The value of routine bone marrow biopsy in patients with diffuse large B-cell lymphoma staged with PET/CT: a Danish-Canadian study.

BACKGROUND: The added diagnostic and prognostic value of routine bone marrow biopsy (BMB) in patients with diffuse large B-cell lymphoma (DLBCL) undergoing positron emission tomography combined with computed tomography (PET/CT) staging is controversial. PATIENTS AND METHODS: Patients with newly diagnosed DLBCL who underwent both staging PET/CT and BMB were retrospectively identified in British Columbia, Aalborg, and Copenhagen. Original written PET/CT and pathology reports were retrospectively reviewed to determine Ann Arbor stage and outcomes, with and without the contribution of BMB. RESULTS: A total of 530 patients were identified: 146 (28%) had focal bone marrow (BM) lesions on PET/CT and 87 (16%) had positive BMB. Fifty-two of 146 patients (36%) with positive PET/CT had a positive BMB [39 DLBCL, 13 indolent non-Hodgkin lymphoma (iNHL)], while 35 of 384 patients (9%) with negative PET/CT had positive BMB (12 DLBCL, 23 iNHL). BMB upstaged 12/209 (6%) of stage I/II patients to stage IV, although this was the case for only 3 (1%) patients with DLBCL in the BMB. PET/CT identified BM involvement by BMB with sensitivity 60%, specificity 79%, positive predictive value 36%, and negative predictive value 91%. Concordant histological involvement of the BM by DLBCL was associated with worse overall survival and progression-free survival than discordant or no involvement in univariate and multivariate analyses. CONCLUSIONS: In patients with DLBCL, staging PET/CT can miss BM involvement with concordant DLBCL (less common) or discordant iNHL (more common). Routine BMB does not add relevant diagnostic or prognostic value over PET/CT alone in the majority of patients with DLBCL.

New avenues for residual stress analysis in ultrathin atomic layer deposited free-standing membranes through release of micro-cantilevers.

The fabrication of thinnest, yet undeformed membrane structures with nanometer resolution is a prerequisite for a variety of Microelectromechanical systems (MEMS). However, functionally relevant thin films are susceptible to growth-generated stress. To tune the performance and reach large aspect ratios, knowledge of the intrinsic material properties is indispensable. Here, we present a new method for stress evaluation through releasing defined micro-cantilever segments by focused ion beam (FIB) milling from a predefined free-standing membrane structure. Thereby, the cantilever segment is allowed to equilibrate to a stress-released state through measurable strain in the form of a resulting radius of curvature. This radius can be back-calculated to the residual stress state. The method was tested on a 20 nm and 50 nm thick tunnel-like ALD Image 1 membrane structure, revealing a significant amount of residual stress with 866 MPa and 6104 MPa, respectively. Complementary finite element analysis to estimate the stress distribution in the structure showed a 97% and 90% agreement in out-of-plane deflection for the 20 nm and 50 nm membranes, respectively. This work reveals the possibilities of releasing entire membrane segments from thin film membranes with a significant amount of residual stress and to use the resulting bending behavior for evaluating stress and strain by measuring their deformation.

Performance comparison of Affymetrix SNP6.0 and cytogenetic 2.7M whole-genome microarrays in complex cancer samples.

The Affymetrix cytogenetic 2.7M whole-genome microarray (Cyto2.7M) detects genomic aberrations. The Cyto2.7M array has increased coverage in regions with cancer-related genes, ~4-fold reduced processing time, and 5-fold reduced input requirements (100 ng) compared to the commonly used Affymetrix SNP6.0 genome-wide microarray (SNP6.0). We set out to compare the performance of these microarrays on cancer samples containing complex genomic changes. We analyzed genomic DNA from 8 lymphoma samples and 1 blood sample using both SNP6.0 and Cyto2.7M microarrays. We compared the arrays with respect to 4 parameters, including detection of copy number variations (CNV), CNV boundaries, the actual copy number (CN) assigned to the aberrations, and loss of heterozygosity. The CN state of selected regions was validated by quantitative PCR. Very high consistency between arrays on all parameters tested was observed, hence only 30 of 224 aberrations disagreed on the CN state, corresponding to a total of ~12 Mb or 0.06% of the analyzed base pairs. Thus, the SNP6.0 and Cyto2.7M arrays are equally well suited to detect genomic aberrations in complex samples such as cancer samples. With reduced processing time and lower input requirements, the Cyto2.7M array enables genomic analysis of samples where only limited DNA is available.

Specific P53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients.

The mechanisms causing resistance to chemotherapeutic drugs in cancer patients are poorly understood. Recent evidence suggests that different forms of chemotherapy may exert their cytotoxic effects by inducing apoptosis. The tumor suppressor gene P53 has a pivotal role inducing apoptosis in response to cellular damage. In vitro investigations have shown intact p53 to play a critical role executing cell death in response to treatment with cytotoxic drugs like 5-fluorouracil, etoposide and doxorubicin. Recently, mutations in the P53 gene were found to confer resistance to anthracyclines in a mouse sarcoma tumor model, and overexpression of the p53 protein (which, in most cases, is due to a mutated gene) was found to be associated with lack of response to cisplatin-based chemotherapy in non-small cell lung cancer. Previous studies have shown mutations in the P53 gene or overexpression of the p53 protein to predict a poor prognosis, but also a beneficial effect of adjuvant radiotherapy or chemotherapy in breast cancer. In this study we present data linking specific mutations in the P53 gene to primary resistance to doxorubicin therapy and early relapse in breast cancer patients.

Increased level of both CD4+FOXP3+ regulatory T cells and CD14+HLA-DR⁻/low myeloid-derived suppressor cells and decreased level of dendritic cells in patients with multiple myeloma.

Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T-cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation of the immune status in patients with MM seems crucial prior to active immune therapy. We evaluated the proportion of both, DC, Treg cells and myeloid-derived suppressor cells (MDSC) in peripheral blood from patients with MM at diagnosis and in remission as well as patients with monoclonal gammopathy of undetermined significance (MGUS). We found that the proportion of both myeloid (m) DC and plasmacytoid (p) DC in patients at diagnosis was lowered compared to control donors, while only the proportion of pDC in patients in remission and with MGUS was significantly lower than in controls. The proportion of CD4+FOXP3+ Treg cells was increased in patients at diagnosis and not in patients in remission or with MGUS. Also, Treg cells from patients with MM were functionally intact as they were able to inhibit proliferation of both CD4 and CD8 T cells. Finally, we observed an increase in the proportion of CD14+HLA-DR⁻/low MDSC in patients with MM at diagnosis, illustrating that this cell fraction is also distorted in patients with MM. Taken together, our results illustrate that, both mDC, pDC, Treg cells and MDSC are affected in patients with MM underlining the fact that the immune system is dysregulated as a consequence of the disease.

Effects of the sea louse Lepeophtheirus salmonis on temporal changes in cortisol, sex steroids, growth and reproductive investment in Arctic charr Salvelinus alpinus.

Groups of mature (5+ year old) Arctic charr Salvelinus alpinus held in sea water were exposed for 34 days to either a high (mean +/-s.e. 0.15 +/- 0.01 sea lice Lepeophtheirus salmonis g(-1) fish mass) (HI), medium (0.07 +/- 0.00 sea lice g(-1) fish mass) (MI) or no [control (C)] sea-lice infection during early stages of gonad development (June to July). Infection with sea lice resulted in increased plasma cortisol concentrations and this was related to intensity of infection; females tended to have higher cortisol concentrations than males at high infection intensities (HI group: female c. 130 ng ml(-1); male c. 80 ng ml(-1)). Plasma osmolality (C c. 330, MI c. 350 and HI c. 415 mOsm) and chloride concentrations (C c. 135, MI c. 155 and HI c. 190 mM) increased significantly with infection intensity, indicating osmoregulatory problems in infected fish. A strong positive relationship between plasma osmolality and cortisol concentration was recorded. Plasma sex-steroid concentrations were influenced negatively by sea-lice infection, particularly in the HI group, and were inversely related to plasma cortisol concentrations. The most heavily infected fish postponed the initiation of reproductive development until exposed to fresh water and timing of ovulation tended to be delayed in these fish. Growth rate and condition were negatively influenced by sea-lice infection and growth rate was inversely related to plasma cortisol concentrations. Sea-lice infection resulted in mortality among females in the HI group, and the proportion of maturing females was lower in the MI group (46%) than in the controls (85%). Egg production in the MI and HI groups was c. 50 and 30% of the C group. Egg size, embryonic survival and fry mass did not differ across groups. Sea lice influence reproductive development and egg production in S. alpinus, and consequently these parasites may influence populations via sublethal effects on broodfish, affecting growth and condition, and their reproductive output.

Response of the Salmon Heart Transcriptome to Pancreas Disease: Differences Between High- and Low-Ranking Families for Resistance.

Pancreas disease caused by salmonid alphaviruses leads to severe losses in Atlantic salmon aquaculture. The aim of our study was to gain a better understanding of the biological differences between salmon with high and low genomic breeding values (H-gEBV and L-gEBV respectively) for pancreas disease resistance. Fish from H- and L-gEBV families were challenged by intraperitoneal injection of salmonid alphavirus or co-habitation with infected fish. Mortality was higher with co-habitation than injection, and for L- than H-gEBV. Heart for RNA-seq and histopathology was collected before challenge and at four- and ten-weeks post-challenge. Heart damage was less severe in injection-challenged H- than L-gEBV fish at week 4. Viral load was lower in H- than L-gEBV salmon after co-habitant challenge. Gene expression differences between H- and L-gEBV manifested before challenge, peaked at week 4, and moderated by week 10. At week 4, H-gEBV salmon showed lower expression of innate antiviral defence genes, stimulation of B- and T-cell immune function, and weaker stress responses. Retarded resolution of the disease explains the higher expression of immune genes in L-gEBV at week 10. Results suggest earlier mobilization of acquired immunity better protects H-gEBV salmon by accelerating clearance of the virus and resolution of the disease.

Effects of gadolinium contrast agents in naïve and nephrectomized rats: relevance to nephrogenic systemic fibrosis.

BACKGROUND: Human nephrogenic systemic fibrosis (NSF) is a rare condition reported in patients with severe renal insufficiency exposed to a gadolinium (Gd)-based contrast agent. An animal model of NSF could help to investigate its mechanisms and lead to prevention and treatment. PURPOSE: To evaluate a possible animal model of NSF using naive and partially nephrectomized rats to induce conditions similar to those in patients at risk of NSF. MATERIAL AND METHODS: Naive rats received intravenous doses of 5 or 10 mmol/kg Omniscan; 5 mmol/kg Magnevist; 1 mmol/kg caldiamide; 1, 2.5, or 5 mmol/kg gadodiamide; 25 micromol/kg GdCl(3); or 25 micromol/kg Gd citrate. Partially nephrectomized rats received 5 mmol/kg Omniscan; 5 mmol/kg Magnevist; 1 mmol/kg caldiamide; 1 mmol/kg gadodiamide; 25 micromol/kg GdCl(3); or 25 micromol/kg Gd citrate. There were three or four animals per group. Clinical signs were recorded during treatment. At termination, clinical biochemistry, histopathology, and tissue Gd and Zn concentrations were investigated. RESULTS: Similar responses to treatment were seen in naive and nephrectomized rats. High doses of gadodiamide were toxic, necessitating early termination of the affected animals. Skin lesions appeared in naive and nephrectomized groups treated with gadodiamide or Omniscan, coinciding with the onset of signs of pruritus, i.e., intensive scratching. The histomorphological features of the skin lesions were also consistent with superficial physical trauma. Dermal fibrosis was not a feature of these skin lesions in any of the groups, i.e., no increased collagen density, CD34+ cells, or increased fibroblasts. This was supported by skinfold measurements that demonstrated no increased skin thickness. Treatment with the gadolinium-based contrast agents and Gd salts resulted in increased Gd content of several tissues. The Gd salts were mainly taken up by the liver and spleen, possibly reflecting formation of insoluble particles and macrophage uptake. Zn tissue concentrations were normal or increased. Other major treatment-related changes included increased serum rat C-reactive protein and histamine; mineralization affecting the dermis, stomach, and blood vessels; and renal proximal tubule vacuolation. CONCLUSION: The visible skin lesions seen in this study appeared to be caused by excessive scratching in response to pruritus. As there was no evidence of dermal fibrosis, the cardinal feature of human NSF, this did not appear to be a model of human NSF.

Quantitative and qualitative assessment of exposure among employees in Norwegian smelters.

OBJECTIVES: To generate a job exposure matrix (JEM) for dust exposure in Norwegian smelters to be used in an epidemiologic study of respiratory diseases and to identify determinants of exposure. METHODS: The arithmetic mean and geometric mean (GM) of 2619 personal dust exposure measurements were applied in constructing the JEM, which was assigned to 2620 employees participating in a respiratory survey including yearly spirometry and a respiratory questionnaire. A qualitative exposure classification was constructed: (i) line operators were those employed full time in the production line, (ii) non-exposed employees were those who did not work in production and (iii) the remainder were classified as non-line operators. RESULTS: In the ferrosilicon alloy and silicon metal production group (FeSi/Si-metal), the median GM of dust exposure was 2.3 mg m(-3) (0.04-5.6) (10-90% percentiles) compared with 1.6 mg m(-3) (0.02-2.3) in the silicomanganese, ferromanganese and ferrochromium production group (SiMn/FeMn/FeCr). Multivariate analyses showed that dust exposure concentration levels decreased significantly with increasing age (FeSi/Si-metal), was significantly lower in females than in males and was significantly higher in current smokers than in never-smokers. Dust exposure concentration levels were also higher in employees reporting previous exposure to dust, fumes and gases than in employees without such previous exposure, though, significant only in the FeSi/Si-metal production group. CONCLUSION: The dust exposure levels of the employees were higher in the FeSi/Si-metal production group than in the SiMn/FeMn/FeCr production group. Age, gender, smoking status and previous exposure were significant determinants of dust exposure and should be evaluated in future analyses of the relationship between health outcomes and dust exposure in this industry.

D-dimer at venous thrombosis diagnosis is associated with risk of recurrence.

Essentials Whether D-dimer at incident venous thromboembolism (VTE) can predict recurrence-risk is unknown. We explored this association in 454 cancer-free patients with a first lifetime VTE. A low D-dimer at first VTE diagnosis was associated with a low recurrence risk. The association was predominant in patients with deep vein thrombosis and unprovoked VTE. Click to hear Dr Cannegieter's presentation on venous thrombosis: prediction of recurrence SUMMARY: Background Venous thromboembolism (VTE) is a common disease with a high recurrence rate. D-dimer measured after cessation of anticoagulant therapy predicts recurrence, and is used to decide on treatment prolongation. However, whether D-dimer measured at first VTE diagnosis can be used to assess recurrence-risk is unknown. Aims To investigate the association between D-dimer, measured at first VTE diagnosis and risk of recurrent VTE. Methods Information on clinical risk factors and laboratory markers were collected in 454 cancer-free patients with a first VTE. Recurrent VTEs and deaths during follow-up (1994-2012) were recorded. Results During a median follow-up of 3.9 years, 84 patients experienced a recurrent VTE. The crude recurrence rate was 1.7 (95% confidence interval [CI], 1.0-2.9) per 100 person-years in the lower quartile of D-dimer (≤ 1500 ng mL-1 ), and 4.9 (95% CI, 3.9-6.1) per 100 person-years in the upper three quartiles combined, yielding an absolute risk difference of 3.2 per 100 person-years. Patients with D-dimer ≤ 1500 ng mL-1 had 54% lower recurrence-risk than patients with D-dimer > 1500 ng mL-1 (HR, 0.46; 95% CI, 0.25-0.82). The association was particularly pronounced among patients with unprovoked events and deep vein thrombosis, showing a 66% (HR, 0.34; 95% CI, 0.15-0.74) and 68% (HR, 0.32; 95% CI, 0.14-0.71) lower recurrence risk among patients with D-dimer ≤ 1500 ng mL-1 , respectively. Conclusions A low D-dimer (≤ 1500 ng mL-1 ) measured at first VTE diagnosis was associated with a low recurrence risk, particularly among patients with DVT and unprovoked events. Our findings suggest that a clinical decision to avoid prolonged anticoagulant treatment could be considered based on low D-dimer at the time of VTE diagnosis.

Carotid endarterectomy: time-trends and results during a 20-year period.

AIM: The aim of this study was to evaluate the results following surgery for carotid artery stenosis in a single institution during a 20-year period. METHODS: In a retrospective study, 556 operations were performed in 496 patients during the period 1983-2002. Comorbidities, mortality, stroke and other surgical and general complications were recorded. Follow-up was performed and data retrieved from medical records, questionnaires, and visits to local hospitals. Data on late mortality were retrieved from the Norwegian Registrar's Office of birth and deaths. RESULTS: The mean age was 66.9 years (range 43-84 years), and 60% were men; 84% had symptomatic carotid artery stenosis. General anesthesia was applied in 95.5%. A shunt was used in 61.3%, and patch angioplasty in 95.1%. Autologous vein patch was used in almost all cases and there were no cases of patch rupture. Postoperative myocardial infarction occurred in 16 (2.9%) of the patients, and 5 were fatal. All types of stroke within 30 days of surgery occurred in 23 (4.1%) including 1 fatal stroke, and 7 patients died of other causes. The total stroke/mortality rate was 5.4%. Patients with previous coronary artery bypass had a favorable outcome regarding long-time survival. In contrast, increasing age, diabetes, renal failure and intermittent claudication predicted reduced long-term survival. No operations were performed for recurrent stenosis. CONCLUSIONS: We have used fairly the same policy regarding operative technique during the 20-year period and the results are in agreement with those presented in large international trials. The long-term results were favorable, and improved over time, probably due to better preoperative evaluation of the patients, better timing of surgery and treatment of comorbidities.

Acute lymphoblastic leukemia in adolescents between 10 and 19 years of age in Denmark--secondary publication.

INTRODUCTION: Data seem to indicate that young adults with acute lymphoblastic leukemia (ALL) have a better survival when treated with pediatric protocols compared with adult ALL protocols. The purpose of the study was to report the clinical characteristics and outcome of all children and young adults 10-19 years of age diagnosed with ALL in Denmark between 1992 and 2001. MATERIAL: The study includes 99 patients 10-19 years of age with ALL in Denmark during a ten year period found in the complete NOPHO (Nordic Society of Pediatric Hematology and Oncology) registry and through the Danish Cancer Registry and local pathology databases. Data were retrieved by reviewing medical charts of the patients. A total of 61 children (10-14 years) treated on pediatric protocols and 38 young adults (15-19 years) were diagnosed with ALL. Data were reported as of January 1st 2005. RESULTS: There were no difference with respect to the distribution of T-ALL, CNS-leukemia, total white blood count and high risk chromosomal abnormalities between the two groups. There was a statistical significant lower event free survival (p<0.01) and lower overall survival (p<0.01) in young adults compared with 10-14 year-old children (0.38 vs 0.60 and 0.47 vs 0.67). There were more transplant-related deaths in the young adults. Higher treatment intensity in children may be an additional explanatory factor. Children received more prednisone, vincristine and high-dose methotrexate than young adults. CONCLUSION: Young adult patients with ALL might benefit from therapy with pediatric NOPHO ALL protocols.

The APC gene I1307K variant is rare in Norwegian patients with familial and sporadic colorectal or breast cancer.

Recently, a T-to-A transversion creating an 8-base mononucleotide tract in the APC gene, resulting in substitution of lysine for isoleucine at codon 1307 (I1307K), was found in a subset of Ashkenazi Jews. This sequence variant was most frequent in colorectal cancer patients with a positive family history of colorectal cancer. To determine whether the I1307K variant plays a role in colorectal or breast cancer predisposition in the Norwegian population, we have analyzed blood samples from 210 colorectal cancer patients and 183 breast cancer patients by PCR and direct sequencing. Thirty-seven of the colorectal cancer patients had a positive family history of cancer. Among the breast cancer patients, 24 had a family history of colorectal cancer and 75 a family history of breast and/or ovarian cancer. Only one colorectal cancer patient who belonged to a Jewish family was found to carry the A variant. Our data show that the I1307K variant is rare in the Norwegian population and should not be viewed as a candidate for susceptibility testing for colorectal cancer.

Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer.

TP53 status [mutations, immunostaining, and loss of heterozygosity (LOH)], expression of c-erbB-2, bcl-2, and histological grading were correlated to the response to doxorubicin monotherapy (14 mg/m2) administered weekly to 90 patients with locally advanced breast cancer. Mutations in the TP53 gene, in particular those affecting or disrupting the loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (P = 0.063 for all mutations and P = 0.008 for mutations affecting L2/L3, respectively). Similarly, expression of c-erbB-2 (P = 0.041), a high histological grade (P = 0.023), and lack of expression of bcl-2 (P = 0.018) all predicted chemoresistance. No statistically significant association between either p53 immunostaining or TP53 LOH and response to therapy was recorded, despite the finding that both were associated with TP53 mutation status (p53 immunostaining, P < 0.001; LOH, P = 0.021). Lack of immunostaining for p53 despite mutation of the TP53 gene was particularly seen in tumors harboring nonsense mutations or deletions/splices (7 of 10 negative for staining compared with 4 of 16 with missense mutations). TP53 mutations (total/affecting L2/L3 domains) were associated with expression of c-erbB-2 (P < 0.001 for both), high histological grade (P = 0.001 and P = 0.025), and bcl-2 negativity (P = 0.003 and P = 0.002). TP53 mutations, histological grade, and expression of bcl-2 (but not LOH or c-erbB-2 expression) all predicted for relapse-free as well as breast cancer-specific survival in univariate analysis (Ps between <0.0001 and 0.0155), but only tumor grade was found to be predictive in multivariate analysis (P = 0.01 and P = 0.0007, respectively). Our data are consistent with the hypothesis that certain TP53 mutations predict for resistance to doxorubicin in breast cancer patients. However, the observation that the majority of patients with TP53 mutations affecting or disrupting the L2/L3 domains with LOH in addition (n = 12) obtained a partial response (n = 4) or stabilization of disease (n = 5) during chemotherapy suggests redundant mechanisms to compensate for loss of p53 function. Our findings are consistent with the hypothesis that other defects may act in concert with loss of p53 function, causing resistance to doxorubicin in breast cancers.

Hospital-related first venous thromboembolism and risk of recurrence.

Essentials Recurrence risk after a hospital-related venous thromboembolism (VTE) is underinvestigated. We explored this association in a cohort of patients with a first VTE from the Tromsø study. Stratification on hospital-related factors revealed considerable differences in recurrence risk. The recurrence risk was high in cases with a VTE related to hospitalization for medical illness. SUMMARY: Background Hospitalization is a well-established risk factor for first venous thromboembolism (VTE), but the risk of recurrence, particularly in patients hospitalized for conditions other than cancer or surgery, has scarcely been investigated. The cumulative incidence of recurrence in hospital-related VTE may be influenced by the competing risk of death. Objectives To investigate the risk of recurrence and mortality among patients with a first hospital-related VTE in models with and without death as a competing event. Methods Information on hospital-related risk factors was collected in 822 patients with a first-lifetime VTE derived from the Tromsø study. Recurrent VTEs and deaths were recorded during follow-up (1994-2012). Results During a median of 2.79 years of follow-up, 132 patients experienced a recurrent VTE. Stratification on hospital-related factors revealed considerable differences in recurrence risk. The 5-year cumulative incidence of recurrence was 27.4%, 11.0% and 20.1% in patients with incident VTEs related to cancer, surgery or other medical illness, respectively, and 18.4% in patients with a non-hospital-related first VTE. The mortality rates were high for all subgroups of hospital-related VTE, except for surgery-related events. Consequently, the cumulative incidence of recurrence dropped in the competing risk analyses, showing a 5-year cumulative incidence of 14.4%, 11.7% and 9.7% in patients with a first VTE related to hospitalization for other medical illness, cancer or surgery, respectively. Conclusions Our findings suggest that patients with incident VTEs related to hospitalization for medical illness other than cancer or surgery have a high recurrence-risk, even in the presence of competing risk of death.

Management of relapsed multiple myeloma: recommendations of the International Myeloma Working Group.

The prognosis for patients multiple myeloma (MM) has improved substantially over the past decade with the development of new, more effective chemotherapeutic agents and regimens that possess a high level of anti-tumor activity. In spite of this important progress, however, nearly all MM patients ultimately relapse, even those who experience a complete response to initial therapy. Management of relapsed MM thus represents a vital aspect of the overall care for patients with MM and a critical area of ongoing scientific and clinical research. This comprehensive manuscript from the International Myeloma Working Group provides detailed recommendations on management of relapsed disease, with sections dedicated to diagnostic evaluation, determinants of therapy, and general approach to patients with specific disease characteristics. In addition, the manuscript provides a summary of evidence from clinical trials that have significantly impacted the field, including those evaluating conventional dose therapies, as well as both autologous and allogeneic stem cell transplantation. Specific recommendations are offered for management of first and second relapse, relapsed and refractory disease, and both autologous and allogeneic transplant. Finally, perspective is provided regarding new agents and promising directions in management of relapsed MM.

Short-term liquid marrow cultures are supported by a mixture of haematopoietic cytokines but do not purge for acute myeloid or lymphoid leukemic marrow cells.

Short-term liquid marrow culture (STLMC) is a potential source for autografting in leukemia. In a preclinical setting, including candidates for autologous marrow transplantation, we have studied STLMC supported by a selected mixture of clinical available recombinant human haematopoietic growth factors. STLMC of leukemic marrow cells were prospectively performed to evaluate the purging effect. Bone marrow cells cultured and supported by the selected mixture of rhIL-3/rhGM-CSF/rhEpo revealed an increased number of day 10-12 cultured cells, parallelled by an increased proliferation rate when compared to unstimulated cultures. The median number of myeloid progenitors recognized as day 7 and day 14 granulocyte-macrophage colony-forming units (day 7/14 GM-CFU) was significantly increased in the supported STLMC to 145/305 from 105/115 per ml culture (n = 7, p < 0.01). Further addition of rhKL did not enhance the numbers of day 7 or day 14 GM-CFUs per ml culture. In no instance was the number of clonogenic cells at the end of culture greater than the input day 0, except in cultures of purified CD34-positive marrow progenitors which resulted in an expansion of late myeloid progenitors. Cytokine-supported cultures of leukemic marrow cells from acute myeloid (n = 14) and lymphoblastic (n = 7) leukemia patients were established at the time of diagnosis. In the supported cultures, the cell number increased for myeloblast but was unchanged for lymphoblast leukemic marrow cells compared to non-supported cultures. Immunophenotypic and cytogenetic studies of selected leukemic cell samples identified unchanged myeloid or slightly reduced frequencies of lymphoblastic leukemic cells at the end of culture. This preclinical study supports the idea that the addition of a mixture of clinical available haemopoietic cytokines to STLMC increases the recovery of detectable myeloid progenitors which may enhance myeloid regeneration after autografting. No substantial selective loss of myeloid leukemic cells was found in the cytokine-supported short-term culture system.

CD34+ megakaryoblastic leukaemic cells are CD38-, but CD61+ and glycophorin A+: improved criteria for diagnosis of AML-M7?

The aim of this flow cytometry study in acute megakaryoblastic leukaemia (AML-M7) was to describe the membrane phenotype of CD34+ progenitor subsets and compare these with the phenotypes expressed by other AML FAB types. Following conventional histopathological diagnosis mononuclear cells from bone marrow and blood were examined in seven patients with AML-M7 and compared with results from 26 sequential patients with AML-M0 to AML-M6. The CD34+ subsets in AML-M7 patients differed from that of patients with AML-M0 to AML-M6 as the CD34+ CD61+ and the CD34+ Glycophorin A+ subsets were median 31% and 20%, respectively, compared to 4% and 2% in the AML-M0 to AML-M6 (P = 0.0005). Only 1% of the CD34+ progenitors were CD34+ CD38+ in AML-M7 compared to 72% in other AML subtypes (P < 0.000). These findings suggest that the CD34+ cell compartment in AML-M7 consists of early lineage-specific progenitors. In conclusion, flow cytometry analysis of CD34+ subsets may improve the diagnostic safety in AML-M7 and consequently the prognostic significance of immunophenotyping in acute leukaemia.

Detection of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies using end sequenced probes.

Translocations involving the immunoglobulin loci are recurring events of B cell oncogenesis. The majority of translocations involve the immunoglobulin heavy chain (IGH) locus, while a minor part involves the immunoglobulin light chain loci consisting of the kappa light chain (IGK) located at 2p11.2 and the lambda light chain (IGL) located at 22q11.2. We characterised BAC clones, spanning the IGK and IGL loci, for detection of illegitimate rearrangements by fluorescence in situ hybridisation (FISH). Within the IGL region we have identified six end sequenced probes (22M5, 1152K19, 2036J16, 3188M21, 3115E23 and 274M7) covering the variable (IGLV) cluster and two probes (165G5 and 31L9) covering the constant (IGLC) cluster. Within the IGK region four probes (969D7, 316G9, 122B6 and 2575M21) have been identified covering the variable (IGKV) cluster, and one probe (1021F11) covering the IGK constant (IGKC) cluster. A series of 24 cell lines of different origin have been analysed for the presence of translocations involving the immunoglobulin light chain loci by dual-colour FISH where the split of the variable cluster and the constant cluster indicated a translocation. Probes established in this study can be used for universal screening of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies.

Quantitation of minimal residual disease in multiple myeloma using an allele-specific real-time PCR assay.

OBJECTIVE: To develop a real-time PCR method, based on the 5'nuclease TaqMan technology, for quantitation of clonal cells in multiple myeloma (MM). MATERIALS AND METHODS: The real-time quantitative PCR method incorporates both an allele-specific oligonucleotides (ASO) primer and an ASO dual-labeled fluorogenic probe (ASO TaqMan probe). The ASO primer and probe corresponded to the complementary determining region 3 (CDR3) of the rearranged immunoglobulin heavy chain gene (IgH). With the use of a sequence detector, PCR product accumulation was measured through the ASO TaqMan probe. The real-time PCR method was compared with flow cytometric quantitation of myeloma plasma cells. RESULTS: The application of the real-time quantitative ASO IgH PCR method is illustrated by a sequential analysis of minimal residual disease (MRD) in bone marrow (BM) samples from myeloma patients undergoing peripheral blood stem cell (PBSC) transplantation. The real-time PCR method was able to quantitate residual malignant cells in BM samples from patients who were considered to be in complete remission. Further, it was illustrated that a potential problem in determining tumor cell content in myeloma BM samples is the heterogeneous infiltration of the marrow. CONCLUSION: The application of the real-time PCR method provides a sensitive, highly specific, and reproducible quantitation of myeloma cells.