Cost-Effective Flow Cytometry Testing Strategies.

Cost-effective flow cytometry (FC) requires development of FC panels focused to common diagnoses and strategies to identify cases where limited FC testing is sufficient. Focused panels include sufficient antibodies and to identify common diseases and appropriate analysis strategies to identify rare diseases that need additional FC testing. Strategies to limit FC testing include the use of algorithms to predict disease probability, with limited FC performed if disease is unlikely. Successful algorithms use easily available parameters, have well-defined rules for use, and are periodically reviewed and updated to maximize efficiency while containing costs.

Synchronous Presentation of Renal Cell Carcinoma and Hodgkin Lymphoma in an Adolescent.

BACKGROUND: Coincidence of renal cell carcinoma (RCC) and hematologic malignancies has been reported in adults but not in children. OBSERVATION: We report a case of a 16-year-old girl in whom RCC was incidentally discovered on the computed tomography scan that was performed to stage her underlying Hodgkin lymphoma. Analysis of constitutional cytogenetics for common genetic aberrations that predispose to RCC did not reveal any mutations or genetic variations. However, cytogenetics on the RCC tumor demonstrated a rare reciprocal translocation between chromosomes 6 and 11, t(6;11)(p21;q12). After undergoing partial nephrectomy with regional lymphadenectomy and treatment with multiagent chemotherapy, patient is cancer-free, now 33 months from end of therapy. CONCLUSIONS: This case highlights the importance for histologic confirmation of a renal mass when concurrently discovered during the diagnostic evaluation of other malignancies.

Transmission of Babesia microti Parasites by Solid Organ Transplantation.

Babesia microti, an intraerythrocytic parasite, is tickborne in nature. In contrast to transmission by blood transfusion, which has been well documented, transmission associated with solid organ transplantation has not been reported. We describe parasitologically confirmed cases of babesiosis diagnosed ≈8 weeks posttransplantation in 2 recipients of renal allografts from an organ donor who was multiply transfused on the day he died from traumatic injuries. The organ donor and recipients had no identified risk factors for tickborne infection. Antibodies against B. microti parasites were not detected by serologic testing of archived pretransplant specimens. However, 1 of the organ donor's blood donors was seropositive when tested postdonation and had risk factors for tick exposure. The organ donor probably served as a conduit of Babesia parasites from the seropositive blood donor to both kidney recipients. Babesiosis should be included in the differential diagnosis of unexplained fever and hemolytic anemia after blood transfusion or organ transplantation.

A unique CD4+ large granular lymphocytosis occurring in patients treated with tumor necrosis factor α inhibitors: report of 2 cases.

We report 2 cases of CD4(+) large granular lymphocyte (LGL) lymphocytosis occurring in patients being treated with a monoclonal antibody against tumor necrosis factor α for underlying autoimmune disorders. CD4(+) LGL lymphocytosis is a rare subset of LGL disease that has previously only been described in patients without underlying autoimmune disorders, and most demonstrate uniform coexpression of CD56 on the atypical T cells. The clinical features, with both cases occurring in patients with autoimmune disease, and immunophenotypic features, with both cases showing dim CD8 coexpression without CD56 in the CD4(+) LGLs, suggest that the reported cases are distinct from those previously described and may represent a novel T-cell LGL lymphocytosis emerging from iatrogenic immune modulation of patients with underlying autoimmune disorders.

Efficient assessment of peripheral blood lymphocytosis in adults: developing new thresholds for blood smear review by pathologists.

BACKGROUND: Peripheral smear review is a critical, but labor intensive adjunct for evaluation of lymphocytosis. Standard practice based on consensus guidelines is to review cases with absolute lymphocyte count (ALC) >5×109/L. We hypothesize that identifying cases for review by applying appropriately adjusted ALC and age discriminators will decrease laboratory workload without compromising patient care. METHODS: 1170 complete blood counts with ALCs >5×109/L analyzed in the core laboratory during a 2-year period were included. Patients were categorized into diagnostic groups based on follow-up criteria. A total of 402 patients with new onset lymphocytosis who met criteria for reactive lymphocytosis (82%) or lymphoproliferative disorder (18%) were used to establish optimal ALC and age thresholds from receiver operating characteristic (ROC) curve analysis. RESULTS: ALC as a discriminator for neoplastic lymphocytosis had an ROC area under the curve (AUC) of 0.732. Selecting cases with ALC >10×109/L enriched the proportion of neoplastic cases in the review pool (90% specificity); however, many cases with ALC below this threshold were also neoplastic (52% sensitivity). For cases with ALC between 5 and 10×109/L, age as a discriminator had an ROC AUC of 0.886. Selecting patients >50 years old in this group for review captured the neoplastic cases while excluding the reactive cases (93% sensitivity, 62% specificity). When applied to a validation cohort, the predictive performance of the thresholds was maintained while reducing smears reviewed by 50%. CONCLUSIONS: We show that modifying the standard 5×109/L ALC smear review threshold through retrospective analysis of institutional data can reduce laboratory workload without compromising quality.

Kaposi sarcoma herpesvirus/human herpesvirus-8-negative effusion-based lymphoma: report of 3 cases and review of the literature.

BACKGROUND: Primary effusion lymphoma (PEL) is a rare subtype of large B-cell lymphoma that arises in body cavities without detectable tumor masses. PEL is universally associated with Kaposi sarcoma herpesvirus (KSHV)/human herpesvirus-8 (HHV8). Despite overlapping features, KSHV/HHV8-negative effusion-based lymphoma is a distinct entity from PEL. To date, 52 cases have been reported. The authors report 3 additional cases received in their laboratory from 2007 to 2012. METHODS: Clinical data, cytomorphologic features, and immunophenotypic features of the 3 cases were described and compared with those reported in the literature. RESULTS: The cells in HHV8-negative effusion lymphoma commonly revealed large cell, immunoblastic morphology and B-cell immunophenotype. The 3 cases demonstrated cytomorphologic and immunophenotypic variability. Cytomorphologically, 1 case contained large, highly atypical cells with a moderate amount of cytoplasm, round nucleus, coarsely granular chromatin, and a single macronucleolus. The other 2 cases had medium to large atypical cells with high nuclear-to-cytoplasmic ratios, slightly irregular to cleaved nuclei, and multiple conspicuous nucleoli. One case had a null phenotype with aberrant cytokeratin expression. B-cell phenotype was established by clonal immunoglobulin heavy-chain rearrangement using polymerase chain reaction, whereas the other 2 cases demonstrated a B-cell phenotype by flow cytometry and immunohistochemical staining. All 3 cases were negative for both HHV8 and Epstein-Barr virus. CONCLUSIONS: HHV8-negative effusion lymphoma exhibits clinical, cytomorphologic, and immunophenotypic variability. Cases with a null-phenotype can be particularly challenging. When effusion lymphoma is suspected, ancillary tests are helpful. Moreover, HHV8 detection is critical in differentiating PEL and HHV8-negative effusion lymphoma, because they have overlapping features yet different prognoses.

Use of tissue microarray and automated quantitative analysis for screening and validation of potential biomarkers in mantle cell lymphoma.

BACKGROUND: Cancer biomarker studies using the combination of tissue microarray and automated quantitative assessment of immunofluorescence (TMA-AQUA) have been successfully performed for various types of human carcinoma, but its performance characteristics have yet to be evaluated in human lymphoma. METHODS: A pilot TMA was constructed containing duplicate 1.5 mm cores from 15 cases of mantle cell lymphoma (MCL), 3 cases of low-grade B-cell lymphoma, and 3 cases of benign lymphoid tissue. Protein expression of c-Myc, Cdc2, Cyclin D1, Ki-67, Mcm2, and p27 by immunofluorescence and chromagenic staining were evaluated by AQUA and visual scoring, respectively. Gene expression of cMYC, CDC2, and CCND1 was determined by quantitative nuclease protection assay. RESULTS: Protein expression between duplicate cores determined by AQUA showed excellent correlation for all markers [correlation coefficient (R)=0.79 to 0.94] and Cyclin D1 expression was significantly higher in MCL cases compared with non-MCL cases (P=0.00019). Overall correlation of AQUA with scoring of chromagenic staining by 2 pathologists was good for all markers (R=0.56 to 0.90), except Cdc2 (R=0.25). Localization of expression to cytoplasmic and/or nuclear compartments was comparable with chromagenic staining patterns for all markers except Ki-67 and Mcm2, where a significant difference between nuclear and cytoplasmic expression could not be appreciated by AQUA, despite clear nuclear localization by chromagenic staining. Correlation of gene expression with protein expression was variable for CDC2, cMYC, and CCND1 (R=0.32, 0.35, and 0.69). CONCLUSIONS: TMA-AQUA has the potential to be successfully used as a high-throughput protein biomarker screening platform for MCL; however, appropriate target protein selection and antibody performance validation are factors that need to be considered.

Bone marrow stromal cells from multiple myeloma patients uniquely induce bortezomib resistant NF-kappaB activity in myeloma cells.

BACKGROUND: Components of the microenvironment such as bone marrow stromal cells (BMSCs) are well known to support multiple myeloma (MM) disease progression and resistance to chemotherapy including the proteasome inhibitor bortezomib. However, functional distinctions between BMSCs in MM patients and those in disease-free marrow are not completely understood. We and other investigators have recently reported that NF-kappaB activity in primary MM cells is largely resistant to the proteasome inhibitor bortezomib, and that further enhancement of NF-kappaB by BMSCs is similarly resistant to bortezomib and may mediate resistance to this therapy. The mediating factor(s) of this bortezomib-resistant NF-kappaB activity is induced by BMSCs is not currently understood. RESULTS: Here we report that BMSCs specifically derived from MM patients are capable of further activating bortezomib-resistant NF-kappaB activity in MM cells. This induced activity is mediated by soluble proteinaceous factors secreted by MM BMSCs. Among the multiple factors evaluated, interleukin-8 was secreted by BMSCs from MM patients at significantly higher levels compared to those from non-MM sources, and we found that IL-8 contributes to BMSC-induced NF-kappaB activity. CONCLUSIONS: BMSCs from MM patients uniquely enhance constitutive NF-kappaB activity in MM cells via a proteinaceous secreted factor in part in conjunction with IL-8. Since NF-kappaB is known to potentiate MM cell survival and confer resistance to drugs including bortezomib, further identification of the NF-kappaB activating factors produced specifically by MM-derived BMSCs may provide a novel biomarker and/or drug target for the treatment of this commonly fatal disease.

Bortezomib-resistant nuclear factor-kappaB activity in multiple myeloma cells.

Bortezomib (Velcade/PS341), a proteasome inhibitor used in the treatment of multiple myeloma (MM), can inhibit activation of nuclear factor-kappaB (NF-kappaB), a family of transcription factors often deregulated and constitutively activated in primary MM cells. NF-kappaB can be activated via several distinct mechanisms, including the proteasome inhibitor-resistant (PIR) pathway. It remains unknown what fraction of primary MM cells harbor constitutive NF-kappaB activity maintained by proteasome-dependent mechanisms. Here, we report an unexpected finding that constitutive NF-kappaB activity in 10 of 14 primary MM samples analyzed is refractory to inhibition by bortezomib. Moreover, when MM cells were cocultured with MM patient-derived bone marrow stromal cells (BMSC), microenvironment components critical for MM growth and survival, further increases in NF-kappaB activity were observed that were also refractory to bortezomib. Similarly, MM-BMSCs caused PIR NF-kappaB activation in the RPMI8226 MM cell line, leading to increased NF-kappaB-dependent transcription and resistance to bortezomib-induced apoptosis. Our findings show that primary MM cells frequently harbor PIR NF-kappaB activity that is further enhanced by the presence of patient-derived BMSCs. They also suggest that this activity is likely relevant to the drug resistance development in some patients. Further elucidation of the mechanism of PIR NF-kappaB regulation could lead to the identification of novel diagnostic biomarkers and/or therapeutic targets for MM treatment.

Overexpression of protein kinase C-{epsilon} in the mouse epidermis leads to a spontaneous myeloproliferative-like disease.

Protein kinase C (PKC)-epsilon, a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mouse lines that overexpress (8- or 18-fold) PKC-epsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate-promotion or by repeated ultraviolet radiation exposures. The susceptibility to the development of SCC was proportional to the level of expression of the PKC-epsilon transgene. We now report that PKC-epsilon FVB/N transgenic mice (line 215) that overexpress in epidermis approximately 18-fold PKC-epsilon protein more than their wild-type littermates spontaneously develop a myeloproliferative-like disease (MPD) in 100% of PKC-epsilon transgenic mice. The MPD was characterized by an excess of neutrophils and eosinophils, resulting in invasion of almost all vital organs of the mouse by 6 months of age. On gross examination these mice present with splenomegaly, hepatomegaly, and severe lymphadenopathy. Examination of the bone marrow revealed almost complete effacement by neutrophils, eosinophils, and their precursors. Furthermore, the spleen and lymph nodes were enlarged and exhibited marked extramedullary hematopoiesis. Complete pathological analysis of the second PKC-epsilon transgenic mouse (line 224) that expresses approximately eightfold PKC-epsilon protein more than their wild-type littermates revealed no remarkable findings in any of the affected organs as seen in line 215. However, peripheral blood analyses of PKC-epsilon transgenic mice indicated significant increases of neutrophils in the circulating blood in both PKC-epsilon transgenic lines. To determine whether there was an imbalance of cytokines in PKC-epsilon transgenic mice (line 215), resulting in aberrant myelopoiesis, we analyzed 17 cytokines in the peripheral blood. This analysis indicated that interleukin-5, interleukin-6, and granulocyte-colony stimulating factor were up-regulated as a function of age. The transgene PKC-epsilon was not detected in any of the affected organs (bone marrow, liver, spleen, lung) We suggest that overexpression of PKC-epsilon in the epidermis may lead to the induction of specific cytokines that may, in a paracrine mechanism, perturb normal hematopoiesis in bone marrow resulting in a granulocytic skew toward that of neutrophils and eosinophils. The susceptibility of PKC-epsilon transgenic mice to the induction of SCC and the spontaneous development of MPD are unrelated.