Svetlana Lagercrantz

Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy

Gene profiling studies of patients with chronic lymphocytic leukemia (CLL) has revealed increased expression of Ror1, a cell surface receptor tyrosine kinase. The aim of present study was to analyze gene and protein expression of Ror1 in CLL cells and normal blood leukocytes. Gene expression analysis reverse transcription‐polymerase chain reaction of ROR1 revealed that all patients with CLL (n = 100) spontaneously expressed ROR1 mRNA whereas enriched blood B and T cells as well as granulocytes from healthy donors (n = 10) were negative. A strong nonphysiological activation signal (PMA/ionomycin) was required to induce expression in vitro in normal lymphocytes. Major genomic aberrations (mutations or truncation) of ROR1 were not observed. Protein expression was analyzed by Western blot using a panel of polyclonal anti‐Ror antibodies as well as flow cytometry. Blood lymphocytes from 18/18 CLL patients, but none of the 10 healthy donors, expressed surface Ror1. The majority of CLL cells exhibited Ror1 surface expression (71% mean; range 36–92%) with a mean fluorescence intensity (MFI) of 20 (range 10–45). The corresponding MFI of CD19 on CLL cells was 26 (range 9–48). There was no difference in the Ror1 protein expression comparing IgVH mutated and unmutated cases as well as progressive and nonprogressive CLL patients. Two different variants of the Ror1 protein, 105 and 130 kDa, were identified. The Ror1 protein expression in patients with CLL but not in normal leukocytes merits further studies of its role in the pathobiology of CLL, which may provide a basis for development of Ror1 directed targeted therapy.

Chromosomal Imbalances in Diffuse Large B-Cell Lymphoma Detected by Comparative Genomic Hybridization

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma. In contrast to many other hematological malignancies, no chromosomal abnormalities with a diagnostic or prognostic value have been identified in DLBCL. Numerical chromosomal imbalances were characterized by comparative genomic hybridization (CGH) performed on 54 DLBCL tumors from a total of 40 patients. The clonal relatedness was demonstrated in 9 of 11 pairs of matched diagnostic tumors and their relapses as determined by IGH gene rearrangement analysis and/or the CGH profiles. Furthermore, immunohistochemical expression analyses of BCL2 and BCL6/LAZ3 were performed on all cases. Copy number changes were detected in 94% of the diagnostic tumor samples and in all of the relapses. Chromosomal losses in diagnostic tumors were preferentially observed at 8p22-pter (29%), 1p34-pter (26%), 6q23-qter (20%), 17p12-pter (17%) and 22q (17%), 9p23-pter (14%), whereas gains were mainly seen in Xq25–26 (43%), 13q22 (26%), 12cen-q14 (20%), 3q24–25 (11%), 7 (11%), and 18q12–21 (11%). Loss of 22q was significantly more commonly seen in the diagnostic tumor samples with more advanced clinical stage in other words, Stage III–IV compared with Stage I–II, and band 18q21 was significantly more often gained in relapses as compared to diagnostic tumors. None of the recurrent alterations were detected as a single abnormality, suggesting that other genetic lesions below the detection level of CGH may be the initiating event in the tumorigenesis of DLBCL. However, the distribution of CGH alterations support the idea of a progression of genetic events where loss of 8p and 9p and gain of 3q, 13q, and 18q would represent relatively early events because they were distributed in tumors with only two abnormalities.

Characterization of 6q deletions in mature B cell lymphomas and childhood acute lymphoblastic leukemia

The study was undertaken with the aim to outline deletion patterns involving the long arm of chromosome 6, a common abnormality in lymphoproliferative disorders. Using a chromosome 6 specific tile path array, 60 samples from in total 49 cases with mantle cell lymphoma (MCL), de novo diffuse large B-cell lymphoma (DLBCL), transformed DLBCL as well as preceding follicular lymphoma (FL), and childhood acute lymphoblastic leukemia (ALL), were characterized. Twenty-six of the studied cases, representing all diagnoses, showed a 6q deletion among which 85% involved a 3 Mb region in 6q21. The minimal deleted interval in 6q21 encompasses the FOXO3A, PRDM1 and HACE1 candidate genes. The PRDM1 gene was found homozygously deleted in a case of DLBCL. Moreover, in two DLBCL cases, an overlapping homozygous deletion was identified in 6q23.3 – 24.1, encompassing the TNFAIP3 gene among others. Taken together, we refined the deletion pattern within the long arm of chromosome 6 in four different types of hematological malignances, suggesting the location of tumor suppressor genes involved in the tumor progression.

Gain of 1q and loss of 9q21.3-q32 are associated with a less favorable prognosis in papillary thyroid carcinoma

In order to approach the genetic mechanisms behind initiation and progression of papillary thyroid carcinoma (PTC) tumorigenesis, we characterized numerical chromosomal imbalances in a panel of 25 PTCs with varying histopathological and clinical features using comparative genomic hybridization (CGH). The most frequently detected imbalance was gain of 9q33‐qter, which was seen in close to 30% of the cases. The commonly occurring regions of loss were assigned to 22q (12%) and 9q21.3‐q32 (12%), while gains preferentially involved the entire X chromosome (20%), 1q (16%), 17q (16%), and 22q (12%). The distribution of CGH alterations supports the idea of a progression of genetic events in the development of PTC, where gain of 9q33‐qter would represent a relatively early event that is followed by loss of 22q and gain of X, 1q, 17q, and 22q. When the detected CGH alterations were compared with the clinical outcome and the histopathological features of the 25 PTC cases, several statistically significant correlations were revealed. The total number of genetic alterations was higher in tumors from patients with aggressive disease as compared to those without signs of aggressiveness. Gain of 1q and loss of 9q21.3‐q32 were exclusively seen in tumors from patients with aggressive disease, and the presence of distant metastases was associated with gain of 1q. A sex‐dependent distribution was also evident for one of the common alterations, with gain of X exclusively seen in male cases. Taken together, the findings identify several candidate locations for tumor suppressor genes and oncogenes that are potentially involved in the establishment and progression of papillary thyroid carcinogenesis.

A novel B-cell line (U-2932) established from a patient with diffuse large B-cell lymphoma following Hodgkin lymphoma.

Little is known about mechanisms leading to secondary non-Hodgkin lymphomas (NHL) in patients treated for Hodgkin lymphoma (HL). Our aim was to characterise in detail a cell line derived from a diffuse large B-cell lymphoma (DLBCL) that had developed in a patient with relapsing HL. The cell line U-2932 was established from ascites in a patient suffering from DLBCL previously treated for HL with multiple chemotherapy regimens. Characterisation was based on morphology, immunophenotype, Epstein-Barr virus (EBV)-status, IgH gene rearrangement status, tumourigenicity, p53 sequencing, and immunohistochemical expression of p53, BCL-2 and BCL-6. The karyotype was investigated using G-banding, comparative genomic hybridisation (CGH) and spectral karyotype (SKY) analysis. This cell line shows typical morphological features of a DLBCL and grows as colonies in nude mice. It expresses a B-cell phenotype with a somatically hypermutated V H 4-39 gene and is negative for EBV. The origin of U-2932 was confirmed by demonstrating an identical V H 4 rearrangement in ascites from the patient. A point mutation of the tumour-suppressor gene p53 was detected in amino acid position 176 and immunohistochemical over-expression of the p53 protein was also demonstrated. U-2932 carries a complex karyotype including high-level amplifications of the chromosomal bands 18q21 and 3q27 and expresses aberrant BCL-2 and BCL-6 immunohistochemically. We were unable to investigate the clonal relationship between the original HL and U-2932. In conclusion, U-2932 is a unique B cell line established from a patient suffering from HL followed by NHL. Overexpression of BCL-2, BCL-6 and p53 may play a role in the tumourigenesis and drug resistance. This cell line may become a useful tool to better understand the mechanisms responsible for development of secondary NHL in patients treated for HL.

Genomic imbalances during transformation from follicular lymphoma to diffuse large B-cell lymphoma

Follicular lymphoma is commonly transformed to a more aggressive diffuse large B-cell lymphoma (DLBCL). In order to molecularely characterize this histiological and clinical transformation, comparative genomic hybridization was applied on 23 follicular lymphoma and 35 transformed DLBCL tumors from a total of 30 patients. The results were also compared with our published findings in de novo DLBCL. Copy number changes were detected in 70% of follicular lymphoma and in 97% of transformed DLBCL. In follicular lymphoma, the most common alterations were +18q21 (33%), +Xq25–26 (28%), +1q31–32 (23%), and −17p (23%), whereas transformed DLBCL most frequently exhibited +Xq25–26 (36%), +12q15 (29%), +7pter-q22 (25%), +8q21 (21%), and −6q16–21(25%). Transformed DLBCL showed significantly more alterations as compared to follicular lymphoma (P=0.0001), and the alterations −6q16–21 and +7pter-q22 were only found in transformed DLBCL but not in follicular lymphoma (P=0.02). Alterations involving +13q22 were significantly less frequent, whereas −4q13–21 was more common in transformed as compared to de novo DLBCL (P=0.01 and P=0.02, respectively). Clinical progression from follicular lymphoma to transformed DLBCL is on the genetic level associated with acquirement of increasing number of genomic copy number changes, with non-random involvement of specific target regions. The findings support diverse genetic background between transformed and de novo DLBCL.

Mantle cell lymphomas with clonal immunoglobulin V(H)3-21 gene rearrangements exhibit fewer genomic imbalances than mantle cell lymphomas utilizing other immunoglobulin V(H) genes.

A preferential use of one particular immunoglobulin variable heavy chain gene, VH3–21, has recently been reported in mantle cell lymphoma, where almost all of these VH3–21+ mantle cell lymphomas showed usage of the same light chain Vλ gene (Vλ3–19) and also had a tendency towards improved prognosis. These findings suggested that VH3–21+ mantle cell lymphomas constitute a distinct subgroup, possibly with antigen stimulation involved in disease pathogenesis. In this study, we applied the comparative genomic hybridization (CGH) method on 37 mantle cell lymphoma tumors in order to investigate if the VH3–21+ tumors are different at the genomic level. Interestingly, VH3–21+ mantle cell lymphomas (n=14) showed significantly fewer genomic aberrations (mean 2.4) compared to non-VH3–21 mantle cell lymphomas (n=23) (mean 4.9). The chromosomal aberrations identified in our study were generally in accordance with previous CGH studies of mantle cell lymphoma; the most frequent aberration was complete or partial loss of chromosome 13, followed by recurrent losses within 6q, 9p, 9q and 11q and frequent gains in 3q, 7p, 8q and 15q. Deletions within 8p and 9p as well as gains in 7p and 15q were found exclusively in the non-VH3–21-utilizing tumors. In summary, VH3–21+ mantle cell lymphomas demonstrated both a lower number and a different spectrum of genomic aberrations than mantle cell lymphoma in general, thus supporting the hypothesis that VH3–21+ mantle cell lymphomas constitute a new subgroup. The findings presented in this report may explain the tendency for a better clinical outcome for patients whose tumors utilize VH3–21.

Molecular cytogenetic characterization of four commonly used cell lines derived from Hodgkin lymphoma

Hodgkin lymphoma (HL) is a malignant lymphoma composed of a minority of neoplastic cells-the Hodgkin and Reed-Sternberg (HRS) cells-and a majority of nonneoplastic inflammatory cells. The low proportion of tumor cells makes genetic studies of primary neoplasia difficult. Therefore, established HL-derived cell lines are commonly used as model systems. Here we have characterized the chromosomal composition of four such cell lines: L-540, DEV, HDLM-2, and CO. Using spectral karyotyping (SKY), reversed DAPI banding, and comparative genomic hybridization (CGH), the karyotypes were characterized and previously unidentified marker chromosomes were resolved. The karyotype for CO was incompatible with the original description but showed striking similarities with the T-ALL-derived cell line CCRF-CEM, suggesting that CO had been cross-contaminated and overgrown prior to arrival at our laboratory. Multiple numerical and structural abnormalities were identified in DEV and HDLM-2, as well as in L-540. Refined composed karyotypes are suggested for the cell lines studied, to be used as references for further studies of lymphoma.

U-2940, a human B-cell line derived from a diffuse large cell lymphoma sequential to Hodgkin lymphoma

Several patterns of association between Hodgkin and non‐Hodgkin lymphomas are recognized, some of which support a common cellular origin or shared transformation events for both malignancies. We describe the U‐2940 cell line derived from a diffuse large B‐cell lymphoma with some features consistent with mediastinal large B‐cell lymphoma, clinically apparent 1 month after the initial course of chemotherapy for Hodgkins disease, fulfilling the criteria for composite malignancies. U‐2940 cells display a mature B phenotype with hypermutated IgH rearrangement typical of germinal/postgerminal center origin. The cell line is negative for Epstein‐Barr virus and no evidence of t(14;18) was found. U‐2940 cells display multiple chromosomal rearrangements similar to recurrent aberrations described in both Hodgkin and non‐Hodgkin lymphomas, also partially shared by U‐2932 derived from a B‐cell lymphoma sequential to Hodgkins disease. The original large B‐cell lymphoma and the U‐2940 cell line bear microsatellite instability, an abnormality associated with particular subtypes of non‐Hodgkin lymphomas and found in tissues involved by Hodgkin lymphoma. Therefore, U‐2940 cells bear several features known to occur in Hodgkin and in non‐Hodgkin lymphomas, leading to the assumption that this cell line may constitute a useful tool to address elective pathways of lymphomagenesis and eventually the Hodgkin and non‐Hodgkin lymphoma association.

Alterations of biomarker profiles after neoadjuvant chemotherapy in breast cancer: tumor heterogeneity should be taken into consideration

Tumor biomarkers including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 are routinely tested in breast cancer patients and their status guides clinical management and predicts prognosis. A few retrospective studies have suggested that neoadjuvant chemotherapy (NAC) in breast cancer may change the status of biomarker expression, which in turn will affect further management of these patients. In this study we take advantage of a relatively large cohort and aim to study the effect of NAC on biomarker expression and explore the impact of tumor size and lymph node involvement on biomarker status changes. We collected 107 patients with invasive breast cancer who received at least three cycles of NAC. We retrospectively performed and scored the immunohistochemistry (IHC) of ER, PR, HER2 and Ki-67 using both the diagnostic core biopsies before NAC and excisional specimens following NAC. HER2 gene status was assessed by fluorescence in situ hybridization for cases with IHC result of 2+. We demonstrated that there was a significant decrease in expression of PR (P = 0.013) and Ki-67 (P = 0.000) in post-NAC specimens compared to pre-NAC core biopsies. In addition, cases with large tumor size (≥2cm) and cases with lymph node metastasis were more frequently to have biomarker changes. Finally we studied cases with HER2 status changes after NAC treatments in detail and emphasized the nature of tumor heterogeneity.