Molecular Perspectives of Diffuse Large B-cell Lymphoma

Case

A 100 year old female was seen for follow-up for her hypertension, mild renal impairment, and fatigue. The patient also stated a three week duration of pain in the area of the right upper quadrant that radiates to her back. No other symptoms or concerns were expressed.

An abdominal CT was performed which showed a 6.6 x 2.1 cm soft tissue mass in the right posterior chest wall that also encases the 11th rib. Given the concern for a malignant process, a core needle biopsy was obtained for histology only.

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H&E, 20x
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H&E, 50x
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CD10
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Ki-67

The H&E stained sections show a diffuse infiltration of atypical lymphoid cells that are large in size with irregular nuclear contours, vesicular chromatin, and some with prominent nucleoli. Frequent apoptotic bodies and mitotic figures were seen. By immunohistochemistry, CD20 highlights the infiltrating cells, which are positive for BCL2, BCL6, and MUM1 (major subset). CD10 is negative within the atypical lymphoid population. CD3 highlights background T-cells. Ki-67 proliferation index is approximately 70%. EBER ISH is negative.

Overall, the findings are consistent with diffuse large B-cell lymphoma, NOS with a non-GCB phenotype by the Hans algorithm.

Discussion

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell lymphoma in adults comprising 30%-40% of new adult lymphomas. Approximately 50% of patients will be cured, even in advanced cases; however, those that fail conventional therapy ultimately succumb to their illness.1 Up to 30% of patients have refractoriness or relapse after initial therapy with rituximab based regimens, particulary R-CHOP (ritixumab, cyclophosphamide, doxorubicin, vincristine, and prednisone).

In the era of new molecular techniques and in the context of the heterogeneous nature of DLBCL, it has become important to accurately assess cell of origin (COO) as this has prognostic implications. With the seminal paper from Alizadeh and colleagues, gene expression profiling (GEP) by a microarray platform produced the concept of germinal center (GCB) versus activated B-cell (ABC) types of DLBCL.2 In the context of prognosis and R-CHOP therapy, the GCB type has a 3 year PFS of 75% as opposed to the ABC type that has a 3 year PFS of 40% (P<.001).3 Although GEP analysis is considered the ideal modality for determining COO, however, given the constraints of most modern hematopathology practices, surrogate immunohistochemical algorithms were developed to aid in COO determination. Of the multiple algorithms, the Hans algorithm is the most widely used and accepted for IHC determination of COO.

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Adapted from Hans et al., Blood, 2004

The COO determination has revealed multiple genetic alterations that are shared between the GCB and ABC phenotype while distinct changes have been identified in each type. Molecular mechanisms at play include, but are not limited to, histone modification, blocks to terminal differentiation, cell cycle activation, PI3K/AKT signaling activation, mTOR pathway activation, as well as a multitude of other signaling cascades. A common shared dysregulated pathway between GCB and ABC types include mutations in CREBBP and EP300, which is in approximately 30% of DLBCL cases and slightly enriched in the GCB group. Mutations/deletions in these genes result in inactivation and alter histone modification subsequently thought to contribute to acetylation of BCL6, which is a key regulatory protein in lymphomagenesis. Up to 33% of DLBCL have mutations in MLL2, which has a broad effect on chromatin regulation and epigenomic alteration. Approximately 35% of DLBCL cases with up to two- to three-fold increase in ABC type cases have genetic alterations in BCL6, particularly chromosomal rearrangements and mutations in the 5’ sequence. Pasqualucci et al also described other factors that lead to BCL6 inactivation, including mutations in MEF2B and FBXO11.4

ABC type DLBCL often displays canonical pathway activation of NF-ƙB signaling, which ultimately promotes survival, proliferation, and inhibition of apoptosis. This potentially is a result of alterations in the CBM signalosome (CARD11, BCL10, and MALT1) with up to 10% of ABC-DLBCL cases having a mutation in CARD11. Another modality of ABC activation is through the B-cell receptor signaling pathway in which 20% of cases harbor a CD79A or CD79B mutation.  Interestingly enough, recurring mutations in MYD88 occur in ~30% of ABC-DLBCLs, which results in upregulation of NF-kB and Janus kinase-signal transducers. Other important genetic alterations include involvement by signaling pathways of spleen tyrosine kinase (SYK), PI3K, Bruton tyrosine kinase (BTK), and protein kinase C-β (PKC-β).

GCB type DLBCL often expresses CD10, LMO2, and BCL6 and has a less understood and distinct pathway when compared to ABC-DLBCL. The most common alterations include t(14;18) IGH-BCL2 (30-40%), C-REL amplification (30%), EZH2 (20%) and PTEN mutations (10%). These changes are almost never seen in ABC-DLBCL.

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Adapted from Pasqualucci et al., Semin Hematol, April 2015

Although the findings in GCB and ABC type DLBCL are described, they are not absolute and multiple studies done by whole exome sequencing (WES) and whole genome sequencing (WGS) have elucidate further complexities and genetic changes. In 2015, data from Novak and colleagues revealed CNAs and mutations that were associated EFS, which also underscored the important 24 month milestone for survival.5 Morin et al in 2013 described 41 novel genes in DLBCL which demonstrated just how complex and heterogeneous DLBCL truly is (see figure below).6

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Adapted from Morin et al., Blood, 2013.

As common as DLBCL is, there is much to be understood not only for lymphomagenesis, but for correct classification and risk stratification. Many targeted therapies have been designed and are in trials at the moment, but given the nature of DLBCL and its heterogeneity, more work on the molecular front is needed. Modalities for assessing COO are currently on the market but are not widely used. Perhaps COO determination by IHC may be an antiquated method, but it is currently the standard by which most pathologists practice. Overall, DLBCL in all its forms is not a uniform entity that can easily be defeated, but requires thought and diligence in achieving a cure.

 

  1. Lohr, JG et al. “Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing,” Proc Natl Acad Sci USA. 2012; 109(10): 3879-3884
  2. Alizadeh AA, et al. “Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling,” Nature 2000, 403:503-11
  3. Sehn, L and Gascoyne, R “Diffuse large B-cell lymphoma: optimizing outcome in the context of clinical and biologic heterogeneity,” Blood. 2015;125(1):22-32
  4. Pasqualucci, L and Dalla-Favera, Riccardo, “The Genetic Landscape of Diffuse Large B Cell Lymphoma,” Semin Hematol. 2015 April; 52(2): 67-76
  5. Novak, AJ et al. “Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma,” Blood Cancer Journal (2015) 5
  6. Morin, R et al. “Mutational and structural analysis of diffuse large B-cell lymphoma using whole-genome sequencing,” 2013;122(7):1256-1265

 

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-Phillip Michaels, MD is a board certified anatomic and clinical pathologist who is a current hematopathology fellow at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. His research interests include molecular profiling of diffuse large B-cell lymphoma as well as pathology resident education, especially in hematopathology and molecular genetic pathology.

Hematopathology Case Study: A 69 Year Old Female with Persistent Monocytosis

Case History

A 69 year old female with a past medical history significant for endometrial adenocarcinoma, traumatic brain injury, atrial fibrillation, hypertension, hyperlipidemia, and persistent monocytosis (absolute monocyte count ranging from 1.6-3.7 K/uL) who had an indeterminate lesion identified in the T5 vertebra, read as “hemangioma, although surrounding edema is worrisome for malignancy” upon staging imaging for history of endometrial carcinoma.

CBC at the time was: WBC 9.8; HGB 12.9; HCT 37.6; PLT 154; MCV 92 fL; MCH 31.7.

Automated differential showed: 43.0 Neutrophils; 34.8 Lymphocytes; 20.0 Monocytes; 1.4 Eosinophils; 0.2 Basophils; 0.6 Immature granulocytes.

Absolute monocyte count was 1.95 K/uL.

This lesion was biopsied and given the findings, a subsequent bone marrow biopsy was performed on 5/4/2017. The bone marrow core biopsy contained multiple compact aggregates of spindle shaped cells with hypogranular cytoplasm, morphologically compatible with atypical mast cells. Within these aggregates, numerous eosinophils are present. By immunohistochemistry, the mast cells are brightly positive for CD117 and mast cell tryptase. Concurrent bone marrow aspirate flow cytometry demonstrated a small population of mast cells that co-expressed CD2 and CD25.

Of note, the patient was found to have a persistent absolute monocytosis. Flow cytometry revealed an abnormal population of monocytes that displayed aberrant phenotypic expression of CD2 and CD56 (subset).

Next-generation sequencing revealed two truncation mutations in the TET2 gene (K988* in 34.6% of the reads and Q1138* in 36.4% of the reads). Cytogenetic analysis revealed a normal female karyotype (46,XX).

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Overall, an immunophenotypically abnormal population of mast cells and monocytes are present in the context of a long-standing absolute monocytosis and the presence of two TET2 truncating mutations, supporting a diagnosis of systemic mastocytosis with an associated hematologic non-mast cell lineage disorder (best classified as chronic myelomonocytic leukemia).

Discussion

A diagnosis of systemic mastocytosis is a combination of clinical, morphologic, immunophenotypic, and molecular analyses, as required by the World Health Organization (WHO 2008). By current consensus guidelines, SM variants are partly distinguished by clinicopathologic criteria referred collectively as B and C findings. B findings include: >30% of bone marrow mast cells (MC) on biopsy and/or serum tryptase levels >200 ng/mL; increased marrow cellularity/dysplasia without meeting diagnostic criteria for another myeloid neoplasm; or enlargement of liver, spleen, or lymph nodes without evidence of organ damage. C findings include: evidence of organ damage caused by a local MC infiltrate, such as abnormal liver function and/or ascites, hypersplenism, cytopenias, large osteolytic lesions/fractures, and malabsorption with weight loss caused by MC infiltrate in the gastrointestinal tract.

Systemic mastocytosis commonly occurs in two types with different clinical courses based upon the aforementioned findings. Indolent SM (ISM) is defined by the absence of C findings. Smoldering SM is a subtype of ISM that displays 2 or more B findings. ISM may become more aggressive and a descriptive term of advanced SM refers to a category including aggressive SM (ASM), mast cell leukemia (MCL), and “SM with an associated myeloid neoplasm.” The latter entity comprises more than 90% of cases that have previously been referred to as SM with an associated hematologic non-mast cell lineage disorder (SH-AHNMD).

ASM and MCL are characterized by organ damage and histologic characteristics. ASM often exhibits multifocal bone marrow infiltration of atypical mast cells that are often spindled in shape with hypogranular or immature morphology. Marked fibrosis often accompanies the infiltrate as well as a KIT D816V mutation. MCL is codified by more than 20% of the marrow aspirate nucleated cells represent by mast cells and on core biopsy, a compact infiltrate is often identified with usually low level fibrosis. In MCL, circulating mast cells are greater than 10% of nucleated cells but according to Gotlib et al., the aleukemic MCL (less than 10% circulating mast cells) is more common.

In the context of our patient, myeloid neoplasms associated with SM are often represented by MDS, MPN, or MDS/MPN overlap disorders, and occasionally AML.

Associated lymphoid or plasma cell neoplasms have been described, but in a much lower frequency.

In accordance with the diagnostic implications, KIT D816V mutational analysis is important therapeutically. Most patients with SM harbor the KIT D816V mutation (>80% in one clinical series; 90-100% in research studies using purified MCs), which is a considered imatinib-resistant mutation. Midostaurin (a second generation TKI) may provide some disease response while nilotinib or dasatinib are usually less likely to lead to a durable response. The rare patients who have a juxtamembrane domain KIT mutation are much more likely to respond to imatinib or masitinib.

For disease response, criteria were first published in 2003 by Valent, et al. In a reiterated version published in 2007, the evaluation of clinical evidence of organ damage (C findings), was the foundation for determining appropriate response. Another facet to determining response was in relation to BM MC burden, serum tryptase level, and organomegaly, which further subcategorized the levels of major response (MR). MR was defined as normalization of 1 or more C findings. In turn, MR was divided into 3 categories:

  1. Complete remission (resolution of MC infiltrates in organs, serum tryptase less than 20 ng/mL, and disappearance of SM-associated organomegaly)
  2. Incomplete remission (decrease in MC infiltrates in organs and/or serum tryptase levels and/or visible regression of organomegaly by >50%)
  3. Pure clinical response (without decrease in MC infiltrates, serum tryptase levels, or organomegaly)

Partial response (PR) is defined as incomplete regression of 1 or more C findings and include good partial response (GPR; >50% regression of 1 or more C findings) and minor response (<50% regression).

Lastly, the Mayo Clinic published revised response criteria in 2010 which established minimal baseline laboratory abnormalities for organ damage to be evaluated in order to allow for more accurate assessment of response to therapy that is clinically more relevant.

Overall, systemic mastocytosis is a rare entity that displays a range of presentations that can be described as indolent up to an aggressive (advanced) phenotype. The hallmarks for diagnosis include histologic, immunophenotypic, molecular, and clinical findings.

 

References

  1. Gotlib, J et al. “International Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) & European Competence Network on Mastocytosis (ECNM) consensus response criteria in advanced systemic mastocytosis,” Blood, 2012.
  2. Horny HP et al. “Mastocytosis,” In: Swerdlow S et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008:53-63
  3. Valent P et al. “Aggressive systemic mastocytosis and related mast cell disorders: current treatment options and proposed response criteria.” Leuk Res. 2003;27(7):635-641.
  4. Pardanani A, et al. “A critical reappraisal of treatment response criteria in systemic mastocytosis and a proposal for revisions. Eur J Haematol. 2010;84(5):371-378.

 

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-Phillip Michaels, MD is a board certified anatomic and clinical pathologist who is a current hematopathology fellow at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. His research interests include molecular profiling of diffuse large B-cell lymphoma as well as pathology resident education, especially in hematopathology and molecular genetic pathology.

Hematology Case Study: An 80 Year Old Male with History of CLL

Case History

80 year old male patient with history of CLL presented to the emergency room with cough and not feeling well. He was diagnosed with CLL 4 years ago; had been asymptomatic and hence had not received any treatment. CBC done in the emergency room revealed a markedly elevated WBC count of 136 K/uL, decreased hemoglobin of 6.4 g/dl and mildly decreased platelet count at 131 K/uL.

Examination of peripheral blood smear revealed marked lymphocytosis (114.91 K/uL). Majority of the lymphocytes were small with round to oval nuclei. Few larger cells with morphology consistent with prolymphocytes were also noted (overall <5%). Further there was increased polychromasia and spherocytes were easily identified. The patient’s blood type was A positive and the antibody screen was positive. Direct antiglobulin test was positive (IgG) and the antibody identification panel was consistent with the presence of a warm autoantibody. His bilirubin and LDH were both elevated at 3.1 g/dl and 574 U/L, respectively.

The findings were consistent with warm immune mediated hemolysis.

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Image 1. Prolymphocyte, smudge cell, and abundant lymphocytes.

Discussion

Autoimmune hemolytic anemia (AIHA) due to the presence of warm agglutinins is mostly always due to the presence of IgG antibodies that react with protein antigens on the red blood cell (RBC) surface at body temperature.

Underlying causes or conditions that may be associated with AIHA include the following:

  • Preceding viral infections (usually in children).
  • Typical AIHA due to the presence of warm agglutinins has been described in patients with HIV infection.
  • Autoimmune and connective tissue diseases (eg, systemic lupus erythematosus, autoimmune lymphoproliferative syndrome).
  • Immune deficiency diseases, such as common variable immunodeficiency.
  • Malignancies of the immune system (eg, non-Hodgkin lymphoma, chronic lymphocytic leukemia [CLL], with a higher incidence in those treated with purine analogs).
  • Prior allogeneic blood transfusion, hematopoietic cell transplantation, or solid organ transplantation

The incidence of autoimmune hemolytic anemia (AIHA) in patients with CLL is difficult to determine with certainty. As many as one-third of patients with CLL may develop AIHA over the course of their illness unrelated to treatment modality. The prevalence increases with disease stage, from a rate of approximately 4 percent in Binet stage A to 10 percent in stages B and C. The incidence of AIHA may be higher following purine analog treatment.

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-Neerja Vajpayee, MD, is the director of Clinical Pathology at Oneida Health Center in Oneida, New York and is actively involved in signing out surgical pathology and cytology cases in a community setting. Previously, she was on the faculty at SUNY Upstate for several years ( 2002-2016) where she was involved in diagnostic work and medical student/resident teaching.

Hematology Case Study: A 12 Year Old Female with Thrombocytopenia.

Case History

A 12 year old female presented with thrombocytopenia. Previous platelet count performed at a different facility showed a platelet count of <100K.  Patient signs show history of bruising, no history of trauma, intermittent epistaxis.

Family history shows no history of anemia or hypothyroidism from either parent. Incidental finding of hypothyroidism was revealed for this patient when laboratory testing was performed.

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Light staining, “gray” platelets.

Laboratory results

DAT: Negative

PT 11.7/INR 1.1

PTT 38.3

Platelet aggregation studies: Decreased response to ADP-Collagen-Epinephrine and Arachidonic Acid. Results of which are consistent with platelet dysfunction due to storage pool defect.

vonWillberand panel shows within range results for Factor 8, vW antigen and vW Ristocetin.

Peripheral blood smear shows light staining (gray) appearance of platelets.

Diagnosis: Gray Platelet Syndrome

 

Discussion

Gray platelet syndrome (GPS) is an inherited platelet disorder that presents with thrombocytopenia and characteristic pale/gray appearance of platelets under light microscopy. This gray appearance of platelets is due to the absence of alpha granules and their constituents.

According to Gunay-Aygun et al., the diagnosis of GPS requires demonstration of the absence or marked reduction of α-granules in platelets observed by electron microscopy (EM). Megakaryocytes also show decreased α-granules. Platelet dense bodies and lysosomes are unaffected. Alpha granules, the most abundant vesicles in platelets, store proteins that promote platelet adhesiveness and wound healing when secreted during platelet activation. Some α-granule proteins (eg, platelet factor 4 and β-thromboglobulin) are synthesized in megakaryocytes and packed into the vesicles, whereas others are either passively (eg, immunoglobulins and albumin) or actively (eg, fibrinogen) taken up from the plasma by receptor-mediated endocytosis. Proteins synthesized in megakaryocytes are markedly reduced in GPS, whereas other α-granule constituents are less affected. Studies of granule membrane-specific proteins have shown that platelets and megakaryocytes of GPS patients have rudimentary α-granule precursors. Therefore, the basic defect in GPS is thought to be the inability of megakaryocytes to pack endogeneously synthesized secretory proteins into developing α-granules. (Gunay-Aygun et al, 2010).

Most patients who present with GPS are characteristically macrothrombocytopenic and the number of megakaryocytes in the bone marrow appears normal. However platelet survival is reduced. This inability of megakaryocytes to survive is due to the alpha granule deficiency of this disorder therefore leading to thrombocytopenia. Myelofibrosis and splenomegaly is also apparent on patients with GPS but severe hemorrhage is unlikely, bleeding tendencies tend to be mild to moderate for GPS.

Most patients had bleeding symptoms from infancy with the average onset of 2 years of age. Average age of diagnosis is 10-14 years of age; some patients who have Gray Platelet Syndrome have presented with initial diagnosis of ITP (idiopathic thrombocytopenic purpura).

Reference

Gunay-Aygun, M., Zivony-Elboum, Y., Gumruk, F., Geiger, D., Cetin, M., Khayat, M., . . . Falik-Zaccai, T. (2010). Gray platelet syndrome: natural history of a large patient cohort and locus assignment to chromosome 3p. Blood, 116(23), 4990-5001. doi:10.1182/blood-2010-05-286534

 

-Written in collaboration with Stephanie Foster, BS MLS

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-Carlo Ledesma, MS, SH(ASCP)CM MT(ASCPi) MT(AMT) is the program director for the Medical Laboratory Technology and Phlebotomy at Rose State College in Midwest City, Oklahoma as well as a technical consultant for Royal Laboratory Services. Carlo has worked in several areas of the laboratory including microbiology and hematology before becoming a laboratory manager and program director.

Hematopathology Case Study: A 7 Year Old Transplant Patient with Neck Swelling

A 7 year old male with a history of restrictive cardiomyopathy status-post orthotopic heart transplant in June, 2010 that was on maintenance doses of tacrolimus and mycophenolate mofetil presented to his primary pediatrician left neck swelling. Starting in January 2017, the patient began with neck pain and swelling in the context of a recent gastrointestinal illness. Per CT report of the neck, a rim enhancing well-defined suppurative level III lymph node measuring 1.4 x 1.2 x 2.1 cm with adjacent soft tissue inflammatory changes extending into the left parapharyngeal space was identified. The patient was subsequently started on antibiotics and was discharged home with some improvement of swelling and pain.

The patient then presented again with continued neck swelling, although painless this time, and the patient’s cardiologist was contacted, who recommended a decrease in tacrolimus dosing. An otolaryngology evaluation was requested and given the concerning findings, the patient was admitted for further work-up, including a biopsy with a lymphoma protocol.

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EBER

 

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Flow Cytometry

 

Results

Flow cytometry revealed a kappa restricted CD10 positive mature B-cell population.

On biopsy examination, a population of monotonous lymphoid cells that are large in size with round to mildly irregular nuclear contours, open chromatin, and multiple inconspicuous nucleoli are present in a diffuse pattern. Abundant apoptotic bodies and mitotic figures are noted and occasional “starry sky” features are present. By immunohistochemistry, BCL6 highlights the neoplastic lymphocytes while BCL2 highlights background T-cells. EBER is negative.

Overall, despite a negative t(8;14) IGH/MYC translocation, the findings are best considered to be of an EBV-negative post-transplant lymphoproliferative disorder with morphologic features consistent with Burkitt lymphoma.

Discussion

Post-transplant lymphoproliferative disorders (PTLD) are a relatively rare complication in a variety of transplants that occurs in 2-10% of post-transplant patients. Overall, following a solid organ transplant (SOT), PTLD development is 1-5% of recipients with the highest incidence in intestinal and multivisceral transplantations (5-20%). Another factor is EBV status of the recipient, for which those that are EBV-naïve and lack cellular immunity to EBV are susceptible to graft-mediated EBV infection and ultimately developing an increased incidence in early PTLD. This population is overrepresented by pediatric transplant recipients1.

The presentation is highly variable and ranges from benign proliferations to overt lymphoproliferative disorders. Classifications for PTLD include early lesions, which are oligo- or polyclonal proliferations of EBV positive B cells have either a predominant infectious mononucleosis-like proliferation or a plasmacytic hyperplasia form. Polymorphic PTLD is a similar concept to the early proliferative lesions but the host architecture of the native structure is disrupted. Lastly, monomorphic PTLD is an entity that fulfills criteria for a non-Hodgkin lymphoma and is diagnosed according to the criteria of non-transplant associated lymphomas. Within pediatric registry studies, monomorphic PTLD accounts for 35-83% of all PTLD cases. B-cell lymphomas, particularly DLBCL, comprise the vast majority of monomorphic PTLD with plasmacytoma and T-cell lymphoproliferative disorders much less common2.

In this particular case, with the patient having been 7 years post-transplant and negative studies for EBV present, it is not surprising that germinal center phenotypic markers are highly expressed, such as CD10 and BCL6, which has been well elucidated by Jagadeesh, et al. Although not many genetic studies have been performed on post-transplant B-cell lymphomas, regardless of EBV status, there is some data demonstrating trisomies of 9 and/or 11 with translocations 8q24.1 (C-MYC), 3q24 (BCL6), and 14q32 (IGH). Rinaldi et al. noticed a lack of genetic lesions characteristic of postgerminal center derivation, such as gain of chromosome 3 (FOXP1, BCL6, and NFKBIZ) and 18q (BCL2 and NFATC1) together with losses of 6q (PRDM1 and TNFAIP3) in post-transplant DLBCL.  A number of DNA mutations have also been described including genes associated with somatic hypermutation (SHM) such as PIM-1, PAX5, C-MYC, and RhoH/TTF. These particular mutations are also found to be independent of EBV status1.

Overall, post-transplant lymphoproliferative disorders occur in a variety of transplant settings across many age groups and can be dependent on EBV and CMV status as well as the type and degree of immunosuppression. Although many variations take place in PTLD, patients with the monomorphic type are diagnosed according to their non-transplant counterparts. Current perspective includes further analysis of molecular and cellular mechanisms incorporated into research projects, which could better aid in prognostic implications and future therapeutics.

  1. Morscio, et al. “Molecular pathogenesis of B-cell posttransplant lymphoproliferative disorder: What do we know so far?” Clinical and Developmental Immunology 2013.
  2. Mynarek, et al. “Posttransplant lymphoproliferative disease after pediatric solid organ transplantation,” Clinical and Developmental Immunology 2013.

 

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-Phillip Michaels, MD is a board certified anatomic and clinical pathologist who is a current hematopathology fellow at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. His research interests include molecular profiling of diffuse large B-cell lymphoma as well as pathology resident education, especially in hematopathology and molecular genetic pathology.

Sickle Cell Anemia

Sickle cell disease is a blood cell disorder that affects a lot of Americans, especially those of African descent. The disease is a result of a genetic defect that changes the structure of hemoglobin. This alteration in hemoglobin causes normally round red cells to become sickle in shape as well as sticky and deformed. As a result, these deformed red cells block perfusion of red blood cells into circulation and vital organs and affects the vasculature resulting in severe pain, organ damage and sometimes stroke.

Management of sickle cell disease includes blood transfusion, hydorxyurea and pain management. The long-term administration of hydroxyurea to stimulate the production of fetal hemoglobin is a form of therapy that provides relief in that fetal hemoglobin essentially protects the red cells from sickling. The transfusion of red cells lowers the amount of sickle hemoglobin levels.

Transplantation of hematopoietic stem cells from HLA-identical siblings can be curative in several nonmalignant hematologic disorders, including aplastic anemia, β-thalassemia major, congenital immunodeficiency disorders, and certain inborn errors of metabolism. Pilot studies of bone marrow transplantation for the treatment of young patients with symptomatic sickle cell disease have demonstrated eradication of the underlying disease with low transplantation-related mortality (Bhatia and Walters, 2007).

The process for successful bone marrow transplant to cure sickle cell involves administration of chemotherapy or immunosuppressive drugs to eradicate all the cells.  Now, doctors have developed a more successful regimen where patients take immunosuppressive drugs with a low dose body irradiation, a treatment much less harsh than chemotherapy. Next, donor cells from a healthy and tissue-matched sibling are transfused into the patient. Stem cells from the donor produce healthy new blood cells in the patient, eventually in sufficient quantity to eliminate symptoms. In many cases, sickle cells can no longer be detected. Patients must continue to take immunosuppressant drugs for at least a year.

In the reported trial, published online in the journal Biology of Blood & Marrow Transplantation, physicians from the University in Illinois Chicago transplanted 13 patients, 17 to 40 years of age, with a stem cell preparation from the blood of a tissue-matched sibling. In a further advance of the NIH procedure, the physicians successfully transplanted two patients with cells from siblings who matched but had a different blood type (Parmet, 2015)

Stem cell transplantation has proven itself to provide cures for a lot of hematologic malignancies, now it is successfully finding its way to cure other hematologic abnormalities, including sickle cell anemia.  This continued advancement in medicine will provide relief to a lot of patients who are suffering from sickle cell disease.

References

  1. Bhatia, M., & Walters, M. C. (2007). Hematopoietic cell transplantation for thalassemia and sickle cell disease: past, present and future. Bone Marrow Transplantation, 41(2), 109-117. doi:10.1038/sj.bmt.1705943
  1. Parmet, S. (2015). Adults with sickle cell disease cured with stem cell transplants. Retrieved March 25,2017, from https://news.uic.edu/cure-for-sickle-cell

 

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-Carlo Ledesma, MS, SH(ASCP)CM MT(ASCPi) MT(AMT) is the program director for the Medical Laboratory Technology and Phlebotomy at Rose State College in Midwest City, Oklahoma as well as a technical consultant for Royal Laboratory Services. Carlo has worked in several areas of the laboratory including microbiology and hematology before becoming a laboratory manager and program director.

Interference in Lab Assays

A 69 year old patient with cirrhosis presented to the ER with fever. Her bilirubin was markedly elevated at 7.4 g/dl and her hemoglobin and hematocrit were measured at 13.4 g/dl and 35.6% respectively with a MCV of 103.2 fl and MCH of 38.5 pg. The next day her H/H were 11.9 g/dl and 31.3 % respectively. While her hemoglobin one day later was 11.9 g/dl, the reported hematocrit was 39.3%. Patient had a bilirubin level of 8.7 g/dl at this time.

The fluctuating numbers together with the discrepancy between hemoglobin and hematocrit over a very short period of time was concerning. We realized that presence of markedly icteric plasma was responsible for these discordant values. Saline replacement and spun crit were performed in order to correct interference by bilirubin. Subsequent measurements of H/H revealed hemoglobin in the range of 12.9 g/dl with a hematocrit of 38% and a MCV of 113 fl. As the bilirubin levels started dropping (in the range of 6.5 g/dl) the hemoglobin level measured by the analyzer fell in the range of 10.3 to 11 g/dl. The instrument (XN-200) gave no error codes and therefore we were able to report out the analyzer results without correction. It was however very important to convey to the clinical team that the H/H values did not truly represent a fall from the previous values. As the two methodologies were different (spun crit and plasma replacement was being no longer performed) the numbers should be interpreted accordingly. Patient was not bleeding actively and did not require any blood transfusion.

Interference occurs when a substance or process falsely alters an assay result. Interferences are classified as endogenous or exogenous. Endogenous interference originates from substances present in the patient’s own specimen. Exogenous interferences are substances introduced into the patient’s specimen. Interference from hemolysis, icterus and lipemia are most frequently studied. Protein interferences are most often associated with paraproteins and predominantly with IgM or IgG and rarely with IgA. Drug interference may be due to the parent drug, metabolite(s) or additives in the drug preparation. Determining if interference is significant requires deviation limits from the original result. Once interferences are identified there is a need to establish procedures for handling affected results as part of the quality system.

Hemoglobin is quantified based on its absorption characteristics. Conditions such as hyperlipidemias, hyperbilirubinemia, a very high white blood cell count, and high serum protein can interfere with this measurement and result in falsely elevated hemoglobin values. When the values of hemoglobin, red cell count, and MCV are affected, MCH and MCHC also become abnormal, since these indices are calculated and are not directly measured. Sometimes a set of spurious values may be the first clue to an otherwise unsuspected clinical condition (e.g., the combination of low hematocrit, normal hemoglobin, and high MCV and MCHC is characteristic of cold agglutinins).

Although one must pay attention to very high amounts of bilirubin within the plasma, most hematology analyzers do not presently demonstrate any interference with bilirubin, at least for concentrations up to 250 mg/l. Above these values attention is however needed.

High serum or plasma bilirubin concentrations can cause spectral interference with assays near the bilirubin absorbance peak of ~ 456 nm. Chemical interference e.g. with peroxidase-catalysed reactions may also occur.

 

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-Neerja Vajpayee, MD, is the director of Clinical Pathology at Oneida Health Center in Oneida, New York and is actively involved in signing out surgical pathology and cytology cases in a community setting. Previously, she was on the faculty at SUNY Upstate for several years ( 2002-2016) where she was involved in diagnostic work and medical student/resident teaching.