monosomy 18, we obtained fluorescence in situ hybridization results (using centromeric probe for chromosome 18) of 22 MM patients who displayed a normal karyotype. Monosomy 18 was present in 3 of 22 patients (14%). Using conventional cytogenetics, we detected monosomy 18 in one patient affected with PCL. Two of four cases with monosomy 18 followed an aggressive course, with overall survival of 1 and 9 months. The remaining two are in follow-up and remain stable. The association of monosomy 18 with IgA subtype predominance and poor prognosis was not observed in this series of MMs and PCLs. Although these results do not confirm our previous hypothesis, further observations of this group of patients (with monosomy 18) regarding malignant transformation is warranted.

Two cases of tetrasomy 9p syndrome with tissue limited mosaicism

Tetrasomy of short arm of chromosome 9 constitutes a clinically recognizable chromosomal syndrome. Isochromosome 9p shows a strong propensity to tissue‐limited mosaicism. It occurs predominantly in peripheral blood cultures, often at a lower frequency or even absent in skin, amniotic fluid or chorionic villous cell cultures. Tissue‐limited nature of mosaicism may render prenatal detection of this condition very difficult. Herein, we report two new cases of mosaic tetrasomy 9p. Conventional cytogenetics (CC) and FISH studies demonstrated a differential expression of the mosaicism in several tissues. We review the literature and discuss the implications of these findings in cytogenetic prenatal diagnosis.

Bone marrow fibrosis in myelodysplastic syndromes: a prospective evaluation including mutational analysis

The biological and molecular events that underlie bone marrow fibrosis in patients with myelodysplastic syndromes are poorly understood, and its prognostic role in the era of the Revised International Prognostic Scoring System (IPSS-R) is not yet fully determined. We have evaluated the clinical and biological events that underlie bone marrow fibrotic changes, as well as its prognostic role, in a well-characterized prospective patient cohort (n=77) of primary MDS patients. The degree of marrow fibrosis was linked to parameters of erythropoietic failure, marrow cellularity, p53 protein accumulation, WT1 gene expression, and serum levels of CXCL9 and CXCL10, but not to other covariates including the IPSS-R score. The presence of bone marrow fibrosis grade 2 or higher was associated with the presence of mutations in cohesin complex genes (31.5% vs. 5.4%, p=0.006). By contrast, mutations in CALR, JAK2, PDGFRA, PDGFRB, and TP53 were very rare. Survival analysis showed that marrow fibrosis grade 2 or higher was a relevant significant predictor for of overall survival, and independent of age, performance status, and IPSS-R score in multivariate analysis.

Co-existence of JAK2 V617F and CALR mutations in primary myelofibrosis

In December 2013 mutations were described in the calreticulin (CALR) gene in 67–71% and 56–88% of patients with JAK2 V617F and MPL negative essential thrombocythemia (ET) and primary myelofibrosis (PMF), respectively [1,2]. Since this discovery, not only have CALR mutations been recommended to be included in the diagnostic algorithm for myeloproliferative neoplasms [3], but also CALR exon 9 mutations have been recognized to have clinical utility, as patients with these mutations have a better outcome than JAK2 V617F positive patients [4,5]. CALR mutations have also been reported to be mutually exclusive from JAK2 V617F or MPL mutations [1,2], so the majority of retrospective studies do not test for CALR mutations in JAK2 V617F or MPL positive patients. Recently, some series have described patients harboring mutations in both JAK2 V617F and CALR genes [5–7], but the true frequency of patients with “double-positive” disease is unknown, as mutations in JAK2 V617F, MPL and CALR are not routinely studied in all patients. For this reason, the objective of this study was to establish the true frequency of patients with double-positive PMF. Peripheral blood or bone marrow samples were obtained from 73 patients (45 males, median age 69 years) diagnosed with PMF according to the World Health Organization (WHO) classification [8]. Written informed consent for sample collection was received from all participants. DNA was extracted from blood or bone marrow samples collected in ethylenediaminetetraacetic acid anticoagulant using a QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and diluted with double-distilled water. In compliance with the Declaration of Helsinki this study was approved by the Institutional Review Board of the Hospital Germans Trias i Pujol. In all patients JAK2 V617F, MPL and CALR mutations were studied. To detect the presence of JAK2 V617F mutation, an allele-specific polymerase chain reaction (PCR) with TaqMan probes was used. We employed Sanger sequencing to detect MPL exon 10 mutations. Moreover we screened for insertions or deletions in the CALR gene with 6-carboxyfluorescein (6-FAM) labeled primers spanning exon 9 as previously described [1]. We confirmed and described the CALR mutation type with Sanger sequencing. A total of 46 out of 73 (63%) patients were JAK2 V617F positive, and among the 27 JAK2 V617F negative patients, five had MPL mutations (18.5%). Twelve patients out of 73 (16.5%) had a CALR mutation (eight type 1, one type 2 and three different from the 36 described). One patient harbored both JAK2 V617F and CALR mutations (c.1142_1144del) (Figure 1), the other 11 patients being JAK2 V617F and MPL negative. The biological implication of this mutation is unknown, as it implies the deletion of one amino acid but the reading frame does not change, and the somatic nature of this alteration could not be confirmed in constitutional DNA as the patient died of acute myocardial infarction in October 2011. In our series, 85% of patients carried a JAK2 V617F, MPL or CALR mutation, and a single patient had JAK2 V617F and CALR double-positive disease, representing 1.4% of the cohort studied. The patient with double-positive disease was an 86-yearold male with hemoglobin of 142 g/L, white blood cell count 11.7  109/L, platelet count 1170  109/L and a history of rectorrhagia. A diagnosis of PMF was made on the basis of the clinical characteristics, morphology and JAK2 V617F mutation status (allele burden of 60.58%). To date, and taking into account the present study, only four patients have been described (two with ET and two with PMF) with concurrent JAK2 V617F and CALR exon 9 mutations [5–7]; however, this percentage could be underLeukemia & Lymphoma, October 2015; 56(10): 2973–2974

Usefulness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

In up to 5–15% of studies of lymphoproliferative disorders (LPD), flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, two clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and nine TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (four IGH, 10 TCRγ), albeit nonconclusive. Of these, 2/4 were eventually diagnosed with B‐cell lymphoma and 3/10 with T‐cell LPD. In eight IGH samples, the results of PCR techniques were noninformative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and nonconclusive results do not exclude lymphoid neoplasms. Interpretation of T‐cell clonality should be based on all the available clinical and analytical data.

Response to erythropoietic stimulating agents in patients with chronic myelomonocytic leukemia

The efficacy of erythropoietic‐stimulating agents (ESA) in chronic myelomonocytic leukemia (CMML) is unknown. Our objective was to analyze erythroid response (ER) and overall survival (OS) in a series of 94 patients with CMML treated with ESA.

Targeted deep sequencing improves outcome stratification in chronic myelomonocytic leukemia with low risk cytogenetic features

Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1, EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations (TET2wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations (ASXL1, EZH2, NRAS and SRSF2) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.

Frequency of ABL gene mutations in chronic myeloid leukemia patients resistant to imatinib and results of treatment switch to second-generation tyrosine kinase inhibitors

BACKGROUND AND OBJECTIVES Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients do not achieve the optimal response or are resistant to TKI. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKI has produced high rates of hematologic and cytogenetic responses in mutated ABL patients. The aim of this study was to determine the type and frequency of ABL mutations in patients who were resistant to imatinib or had lost the response, and to analyze the effect of second-generation TKI on their outcome. PATIENTS AND METHODS The presence of ABL mutations in 45 CML patients resistant to imatinib was evaluated by direct sequencing and was correlated with the results of the cytogenetic study (performed in 39 cases). The outcome of these patients after therapy with nilotinib or dasatinib was analyzed. RESULTS ABL mutations were detected in 14 out of 45 resistant patients. Patients with clonal cytogenetic evolution tended to develop mutations more frequently than those without clonal evolution. Nine out of the 15 patients with ABL mutation responded to a treatment switch to nilotinib (n=4), dasatinib (n=2), interferon (n=1) or hematopoietic stem cell transplantation (n=2). CONCLUSION The frequency of ABL mutations in CML patients resistant to imatinib is high and is more frequent among those with clonal cytogenetic evolution. The change to second-generation TKI can overcome imatinib resistance in most of the mutated patients.