How I treat diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is usually treated with chemoimmunotherapy in curative intention at initial diagnosis. Novel agents have improved the prognosis of high-risk patients in the front-line and relapsed setting and more accurate prognostic tools enable less intensive treatment for low-risk patients, while maintaining their good prognosis. Here, we summarize our approach to DLBCL patients in the first-line setting according to their risk profile and other common challenges in clinical practice. We recommend an abbreviated course of chemoimmunotherapy in low-risk patients and a negative interim positron emission tomography. For patients with higher-risk disease, a new combination treatment with polatuzumab vedotin has been approved and is a new option in these patients. We also discuss our approach to patients with high risk for subsequent central nervous system involvement, with leg-type lymphoma or with severe comorbidities.

Clonal evolution in diffuse large B-cell lymphoma with central nervous system recurrence.

BACKGROUND: The prognosis of patients with secondary central nervous system lymphoma (SCNSL) is poor and despite massive advances in understanding the mutational landscape of primary diffuse large B-cell lymphoma (DLBCL), the genetic comparison to SCNSL is still lacking. We therefore collected paired samples from six patients with DLBCL with available biopsies from a lymph node (LN) at primary diagnosis and the central nervous system (CNS) at recurrence. PATIENTS AND METHODS: A targeted, massively parallel sequencing approach was used to analyze 216 genes recurrently mutated in DLBCL. Healthy tissue from each patient was also sequenced in order to exclude germline mutations. The results of the primary biopsies were compared with those of the CNS recurrences to depict the genetic background of SCNSL and evaluate clonal evolution. RESULTS: Sequencing was successful in five patients, all of whom had at least one discordant mutation that was not detected in one of their samples. Four patients had mutations that were found in the CNS but not in the primary LN. Discordant mutations were found in genes known to be important in lymphoma biology such as MYC, CARD11, EP300 and CCND3. Two patients had a Jaccard similarity coefficient below 0.5 indicating substantial genetic differences between the primary LN and the CNS recurrence. CONCLUSIONS: This analysis gives an insight into the genetic landscape of SCNSL and confirms the results of our previous study on patients with systemic recurrence of DLBCL with evidence of substantial clonal diversification at relapse in some patients, which might be one of the mechanisms of treatment resistance.

Effects of Implementing a Comprehensive Opioid Reduction Protocol on Overall Opioid Prescribing Among Patients with Chronic, Non-Cancer Pain in a Rural Family Medicine Clinic: A Controlled Cross-over Trial.

BACKGROUND: The opioid crisis presents many challenges for family practice providers in rural communities who treat patients with chronic non-cancer pain (CNCP). Unfortunately, evidence for effective opioid reduction strategies is sparse. We evaluated the effects of implementing a comprehensive opioid reduction protocol on overall opioid prescribing among patients with chronic non-cancer pain in our rural family medicine clinics. METHODS: We compared mean daily milligrams morphine equivalent (MME) prescribed to patients with CNCP in our rural family medicine clinic (n = 93) with another matched clinic (n =93) after implementation of our comprehensive protocol. We also compared mean daily MME prescribed to our patients with CNCP before and after implementation of the protocol. In a subsequent cross over phase, we examined the effects of the protocol when applied to the original control group patients. RESULTS: Mean daily MME in the intervention clinic (29.77) was significantly lower than the control clinic (93.2) after the intervention (t = 6.03; P < .00). Mean daily MME in the intervention group was significantly lower after implementation of the protocol (29.77) than before the protocol (MME 80.34) (t = 5.889; P < .00). After crossover, the mean daily MME was significantly lower (14.34) in the original control group than prior to the cross over intervention (85.68); (t = 8.19; P = .00). DISCUSSION: Our comprehensive opioid reduction protocol led to significant reductions in opioid prescribing in our rural family medicine clinics. Future studies should include important qualitative outcome measures such as patient function.

Correction: The influence of FCGR2A and FCGR3A polymorphisms on the survival of patients with recurrent or metastatic squamous cell head and neck cancer treated with cetuximab.

In the abstract and in other parts of the manuscript the authors wrote that the mutation rs396991 causes a valine (V) to phenylalanine (F) substitution at position 157. However, the correct codon number is 158. These errors have not been fixed in the original Article.

Alterations of sex determination pathways in the genital ridges of males with limited Y chromosome genes†.

We previously demonstrated that in the mouse only two Y chromosome genes are required for a male to produce an offspring with the help of assisted reproduction technologies (ART): testis determinant Sry and spermatogonial proliferation factor Eif2s3y. Subsequently, we have shown that the function of these genes can be replaced by transgenic overexpression of their homologs, autosomally encoded Sox9 and X-chromosome encoded Eif2s3x. Males with Y chromosome contribution limited to two (XEif2s3yOSry), one (XEif2s3yOSox9 and XOSry,Eif2s3x), and no genes (XOSox9,Eif2s3x) produced haploid germ cells and sired offspring after ART. However, despite successful assisted reproductive outcome, they had smaller testes and displayed abnormal development of the seminiferous epithelium and testicular interstitium. Here we explored whether these testicular defects originated from altered pro-testis and pro-ovary factor signaling in genital ridges at the time of sex determination. Timed pregnancies were generated to obtain transgenic XEif2s3yOSry, XEif2s3yOSox9, XOSry,Eif2s3x, XOSox9,Eif2s3x, and wild-type XX and XY fetuses at 12.5 days post coitum. Dissected genital ridges were assessed for their morphology and anatomy, and expression of pro-testis and pro-ovary transcripts. All transgenic males displayed incomplete masculinization of gonadal shape, impaired development of testicular cords and gonadal vasculature, and decreased expression of factors promoting male pathway. Fetal gonad masculinization was more effective when sex determination was driven by the Sry transgene, in the presence of Y chromosome genes, and to a lesser extent a double dosage of X genes. The study adds to the understanding of the role of Y chromosome genes and their homologs during sex determination.

The influence of FCGR2A and FCGR3A polymorphisms on the survival of patients with recurrent or metastatic squamous cell head and neck cancer treated with cetuximab.

FCGR2A-H131R and FCGR3A-V157F are single-nucleotide polymorphisms known to influence the outcome of patients treated with rituximab, cetuximab and trastuzumab. We investigated the impact of these polymorphisms on the clinical outcome of 103 patients with recurrent or metastatic squamous cell carcinoma of the head and neck treated with a platinum compound, fluorouracil and cetuximab as palliative first-line therapy. The survival of patients with FCGR2A-131H/H and/or FCGR3A-157V/V genotypes was significantly longer compared with patients carrying 131R and 157F alleles (median progression-free survival (PFS): 5.5 vs 4.1 months, P=0.02; median overall survival: 10.2 vs 7.2 months, P=0.04). In multivariate analysis, the FCGR2A and 3A genotypes as well as the time between initial diagnosis and relapse of disease not amenable to curative therapy remained the only independent prognostic factors for PFS. The results are in line with previous reports in colorectal cancer patients and confirm the possible value of genetic polymorphisms of immunocompetent cells for the success of cetuximab treatment.

Red blood cell alloimmunization in 184 patients with myeloid neoplasms treated with azacitidine - A retrospective single center experience.

Alloimmunization to Red Blood Cell (RBC) antigens frequently occurs in patients with myeloid neoplasms (AML, MDS and CMML) and potentially poses the patient at risk for delayed hemolytic transfusion reactions and limited supply of compatible RBC-units. However, there is comparatively little data on transfusion associated characteristics in this patient cohort. We therefore retrospectively analyzed transfusion requirements and clinical outcomes of 184 patients with myloid neoplasms treated with azacitidine at the Paracelsus Medical University Salzburg, which were included in the Austrian Registry of Hypomethylating Agents. The mean blood component requirements for AML, MDS and CMML were 39.8, 67.4 and 31.4 RBC units and 31.7, 27.6 and 19.1 platelet (PLT) units respectively. In spite of an extended and stringent RBC unit matching policy (ABO, RhD, RhCcEe and K antigens), 20 (11%) patients formed at least one alloantibody ("allo-group"), whereas 164 patients (89%) did not ("non-allo-group"). The most frequent antibody specificity was anti-E, followed by anti-Wra -Lua, -D, -C and -Jka. Alloimmunization was associated with higher numbers of transfused RBC units (68 vs. 38; p=0.001), as well as with longer time under transfusion (16.7 vs. 9.4 months; p=0.014). Median overall survival (OS) did not differ significantly between the "allo"- and "non-allo-group".

Testicular abnormalities in mice with Y chromosome deficiencies.

We recently investigated mice with Y chromosome gene contribution limited to two, one, or no Y chromosome genes in respect to their ability to produce haploid round spermatids and live offspring following round spermatid injection. Here we explored the normalcy of germ cells and Sertoli cells within seminiferous tubules, and the interstitial tissue of the testis in these mice. We performed quantitative analysis of spermatogenesis and interstitial tissue on Periodic acid-Schiff and hematoxylin-stained mouse testis sections. The seminiferous epithelium of mice with limited Y gene contribution contained various cellular abnormalities, the total number of which was higher than in the males with an intact Y chromosome. The distribution of specific abnormality types varied among tested genotypes. The males with limited Y genes also had an increased population of testicular macrophages and internal vasculature structures. The data indicate that Y chromosome gene deficiencies in mice are associated with cellular abnormalities of the seminiferous epithelium and some changes within the testicular interstitium.

Ristocetin-induced platelet aggregation for monitoring of bleeding tendency in CLL treated with ibrutinib.

Bleeding because of impaired platelet function is a major side effect of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. We quantitatively assessed ristocetin-induced platelet aggregation (RIPA) in 64 patients with chronic lymphocytic leukemia (CLL) under ibrutinib at 287 time points. Eighty-seven bleeding episodes in 39 patients were registered (85 Common Toxicity Criteria (CTC) grade 1 or 2, 2 CTC grade 3) during a median observation period of 10.9 months. At times of bleeding, RIPA values were significantly lower (14 vs 28 U; P<0.0001). RIPA was impaired in patients receiving concomitant antiplatelet therapy or anticoagulation (14 vs 25 U, P=0.005). A gradual decline of median RIPA values was observed with increasing bleeding severity. Importantly, no CTC grade 2 or 3 bleeding were observed with RIPA values of >36 U. Sequential monitoring indicated a decrease of RIPA values from a median of 17 to 9 U within 2 weeks after initiation of treatment as well as an increase above the critical threshold of 36 U within 7 days when ibrutinib was paused. Low RIPA values were similar during treatment with another BTK inhibitor, CC292. Quantitative assessment of platelet function is a practical tool to monitor bleeding tendency under BTK-inhibitor therapy.

Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction.

The mammalian Y chromosome is considered a symbol of maleness, as it encodes a gene driving male sex determination, Sry, as well as a battery of other genes important for male reproduction. We previously demonstrated in the mouse that successful assisted reproduction can be achieved when the Y gene contribution is limited to only two genes, Sry and spermatogonial proliferation factor Eif2s3y. Here, we replaced Sry by transgenic activation of its downstream target Sox9, and Eif2s3y, by transgenic overexpression of its X chromosome-encoded homolog Eif2s3x. The resulting males with no Y chromosome genes produced haploid male gametes and sired offspring after assisted reproduction. Our findings support the existence of functional redundancy between the Y chromosome genes and their homologs encoded on other chromosomes.

Depletion of CLL-associated patrolling monocytes and macrophages controls disease development and repairs immune dysfunction in vivo.

Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eµ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6C(low) patrolling or nonclassical phenotype. In addition, the percentage of MHC-II(hi) dendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-α and CXCL9. In vivo myeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Sry-Independent Overexpression of Sox9 Supports Spermatogenesis and Fertility in the Mouse.

The Y chromosome gene Sry is responsible for sex determination in mammals and initiates a cascade of events that direct differentiation of bipotential genital ridges toward male-specific fate. Sox9 is an autosomal gene and a primary downstream target of SRY. The activation of Sox9 in the absence of Sry is sufficient for initiation of male-specific sex determination. Sry-to-Sox9 replacement has mostly been studied in the context of sex determination during early embryogenesis. Here, we tested whether Sry-to-Sox9 replacement affects male fertility in adulthood. We examined males with the Y chromosome carrying a deletion removing the endogenous Sry, with testes determination driven either by the Sox9 (XY(Tdym1)Sox9) or the Sry (XY(Tdym1)Sry) transgenes as well as wild-type males (XY). XY(Tdym1)Sox9 males had reduced testes size, altered testes shape and vasculature, and increased incidence of defects in seminiferous epithelium underlying the coelomic blood vessel region when compared to XY(Tdym1)Sry and XY. There were no differences between XY(Tdym1)Sry and XY(Tdym1)Sox9 males in respect to sperm number, motility, morphology, and ability to fertilize oocytes in vitro, but for some parameters, transgenic males were impaired when compared to XY. In fecundity trials, XY(Tdym1)Sry, XY(Tdym1)Sox9, and XY males yielded similar average numbers of pups and litters. Overall, our findings support that males lacking the testis determinant Sry can be fertile and reinforce the notion that Sry does not play a role in mature gonads. Although transgenic Sox9 overexpression in the absence of Sry results in certain testicular abnormalities, it does not translate into fertility impairment.

C-reactive protein level is a prognostic indicator for survival and improves the predictive ability of the R-IPI score in diffuse large B-cell lymphoma patients.

BACKGROUND: High levels of C-reactive protein (CRP), an acute phase protein, proofed being associated with decreased clinical outcome in small-scale studies in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to evaluate the prognostic impact of pretreatment CRP levels on overall survival (OS) and disease-free survival (DFS) in a large bicentre study of DLBCL patients. METHODS: Data from 477 DLBCL patients, diagnosed and treated between 2004 and 2013 at two Austrian centres, were evaluated retrospectively. The prognostic influence of CRP and other factors, including age, tumour stage, and revised International Prognostic Index (R-IPI) on 5-year OS and 5-year DFS, were studied by Kaplan-Meier curves as well as univariate and multivariate Cox regression models. Influence of CRP on the predictive accuracy of the R-IPI score was determined by the Harrell concordance index. RESULTS: Kaplan-Meier curves revealed elevated CRP as a factor for decreased 5-year OS and DFS in DLBCL patients (P<0.001, log-rank test). An independent significant association between high CRP levels and poor clinical outcome in multivariate analysis for 5-year OS (HR=1.51, CI 95%=1.04-2.20, P=0.031) and for DFS (HR=1.91, CI 95%=1.28-2.85, P=0.002) was found. The estimated concordance index was 0.75 using the original R-IPI score and 0.79 when CRP was added. CONCLUSIONS: In the present study, we demonstrated high CRP levels at diagnosis of DLBCL as an independent poor prognostic factor for clinical outcome. Adding CRP to the well-established prognostic models such as the R-IPI score might improve their predictive ability.

Increased body mass index is associated with improved overall survival in diffuse large B-cell lymphoma.

BACKGROUND: Obesity is a well-known risk factor for the development of several types of cancer including lymphomas, but its influence on the course of disease is fairly unknown. Recently, a retrospective cancer registry analysis demonstrated significantly prolonged survival for overweight and obese patients with diffuse large B-cell lymphoma (DLBCL). The study population almost exclusively consisted of male US American patients of lower socioeconomic status and one-fifth of patients received cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) chemotherapy without rituximab. Therefore, it remains unclear if these results can be extrapolated to the general DLBCL population. PATIENTS AND METHODS: This retrospective single-center analysis included 183 unselected DLBCL patients who were treated with rituximab and standard-dosed anthracycline-based chemoimmunotherapy as first-line therapy between January 2004 and December 2012. Patients were stratified by body mass index (BMI) into 'low BMI' (<25.0 kg/m(2)) and 'high BMI' (≥25.0 kg/m(2)). RESULTS: The two groups were well balanced regarding age, performance score, international prognostic index, B-symptoms and extranodal involvement. However, there was a trend for male sex (P = 0.053) and higher-stage disease (P = 0.066) in the high-BMI group. Patients with higher BMI had significantly longer overall survival (OS; hazard ratio [HR] 0.546; P = 0.035) with 80.9% of patients alive at 3 years versus 64.2% in the low-BMI group. BMI was also an independent prognostic factor for OS in multivariate analysis (HR 0.557; P = 0.043). CONCLUSION: We could show a significant association between overweight/obesity and improved OS in an unselected DLBCL cohort.

[Autoimmune hemolysis accompanied by thrombopenia: consumption or myelodysplasia?]. / Thrombozytopenie bei Autoimmunhämolyse: Verbrauch oder Myelodysplasie?

HISTORY: A 77-year-old patient with a known autoimmune hemolysis for years was treated with steroids, rituximab, cyclophosphamid, cyclosporin A. Because of accompanying thrombopenia he received eltrombopag and underwent splenectomy but without lasting effect. After 3 years he presented with decreased leukocytes and worsening of thrombopenia. INVESTIGATIONS: A peripheral blood count showed moderate pancytopenia without blasts or left shift. Bone marrow biopsy revealed myelodysplasia with excessive blasts, cytogenetics showed partial trisomy 18q. TREATMENT AND COURSE: Because of the high risk of transformation to acute myeloid leukemia treatment with 5-azacytidine was started. Thrombopenia rapidly improved, but after an infectious complication treatment was paused for several months due to patient wish. Treatment was started again after 11 months because of progressive thrombopenia and resulted in a rapid hematological improvement. CONCLUSION: The new diagnosis of a myelodysplastic syndrome should be considered in the case of new cytopenia even in the presence of an already established hematological disease.

Actinomycin D induces p53-independent cell death and prolongs survival in high-risk chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most prevalent lymphoid malignancy in the elderly of the Western world. Although treatment options have improved over the past two decades, 10-15% of patients still have a poor prognosis and are often resistant to therapy. Aberrations in the p53 pathway, such as a deleted (del17p13) or mutated p53 gene, are highly enriched in this class of patients. In an extensive screen for p53-independent apoptosis inducers, actinomycin D was identified from 1496 substances and shown to induce apoptosis in primary CLL cells derived from high-risk patients including those with aberrant p53, revealing a novel p53-independent mechanism of action. Both pro-survival genes BCL2 and MCL1 are targeted by actinomycin D, in contrast to fludarabine the backbone of current treatment schedules. In the well-established TCL1 transgenic mouse model for high-risk CLL, actinomycin D treatment was more effective in reducing tumor load than fludarabine, with no evidence of resistance after three treatment cycles and an overall survival increase of over 300%. Tumor load reduction was coupled to BCL2 downregulation. Our results identify the clinically approved compound actinomycin D as a potentially valuable treatment option for CLL high-risk patients.