hematopathology – Page 2

The Story of the Mott Cell, COVID-19 and the Cute Little Mouse

I have worked in hematology for many years, and there are certain things that never fail to excite technologists. Working in New Hampshire, it was always exciting to sickle cells or malaria, something common to some, but not common in our patient population. I now work in Baltimore, and see sickle cells nearly every day, and we come across malaria not too infrequently, but we still share good examples and save them for training. When we see something different or unusual, we always share the finding. Cells may need to be sent to the pathologists for a pathology review, and we always check back to see the pathologist’s identification and comments. Medical Technologists by nature are a curious bunch, and we always want to see ‘cool’ things. I wrote a blog two years ago about the only patient I have ever seen with Trypanosoma (Hematology Case Study: The Race to Save a 48 Year Old Man from a Rare Disease). Last month I wrote about Blue-green cytoplasmic inclusions (COVID-19 Patients with “Green Crystals of …” STOP! Please Don’t Call Them That). So, when I saw something else ‘cool’ and different on a peripheral smear, and then saw it AGAIN, on another patient, and saw other techs here in the US and in other countries were also mentioning these, because it’s my nature, I got curious.

When I write these blogs, I often feel a little bit like the mouse in the children’s story “If You Give a Mouse a Cookie”, by Laura Joffe Numeroff. It’s about an adorable little mouse who asks for a cookie, and then decides he needs a glass of milk to go with it, and then he needs a straw, and it goes on and on, in a circle, back to the beginning. Maybe it’s that the mouse is a little ADD, but I like to believe that he’s just creative and curious. I start with an idea, and often go off on many tangents before a blog is finished and comes back to where I started.. When I started writing this, it was because I saw an interesting cell, and I started exploring, and found that others had seen them, too. Then I started looking through my textbooks for references and information, and searched for recent research or studies, and then I wanted to find out more… just like that mouse.

There are some things that we learn about in school and we may see on CAP surveys, but no matter where you work, they are still rarely seen, so are a novelty. Mott cells are one of these things. I have a collection of Hematology texts from grad school and years of teaching Hematology. Several of these don’t even mention Mott cells, but, when they do, it’s barely a sentence in a discussion of plasma cells. I happen to have a very old copy of Abbott Laboratories “The Morphology of Human Blood Cells” . The one with the red cover, from 1975. The term Mott cell does not appear in this manual, but they do show pictures and describe “Plasma cells with globular bodies (Grape, Berry or Morula cells)”, and describe these globules as “Russell bodies”.¹ So some of us who have been working in the field for many years may remember Russell bodies and Morula cells, or Grape cells, even if the term Mott cell is not familiar. Regardless of what we or textbooks call them, they tend to trigger a memory because the images are so unique.

So, again, I’m a bit like that mouse and getting distracted with the background. Why am I writing this blog? In recent months I have seen cells identified as plasmacytoid lymphocytes and Mott cells in several hospitalized patients. I have heard reports of these cells in other facilities as well. So, like a good medical technologist, I got curious about Mott cells. What are they, and what is their significance? And why are we seeing more of these now?

Mott Cells are named after surgeon F.W. Mott. In the 1890’s, William Russell first observed these cells with grape like globular inclusions, but did not recognize what the inclusions were or their significance. Russell examined the cytoplasmic globular inclusions and assumed that these cells were fungi. Ten years later, Mott described cells he called morular cells. He recognized that these cells were plasma cells and the inclusions were indicative of chronic inflammation. Thus, today we refer to these cells as Mott cells, morular cells or grape cells, and the inclusions as Russell bodies.²

Hematology texts describe Mott cells as morphologic variations of plasma cells packed with globules called Russell bodies. We know that plasma cells produce immunoglobulin. When the plasma cells produce excessive amounts of immunoglobulin, and there is defective immunoglobulin secretion, it accumulates in the endoplasmic reticulum and golgi complex of the cells, forming Russell bodies. Russell bodies are eosinophilic, but in the staining process the globulin may dissolve and they therefore appear to be clear vacuoles in the cell under the microscope. Thus, a plasma cell with cytoplasm packed with these Ig inclusions is called a Mott cell.

Mott recognized that these atypical plasma cells were present in inflammation. Plasma cells are not typically seen on peripheral blood smears and constitute less than 4% of the cells in a normal bone marrow. Yet, on occasion, we can see plasma cells, including Mott cells, on peripheral blood smears in both malignant and non-malignant conditions. Mott cells are associated with stress conditions occurring in a number of conditions including chronic inflammation, autoimmune diseases, lymphomas, multiple myeloma, and Wiskott–Aldrich syndrome.³

So, why are we seeing an increased mention of Mott cells now? We seem to be seeing these on patients testing positive for SARS-CoV-2. I have seen cells on patients at my facility that resemble Mott cells. I belong to a Hematology Interest group and over the past few months I have seen several people post pictures of Mott cells, cells with Russell bodies, and plasmacytoid lymphocytes identified on peripheral blood smears of COVID-19 patients. Other techs chimed in with comments that they have also seen these cells recently. I have even seen a comment propose that these cells are indicative of COVID-19 infection.

SARS-CoV-2 definitely causes inflammatory processes and stress conditions in the body, so it makes sense that we may see these cells in COVID-19 positive patients.

Figure 1 shows a Mott cell on an image from Parkland Medical Center Laboratory, Derry, NH. A Mott cell was identified by pathologist in a male patient who tested negative for COVID-19 at the time the sample was drawn, and subsequently tested positive. Mariana Garza, a Medical Technologist working at Las Palmas Medical Center in El Paso, TX shared a case of a 59 year old diabetic male, diagnosed with COVID-19. The patient’s WBC was 31 x 10³/μL. Two Mott cells were identified by pathologist on his differential. So, the curious little mouse in me researched some more.

Image 1. Mott cell. Photo courtesy Parkland Medical Center, Laboratory, Derry, NH.

Several published research papers have studied morphologic changes in peripheral blood cells in COVID-19 patients. As we now know, SARS-CoV-2 affects many organs including the hematopoietic and immune systems. A study in Germany showed that COVID-19 patients exhibited abnormalities in all cell lines; white blood cells, red blood cells and platelets. Increased WBC counts were seen in 41% of samples in their study. Differentials performed on study patients showed lymphocytopenia in 83%, and monocytopenia in 88%. Red blood cell morphology changes were noted. Platelet counts ranged from thrombocytopenia to thrombocytosis, but giant platelets were noted across the board.⁴

Mott cells are indicative of chronic inflammation and may have significance in association with COVID-1. In the above mentioned study, aberrant lymphocytes were noted in 81% of patients who were SARS-CoV-2 positive, and observable in 86% of the same patients after they tested negative. The paper shows plasmacytoid lymphocytes and Mott cells amongst these aberrant lymphocytes. Moreover, morphologic changes in neutrophils, such as a left shift and pseudo‐Pelger‐Huët anomaly, decreased after virus elimination but changes in lymphocytes, indicators of chronic infection, remained.⁴

Another study also reported reactive or plasmacytoid lymphocytes and Mott cells observed in peripheral blood.^4,5 Researchers at Northwick Park Hospital, UK, presented a case study of a 59 year old male with COVID-19 with a normal WBC and thrombocytosis. His differential revealed lymphocytopenia. His differential also showed lymphoplasmacytoid lymphocytes and Mott cells. In their conclusions it is stated that “In our experience, the lymphocyte features illustrated above are common in blood films of patients presenting to hospital with clinically significant Covid‐19. The observation of plasmacytoid lymphocytes supports a provisional clinical diagnosis of this condition.”⁵

Can these variant plasma cells, along with other commonly seen morphological changes, be used as part of a diagnostic algorithm for SARS-Cov-2 infection? As we see more COVID-19 patients there will be more, larger studies done and more Mott cells identified. Some disorders, such as Epstein Barr Virus and Dengue Fever are characterized by distinct viral changes in cells. However, since Mott cells can be seen in many conditions, these alone could not be considered diagnostic, but the indications are that these cells, along with the entire differential and morphological patterns, could prove to be a straightforward and easy to perform supplementary diagnostic tool. More, larger studies need to be done. It was concluded in the German study, that this pattern of morphologic changes in cells could be further investigated and validated with a larger blinded study, and that this information could lead to the development of a morphologic COVID‐19 scoring system.⁴In the meantime, keep an eye out for Mott cells. These should not be ignored and should be in some way noted because they may be of future diagnostic use. That’s all or now, folks! Something to dig deeper into in another blog! The mouse strikes again!

Many thanks to Nikki O’Donnell, MLT, Parkland Medical Center, Derry, NH and Mariana Garza, MT, Las Palmas Medical Center in El Paso, TX for sharing their case studies and photos.

Becky Socha MS, MLS(ASCP)^CMBB

References

Diggs, LAW, Sturm, D, Bell,A. The Morphology of Human Blood Cells, Third edition. Abbott Laboratories. 1975.
ManasaRavath CJ, Noopur Kulkarni, et al. Mott cells- at a glance. International Journal of Contemporary Mudeical Research 2017;4(1):43-44.
Bavle RM. Bizzare plasma cell – mott cell. J Oral Maxillofac Pathol. 2013;17(1):2-3.doi: 10.4103/0973-029X.110682.
Luke, F, Orso, E, et al. Coronavirus disease 2019 induces multi‐lineage, morphologic changes in peripheral blood cells:eJHaem. 2020;1–8.
Foldes D, Hinton R, Arami S, Bain BJ. Plasmacytoid lymphocytes in SARS-CoV-2 infection (Covid-19). Am J Hematol. 2020;1–2. https://doi.org/10.1002/ajh.
Numeroff, Laura. If You Give a Mouse a Cookie, 1985.

-Becky Socha, MS, MLS(ASCP)^CMBB^CMgraduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

COVID-19 Patients with “Green Crystals of …” STOP! Please Don’t Call Them That

Blue-green cytoplasmic inclusions in neutrophils and monocytes are a novelty in hematology. It is rare to see these inclusions on peripheral smears, and when we do, there is excitement, but sadness too, because, when noted, they usually indicate a poor prognosis and impending death. Thus, we have heard them called “green crystals of death” or “death crystals.” I know I would not want to read a family member’s medical chart and see reference to “death crystals.” It’s an insensitive term, and one the medical community is trying to discourage. And, in fact, though it typically does indicate a poor prognosis, not all cases lead to death. In published reports, it has been shown that short term mortality in patients with these crystals is about 60%.¹

These rare inclusions are refractile and irregular in shape, and are found in neutrophils, and occasionally in monocytes. Color seems to be subjective here. They call them green when inclusions in photos or cells I am looking at look very blue to me. The color perceived may depend on the type of stain (Giemsa, Wright or Wright-Giemsa) used and how fancy we get in color names and descriptions. Or, maybe I’m just color blind! Some people (like my husband) are “lumpers” and call anything blue-green, blue, or green, but don’t recognize subtleties of colors. Thus, I guess to make everyone happy, or to compromise, the blue-green description may fit them best.

Image 1. Blue-green inclusions seen in neutrophils. Photos courtesy of Alana D. Swanson. UMMC

These blue-green inclusions were originally reported in patients with hepatic injury and failure. Laboratory results include elevations in AST, ALT and LDH. More recently, there have been cases with no evidence of hepatic injury. Researchers are now finding that these crystals can occur in patients with tissue injury other than liver, and in patients with multiorgan failure. In patients with no liver injury, what is a common factor is that LDH is elevated, indicating tissue injury. Additionally, along with these crystals, lactic acid levels can be used as a predictor of survival. Higher levels of lactic acidosis at the time crystals are noted is a negative predictor of survival.²

In trying to determine the clinical significance of these crystals, they have been subject to a number of different stains to determine their content. The association with hepatic failure led researchers to hypothesize that the crystals were a bile product in circulation. Since then, the crystals have been found to be negative in bile stains. When stained with other stains, Oil Red O showed positive in neutrophils, indicating high lipid content. The inclusions did not stain positive with iron stain or myeloperoxidase. Acid fast stains showed the inclusions to be acid fast positive.³ These crystals also show an interesting similarity to sea-blue histiocytes, which further associates them with tissue injury. After analysis, it is now thought that these crystals contain lipofuscin-like deposits representing lysosomal degradation products, and may be present in multiple types of tissue injury.²

With the current pandemic, I have seen reports of these crystals in COVID-19 patients. I have heard of fellow technologists seeing these, and a recent paper described the first reported cases in patients with COVID-19. These recent incidences may lead to new information about exactly what clinical significance they hold. About one third of COVID-19 patients have elevated ALT and AST, though it is not yet clear whether the liver dysfunction is directly caused by the virus, due to sepsis, or other complications of patient comorbidities. Many COVID-19 patients have mild disease, yet some develop severe pneumonia, respiratory complications, and multiorgan failure. Mortality is increased in these severely affected patients. To better understand and manage treatment for COVID-19, physicians seek to identify biological indicators associated with adverse outcomes.¹

In a New York City study, Cantu and colleagues reported on six COVID-19 patients who presented with blue-green crystals in neutrophils and/or monocytes. All six patients had an initial lymphocytopenia, and significantly elevated AST, ALT, LDH and lactic acid at the time the crystals were noted. All of the patients had comorbidities, yet only two of the six presented with acute liver disease. Interestingly, in the six cases reported on in the study, only one had blue-green inclusions reported from the original manual differential. The others were found retrospectively when correlating the cases with patients known to have elevated ALT and AST. All patients died within 20 days of initial diagnosis.¹

The consensus of several papers in the last few years is that these crystals are being underreported. As seen in the above study, the crystals were originally seen in just one of the six patients. A look back revealed the other cases. With an increase in COVID-19 cases in our facilities, these blue-green crystal inclusions may be a novelty that is wearing off. We may see a rise in their presence, and need to be able to recognize and report them. This information is important to report if clinicians are to use these crystal inclusions along with acute transaminase and lactic acid elevations to predict poor patient outcomes.

Clinicians, hematologists, and laboratory technologists should be educated and have a high level of awareness of these inclusions. The University of Rochester conducted a study a few years ago that noted that, because these crystals are rare, techs may not be on the lookout for them. Once techs see them, they seem to be on the alert and more are reported. The hospital instituted an “increased awareness” campaign, which resulted in an increase in detection. This revealed cases that were not related to liver injury, including patients with metastatic cancer and sepsis. However, an important correlating factor was that all of the patients had mild to severe elevations in liver enzymes. With more awareness, we are starting to see them in patients without hepatic injury, but with other inflammation and tissue injury.⁴

Image 2. Blue- green crystal inclusions seen in a patient diagnosed with sepsis and multiorgan failure. Photo courtesy of Karen Cable, YRMC.

Let’s raise our level of awareness of these maybe-not-so-rare crystal inclusions. And, please be sure to call them by their preferred name, blue-green neutrophil inclusions! Let’s not talk about death crystals or crystals of death.

Many thanks to my colleague Alana D. Swanson, MLS(ASCP)^CM, University of Maryland Medical Center and Karen Cable, Hematology Section Lead, Yavapai Regional Medical Center, Arizona, for the photos used in this blog.

References

Cantu, M, Towne, W, Emmons, F et al. Clinical Significance of blue-green neutrophil and monocyte cytoplasmic inclusions in SARS-CoV-2 positive critically ill patients. Br J Haematol. May 26, 2020.
Hodgkins, SR, Jones, J. A Case of Blue-Green neutrophil inclusions. ASCLS Today. 2019;32:431.
Hodgson, T.O., Ruskova, A., Shugg, C.J., McCallum, V.J. and Morison, I.M. Green neutrophil and monocyte inclusions – time to acknowledge and report. Br J Haematol, 2015;170: 229-235.
Patel,N, Hoffman,CM, Goldman,BJ et al. Green Inclusions in Neutrophils and Monocytes are an Indicator of Acute Liver Injury and High Mortality. Acta Haematol. 2017;138:85-90

Hematopathology and Molecular Diagnostics Case Study: A 63 Year Old Man with Fatigue

The following case is an interesting overlap of Hematopathology and Molecular Diagnostics, and shows the utility of sequencing to detect a cancer before biopsy could.

A 63 year old gentleman presented to a heme/onc physician with six months of intractable anasarca, fatigue, and a recent mild thrombocytopenia (Table 1). They were otherwise in healthy condition. The physician initiated a lymphoma work-up that included a bone marrow biopsy. The tests were negative for M-protein.

Table 1. Summary of symptoms and relevant abnormal labs.

The bone marrow biopsy was somewhat limited, but the core contained multiple marrow elements. After a thorough review by a Hematopathologist, no evidence of dysplasia or other irregularities could be detected (Image 1). Flow cytometry detected no aberrant blast population. Cytogenetics detected 20del [16/20] and 5del [3/20]. These findings did not clearly indicate a specific diagnosis.

Image 1. 40x view of the bone marrow specimen at the initial presentation. No evidence of dysplasia was found.

As the clinical suspicion for a malignancy was high, the bone marrow specimen was sent for sequencing on a 1385-gene panel test. The test included tumor-normal matched DNA sequencing (“tumor” sample: bone marrow, normal: saliva), RNA whole transcriptome sequencing on the bone marrow, and Copy Number Variant (CNV) analysis. Tumor-normal matched sequencing helps rule out variants that are normal and present in the patient.

Somatic mutations were determined as those that were present in the “tumor” sample and not in the matched normal sample. The somatic variants found are listed below with their variant allele frequency (VAF) in parenthesis. Recall that a VAF of 40% means that a mutation is present in the heterozygous state in 80% of cells.

IDH2 (p.R140Q, 46%)
SRSF2 (p.P95T, 51%)
CBL (p.R499*, 47%)
KRAS (p.K117N, 12%)

Figure 1. View of IGV, which displays the NGS reads for IDH1 along with the variant allele highlighted in red. The color of the bars indicates the direction of the reads (forward in red and reverse in blue). This reflects the allele frequency of approximately 50%.

The mutations in these genes are commonly found in myeloid cancers including myselodysplastic syndrome. Activating mutation in IDH2 (isocitrate dehydrogenase 2) increase the production of the oncometabolite 2-HG, which alters methylation in cells taking them to an undiffereitiated state. SRSF2 (Serine And Arginine Rich Splicing Factor 2) is a part of the spliceosome complex, which regulates how sister chromatids separate from each other. Failures in the proper function of the complex creates genomic instability. CBL (Casitas B-lineage Lymphoma) is a negative regulator of multiple signaling pathways, and loss of function mutations (as seen here) lead to increased growth signals through several tyrosine kinase receptors. KRAS (Kirsten RAt Sarcoma virus) is an upstream mediator of the RAS pathway, which acquires mutations that lead to constitutive activation and sends growth signals to cells causing them to proliferate.

Furthermore the CNV analysis also found the heterozygous loss of chromosome 20 as reported in cytogenetics. CNV analysis did not detect chromosome 5 deletion, as it was below the limit of detection (20% for CNV analysis).

Figure 2. This plot shows the normalized read frequency of genes across each of the chromosomes is shown here. The drop at chromosome 20 is shown in a pale brown color on the right side of the graph. This is consistent with the cytogenetic findings. The loss of 5q isn’t seen as it is below the limit of detection of 30%.

These mutations are all individually common in MDS, but the co-occurance of each gives very strong evidence that MDS is the diagnosis (Figure 3). There have also been studies that provide prognostic implications for several of the genetic mutations present. Some mutations like SRSF2 or CBL at high VAF (>10%) indicate a poor prognosis, but mutations in IDH2 or TP53 at any frequency have not only a high chance of progression, but also a faster time to onset of disease. Another non-genetic risk factor for developing MDS is an elevated RDW, which we saw in our patient.

All of these high-risk factors together led us to push for a diagnosis of MDS based off of molecular findings, and the patient was started on treatment with Azacitadine. Our assessment was confirmed 3 months later when, the patient’s follow up bone marrow biopsy showed significant progression with megakaryocytic and erythroid dysplasia and hyperplasia and reticulin fibrosis MF2 (Image 2). Aberrant blasts were detected (1-2%), but not elevated. This demonstrates how molecular findings predicted and predated the patient’s rapid progression to morphologic disease.

Image 2. Dysplastic, hyperplastic megakaryocytes and erythroid lineage.

In summary, multiple molecular mutations indicative of MDS were found in a symptomatic patient’s unremarkable bone marrow biopsy months before a rapid progression to MDS.

References

Steensma DP, Bejar R, Jaiswal S et al. Blood 2015;126(1):9-16.
Sellar RS, Jaiswal S, and Ebert BL. Predicting progression to AML. Nature Medicine 2018; 24:904-6.
Abelson S, Collord G et al. Prediction of acute myeloid leukemia risk in healthy individuals. Nature 2018; 559:400-404.
Desai P, Mencia-Trinchant N, Savenkov O et al. Nature Medicine 2018; 24:1015-23.
Becker PM. Clonal Hematopoiesis: The Seeds of Leukemia or Innocuous Bystander? Blood.2016 13(1)

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

A 66 Year Old Male with Diarrhea, Weight Loss, and Night Sweats

Case History

A 66 year old man with past medical history of recently diagnosed Clostridioides difficile colitis presented to emergency department with diarrhea, weight loss of 52 pounds in 4 months, and occasional night sweats. CT imaging revealed dilation of small bowel with thickened mucosal folds. The duodenum was subsequently biopsied to reveal diffuse intestinal lymphangiectasia containing PAS positive and Congo red negative eosinophilic material and lamina propria foamy macrophages. Laboratory investigations revealed normocytic anemia, proteinuria, and peripheral IgM kappa monoclonal gammopathy.

Biopsy Findings

Bone marrow aspirate shows increased plasma cells and mast cells. H&E stained sections demonstrate a normocellular bone marrow with trilineage hematopoiesis and involvement by 35% plasma cells. By immunohistochemistry, CD138 highlights clusters of plasma cells that predominantly express kappa light chain restriction.

FISH and Mutation Analysis

FISH demonstrated loss of chromosome 11 and gain of chromosome 15, which was consistent with plasma cell dyscrasia. MYD88 mutation analysis did not detect the mutation.

Diagnosis

The findings of the patient’s normocytic anemia, IgM monoclonal gammopathy, and intestinal lymphangectasia with an associated plasma cell dyscrasia involving the bone marrow favor a lymphoplasmacytic lymphoma/Waldenström macroglobulinemia.

Discussion

Waldenstrom macroglobulinemia (WM) is a malignant B-cell lymphoproliferative disorder characterized by lymphoplasmacytic infiltration of the bone marrow and peripheral IgM monoclonal gammopathy.¹ It is rare with an overall incidence of 3 per million persons per year, accounting for 1-2% of hematologic cancers.¹ It occurs predominantly in Caucasian males, with a median age of 63-68 years old at diagnosis.^1-3

Patient may be asymptomatic for years and require observation or experience a broad spectrum of signs and symptoms. These symptoms may be attributable to the tumor infiltration of the bone marrow and lymphoid tissues, IgM circulating in the blood, and IgM depositing into tissues. The most common clinical presentation of WM is fatigue and nonspecific constitutional symptoms, such as fever, night sweats, and weight loss, due to normochromic, normocytic anemia. 20-30% of patients may exhibit lymphadenopathy and hepatosplenomegaly due to infiltration of peripheral tissues. High concentration of IgM in the circulation may lead to hyperviscosity, resulting in oronasal bleeding, gingival bleeding, blurred vision due to retinal hemorrhages, and neurological symptoms, including headache, ataxia, light-headedness, dizziness, and rarely, stroke.^2-3 The gastrointestinal manifestations are rare; however, IgM monoclonal protein may deposit into the lamina propria of the GI tract, causing diarrhea, steatorrhea, and GI bleeding.⁴ Other IgM-related manifestations include cold agglutinin hemolytic anemia, cryoglobulin, and amyloid deposition in tissues.³

Diagnosis of WM includes evidence of IgM monoclonal gammopathy and at least 10% of bone marrow infiltration by lymphoplasmacytic cells.⁵ Monoclonal gammopathy can be detected by the monoclonal spike, or M-spike, on serum protein electrophoresis.³ Serum immunofixation may be performed to identify the type of monoclonal protein and the type of light chain involved.³ In terms of immunophenotype, neoplastic cells express surface IgM, cytoplasmic Igs, CD38, CD79a, and pan B-cell markers (CD19, CD20, and CD22). CD10 and CD23 are absent. Expression of CD5 occurs in approximately 5-20% of cases.⁶Recent studies have reported two most common somatic mutations in WM, which are MYD88 L265P mutations (90-95% of cases) and CXCR4 (30–40% of cases).⁷ Absence of these mutations, however, do not completely exclude the diagnosis of WM.

The International Staging System for WM identifies five factors associated with adverse prognosis, including age older than 65, hemoglobin < 11.5g/dL, platelet count < 100K/μL, beta-2-microglobulin > 3mg/L, and monoclonal IgM concentration > 7g/L.³ Patients younger than the age of 65 years with 0 or 1 of these factors are in the low-risk category with a median survival of 12 years.³ In contrast, patients with 2 or more risk factors are in the intermediate- and high-risk categories and have a median survival of almost 4 years.³

Management of WM depends on the patient’s clinical manifestations.Furthermore, patients with minimal symptoms should be managed with rituximab, whereas patients with severe symptoms related to WM should receive more aggressive treatment, including dexamethasone, rituximab and cyclophosphamide. Hyperviscosity syndrome is an oncologic emergency that requires removal of excess IgM from the circulation via plasmapheresis.⁸

References

Neparidze N, Dhodapkar MV. Waldenstrom’s Macroglobulinemia: Recent advances in biology and therapy. Clin Adv Hematol Onco. 2009 Oct;7(10): 677-690.
Leleu X, Roccaro AM, Moreau AS, Dupire S, Robu D, et al. Waldenstrom Macroglobulinemia. Cancer Lett. 2008 Oct;270(1):095-107.
Tran T. Waldenstrom’s macroglobulinemia: a review of laboratory findings and clinical aspects. Laboratory Medicine. 2013 May;44(2):e19-e21.
Kantamaneni V, Gurram K, Khehra R, Koneru G, Kulkarni A. Distal illeal ulcers as gastrointestinal manifestation of Waldenstrom Macroglbulinemia. 2019 Apr; 6(4):pe00058.
Grunenberg A, Buske C. Monoclonal IgM gammopathy and Waldenstrom’s macroglobulinemia. Dtsch Arztebl Int. 2017 Nov;114(44):745-751.
Bhawna S, Butola KS, Kumar Y. A diagnostic dilemma: Waldenstrom’s macroglobulinemia/plasma cell leukemia. Case Rep Pathol. 2012;2012:271407.
Varettoni M, Zibellini S, Defrancesco I, Ferretti VV, Rizzo E, et all. Pattern of somatic mutations in patients with Waldenstrom macroglobulinemia or IgM monoclonal gammopathy of undetermined significance.
Oza A, Rajkumar SV. Waldenstrom macroglobulinemia: prognosis and management. Blood Cancer Journal. 2015;5:e394.

-Jasmine Saleh, MD MPH is a pathology resident at Loyola University Medical Center with an interest in dermatopathology and hematopathology. Follow Dr. Saleh on Twitter @JasmineSaleh.

–Kamran M. Mirza, MD, PhD, MLS(ASCP)^CM is an Assistant Professor of Pathology and Laboratory Medicine, Medical Education and Applied Health Sciences at Loyola University Chicago Stritch School of Medicine and Parkinson School for Health Sciences and Public Health. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018 and placed #5 in the #PathPower List 2019. Follow him on twitter @kmirza.

Hematopathology Case Study: An 80 Year Old Man with Rapid Onset Cervical Adenopathy

Case History

An 80 year old man presented with rapid onset of cervical adenopathy over a period of few months. The largest lymph node measuring 6 cm was biopsied and sent for histopathological evaluation.

Biopsy Findings

Sections from the lymph node showed effacement of the lymph node architecture by a fairly monotonous population of medium to large sized lymphoid cells arranged in vague nodular pattern. Focally, a starry sky pattern was observed. The cells were 1.5-2 times the size of an RBC, with high N:C ratio, irregular angulated nuclei and small nucleoli. A high mitotic rate of 2-3 mitoses/hpf was seen.

Immunohistochemistry

Immunohistochemical stains showed that the lymphoma cells were positive for CD20, CD5, SOX-11, and negative for Cyclin D1, CD10, CD23, CD30, BCL-1, and BCL-6. Ki67 index was about 70%.

Diagnosis

A diagnosis of Mantle cell lymphoma, pleomorphic variant was made.

Discussion

Mantle cell lymphoma is a peripheral B cell lymphoma, occurring in middle aged or older adults, with a male: female ratio of 7:1. Although Cyclin D1 expression is considered a hallmark of mantle cell lymphoma, yet about 7% cases are known to be Cyclin D1 negative. In these cases, morphological features and SOX-11 positivity helps in establishing a definitive diagnosis.

Differential Diagnosis

In the assessment of morphological features of lymphoma, the cell size is an important starting point. In this case, the lymphoma cells ranged from medium to large sized. The following differential diagnoses were considered:

Burkitt lymphoma

This case showed a “starry sky” pattern focally. A medium sized population of cells, high mitotic rate and a high Ki67 index (70%) favoured a Burkitt lymphoma. However, although commonly seen in Burkitt lymphoma, a “starry sky” pattern is not specific for this type of lymphoma. Also, the lack of typical “squaring off” of nuclei, basophilic cytoplasmic rim were against the diagnosis of Burkitt lymphoma. The nuclei in this case showed 0-1 small nucleoli, unlike the typical basophilic 2-3 prominent nucleoli of Burkitt lymphoma. Moreover, Ki67 index, even though high was not enough for Burkitt lymphoma where it approaches 100%. The cells were negative for CD10 and Bcl-6, which are almost always found in a Burkitt lymphoma. Hence, a diagnosis of Burkitt lymphoma was ruled out.

Diffuse Large B cell Lymphoma

The presence of interspersed large cells with nucleoli, irregular nuclei, high mitotic rate, and a high Ki67 index with a history of very rapid enlargement of lymph node suggested a diagnosis of Diffuse Large B cell lymphoma. However, the scant cytoplasm, lack of bizarre cells, and absence of CD10, BCl-2, BCl-6 were against a diagnosis of DLBCL.

Lymphoblastic lymphoma

A diagnosis of lymphoblastic lymphoma was favoured by the irregularly angulated nuclei, and presence of nucleoli. However, the cells of lymphoblastic lymphoma have a more delicate nuclear chromatin, higher mitotic rate as against the relatively condensed chromatin and the low to high variable mitotic rate of Mantle cell lymphoma. Also, lymphoblastic lymphomas are more commonly of the T cell subtype and occur commonly in younger individuals. In this case, B cell markers were positive (CD 20), and the patient was 80 year old, disfavouring a lymphoblastic lymphoma. The blastoid variant of mantle cell lymphoma is practically indistinguishable from lymphoblastic lymphoma, except that it is Tdt negative.

Cyclin D1 negativity in Mantle cell lymphoma

In the cases of Cyclin D1 negative mantle cell lymphomas, morphology plays a critical role in coming to a diagnosis of mantle cell lymphomas. In this case, points that favoured the diagnosis of mantle cell lymphoma were clinical features such as older age (80 years), and male gender, and morphological features such as a vaguely nodular pattern of growth, irregular nuclei, and 0-1 small nucleoli. Due to the presence of variably sized cells with distinct nucleoli, a pleomorphic variant was considered. Even though Cyclin D1 was found to be negative, the cells were positive for SOX-11.

SOX-11 is a transcription factor that is not normally expressed in B cells, but is sensitive and fairly specific for mantle cell lymphomas. It is important to note that SOX-11 is also positive in 25% Burkitt lymphoma, 100% lymphoblastic lymphoma, and 66% T-prolymphocytic leukemia. Herein lies the importance of recognising morphological features, as all of these lymphomas that may express SOX-11 were ruled on the basis of morphology. A more specific antibody, MRQ-58 may be used for greater specificity. The presence of SOX-11 is considered a specific biomarker for Cyclin-D1 negative mantle cell lymphomas. In these cases, there is upregulation of Cyclin D2 or D3 that may substitute for Cyclin D1 upregulation. But, immunohistochemical detection of Cyclin D2 or D3 is not helpful for establishing a diagnosis, as other lymphomas are commonly positive for these markers. Hence, it is important to perform SOX-11 immunohistochemistry to diagnose the Cyclin D1 negative variant of mantle cell lymphoma.

SOX-11 can be used not just for the diagnosis, but also for determining prognosis of mantle cell lymphoma. Indolent MCL usually lack SOX-11 expression. The pattern of SOX-11 staining has also been used a marker of prognosis. Cytoplasmic expression of MCl, seen in only a few cases was associated with a shorter survival as compared to the more common nuclear staining of SOX-11.

Conclusion

In this age, lymphoma diagnosis relies heavily on the use of immunohistochemical markers. However, this case highlights the importance of morphological features in diagnosing lymphomas with unusual immunohistochemical marker profile. Although, this case was negative for Cyclin D1, considered a hallmark of Mantle cell lymphoma, yet, the combination of morphological features with SOX-11 staining helped in clinching the diagnosis. To avoid a misdiagnosis, it would be prudent to perform SOX-11 staining in all lymphoma cases morphologically resembling MCL, but lacking Cyclin-D1.

-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.

Hematopathology Case Study: A 69 Year Old Male with Weight Loss and Generalized Lymphadenopathy

Case History

The patient is a 69 year old male who presented to the hospital with a 3-month history of drenching night sweats, weight loss, fatigue, and generalized lymphadenopathy. He also endorsed a very itchy rash all over his body. He denied smoking. There was no other relevant social or family history.

Physical examination confirmed diffuse lymphadenopathy, hepatosplenomegaly and a mild diffuse skin rash. Notably, there was a 2.5 cm level-1 lymph node palpated in the left neck. This was subsequently biopsied.

Biopsy

Biopsy of the level-1 neck lymph node revealed a 2.3 x 1.5 x 1.2 cm mass pink-tan and firm mass. Sectioning revealed a glossy white-tan cut surface. H&E staining revealed a polymorphic lymphocytic infiltrate of in the interfollicular zones. The infiltrating lymphocytes ranged from small to large cells with abundant cytoplasm, eosinophils, and plasma cells. There was also a notable increase in the number of high endothelial vessels lined by lymphocytes with irregular nuclear borders and clear cytoplasmic zones.

Image 1. Polymorphic infiltrate of small, mature appearing lymphocytes (A), with prominent blood vessels and clear cytoplasm (B). Most of these cells were CD3 positive T cells (C) with expanded CD21 positive FDC meshworks (D) and scattered CD30 positive immunoblasts (E)

Further characterization by immunohistochemical staining showed the majority of the interfollicular cells to be CD3 and CD5 expressing T cells. These were a mix of CD4 and CD8 positive cells but with marked CD4 predominance. CD7 appeared positive in a smaller population of T-cells compared to CD3 (consistent with loss of this pan-T-cell marker). Varying numbers of the interfollicular cells were positive for CD10, BCL-6, CXCL-13, and PD-1 with a strong positivity for ICOS, phenotypically consistent with an expansion of Tfh (T-follicular helper cell) cells.

Interspersed between the T cells were numerous CD20 positive cells with prominent nucleoli that also revealed CD30 positivity. CD21 staining revealed expanded follicular dendritic cell meshworks. EBER ISH was positive in a rare subset of cells. Kappa and lambda ISH showed an increased number of polytypic plasma cells.

Flow Cytometry showed the presence of a small population of T-cells that were CD4 positive but CD3 negative. There was no evidence of B-cell clonality. TCR-G PCR was positive.

A final diagnosis of Angioimmunoblastic T-cell lymphoma (AITL) was rendered.

Discussion

AITL is a relatively rare neoplasm of mature T follicular helper cells, representing about 1-2% of all non-Hodgkin lymphomas. It is; however, one of the more common subtypes of peripheral T-cell lymphomas, accounting for 15-30% of this subgroup. The condition was first reported in 1974 in Lancet as a non-neoplastic abnormal immune reaction¹. It was first recognized as a distinct clinical entity in in 1994 in the Revised European American Lymphoma Classification². The disease shows a geological preference to Europe (28.7%) over Asia (17.9%) and North America (16%). AITL occurs primarily in middle aged and elderly individuals and shows a slight predominance of males over females.

The disease has a strong association with EBV infection, but the neoplastic T-cells are almost always EBV negative, creating an interesting question of EBV’s function in the etiology of AITL. AITL most often presents late in the disease course with diffuse systemic involvement, including hepatosplenomegaly, lymphadenopathy and other symptoms such as rash with pruritis and arthritis. Lab findings include cold agglutinins, rheumatoid factor and anti-smooth muscle antibodies. There also tends to be immunodeficiency secondary to the neoplastic process. The clinical course of AITL is variable, but the prognosis is poor, with the average survival time after diagnosis being < 3 years. The histological features and genetic findings have not been found to impact clinical course.

Microscopically, AITL presents with either partial or total effacement of the normal lymph node architecture with perinodal infiltration. The cells of AITL are small to medium-sized lymphocytes with clear to pale cytoplasm, distinct cell membranes and very minimal cytological atypia. These cells often congregate around the high endothelial venules. The T-lymphocytes are present in a largely polymorphous inflammatory background of other lymphocytes, histiocytes, plasma cells and eosinophils. There are 3 overlapping sub-patterns of AITL. The first of these is similar to a reactive follicular hyperplasia, and can only be distinguished from normal hyperplasia by use of immunohistochemical stains to differentiate the neoplastic cells from normal reactive cells. The second pattern has retained follicles, but they show regressive changes. The third pattern has completely or sub totally effaced. These three patterns seem to be on a spectrum with one another, given that progression from the first to the third pattern has been seen on consecutive biopsies in the same patient.

Cytologically, AITL cells express pan-T-cell markers including CD2, CD3 and CD5 and the vast majority are CD4 positive. CD3 may be quantitatively decreased or absent by flow cytometry. There are a variable number of CD8 positive T-cells. The tumor cells also show the immunophenotyping of normal T follicular helper cells including CD10, CXCL13, ICOS, BCL6 and PD1 in 60-100% of cases. CXCL13 and CD10 are the most specific, whereas PD1 and ICOS are the most sensitive.

References

Horne, C., Fraser, R., & Petrie, J. (1974). Angio-Immunoblastic Lymphadenopathy With Dysproteinemia. The Lancet, 304(7875), 291. doi:10.1016/s0140-6736(74)91455-x
Harris, N.l. “A Revised European-American Classification of Lymphoid Neoplasms: a Proposal from the International Lymphoma Study Group.” Current Diagnostic Pathology, vol. 2, no. 1, 1994, pp. 58–59., doi:10.1016/s0968-6053(00)80051-4.
Swerdlow, Steven H. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer, 2017.
“Angioimmunoblastic T Cell Lymphoma.” Pathology Outlines – PathologyOutlines.com, http://www.pathologyoutlines.com/topic/lymphomanonBAITL.html.

-Zachary Fattal is a 4th year medical student at the Central Michigan University College of Medicine. He is pursuing a career in pathology and has a special interest in hematopathology, cytopathology and blood bank/transfusion medicine. You can follow him on Twitter @Paraparacelsus.

–Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor of Pathology and Medical Education at Loyola University Health System. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018. Follow him on twitter @kmirza.

Hematopathology Case Study: A 36 Year Old Woman with an Incidental Neck Mass

Case History

A 36 year old female underwent thyroidectomy for multinodular goitre that led to the fortuitous discovery of a neck mass. The neck mass specimen submitted comprised two lymph nodes measuring 2.2 cm and 1.3 cm in the greatest dimensions, with a fleshy tan cut surface.

Biopsy Findings

H&E stained sections revealed numerous non-necrotizing granulomas effacing and replacing normal lymph node architecture. These consisted of pale epithelioid histiocytes and Langhans type of giant cells. The granulomas lacked a peripheral rim of lymphocytes. AFB and GMS stains were negative for microorganisms

Diagnosis

A diagnosis of non-necrotizing granulomatous lymphadenitis was rendered noting that in the correct clinical context the findings could represent sarcoidosis.

Discussion

Granulomatous inflammation is a special type of chronic inflammatory response characterised by the formation of discrete collections of histiocytes called granulomas. Activated histiocytes appear as epithelioid cells with round to oval nuclei, often with irregular contours and abundant granular eosinophilic cytoplasm with indistinct cell borders. They may coalesce to form multinucleated giant cells. When found in the lymph node, the reaction pattern is called granulomatous lymphadenitis. It can be caused by a variety of different conditions, and therefore, requires thorough workup to come to a conclusive diagnosis.

On the basis of presence or absence of necrosis, granulomatous lymphadenitis can be classified as necrotizing or non-necrotizing. Additionally, the presence of an abscess, usually central, indicates a suppurative lymphadenitis.

Non-necrotizing granulomatous lymphadenitis:

Sarcoidosis lymphadenitis is the prototype of non-necrotizing granulomatous lymphadenitis. It shows the presence of discrete granulomas without a peripheral rim of lymphocytes, called “naked granulomas”. The early phase shows follicular hyperplasia and sinus histiocytosis, followed by appearance of epithelioid cell nodules toward the end of this phase. The peak phase shows well-demarcated granulomas composed of epithelioid cells with scattered multinucleated giant cells observed throughout the lymph node. Granulomas may occasionally coalesce. In the late phase, increased collagen fibers result in fibrosis and hyalinization. There are no neutrophils and it is uncommon to find small foci of central necrosis. Numerous inclusions such as asteroid, Schaumann, or Hamazaki-Wesenberg bodies can be seen. In this case, we observed well-demarcated granulomas throughout the lymph node, typical of the peak phase without any caseous necrosis or suppuration.

Other causes of granulomatous lymphadenitis can be ruled out as follows.

Sarcoid-like lymphadenitis: It shows a similar pattern of non-necrotizing lymphadenitis like sarcoidosis. However, classically sarcoid like reaction shows scattered small epithelioid granulomas with sparsely arranged epithelioid cells. The border of the granulomas is usually obscure. The CD4:CD8 ratio ranges from 0.8 to 2.25 while in sarcoidosis, it is >3.5. These findings help distinguish sarcoid-like lymphadenitis from sarcoidosis.

Sarcoid-like adenitis may be seen in numerous conditions such as carcinoma, Toxoplasmosis, fungal infections, tuberculosis, immunocompromised states, pneumoconiosis etc. The fact that tuberculosis and fungal infections can present with a non-necrotizing granulomatous lymphadenitis highlights the importance of performing fungal (PAS & GMS) and AFB (Ziehl Neelson) stains in non-necrotizing lymphadenitis as well. In this case, the granulomas had distinct borders, numerous epithelioid cells, no organisms were identified on special stains, nor was there any history of immune compromise; ruling out a sarcoid-like reaction.

Berylliosis: The lymph node picture in Berylliosis is identical to that of sarcoidosis. We may even see asteroid bodies or Schaumann bodies. A diagnosis can be established by eliciting a history of chronic exposure to Beryllium. Beryllium lymphocyte proliferation test (BeLPT) is a test that measures Beryllium sensitization and is very specific for Beryllium exposure. There was no known history of exposure to Beryllium in this case.

Toxoplasmosis: A classic triad of follicular hyperplasia, small granulomas composed of epithelioid cells within and around hyperplastic follicles and, monocytoid B cell hyperplasia, is observed in toxoplasmosis lymphadenitis. This case did not show follicular hyperplasia, ruling out toxoplasmosis.

Necrotizing granulomatous lymphadenitis

Even though we did not find any necrosis in this case, yet, it is worthwhile to review briefly the various causes of necrotizing lymphadenitis.

Non-suppurative

Tuberculosis: Histology of a tuberculous lymph node is characterised by central caseous necrosis surrounded by an epithelioid cell layer. The outermost layer is comprised of lymphocytes and fibrosis. Plasma cells are not observed. Diagnosis can be established by performing an AFB stain that demonstrates acid fast rod shaped bacteria in the areas of necrosis. Organisms can also be detected by PCR.

BCG lymphadenitis: About 0.7 to 2.3% of BCG vaccinated children may develop BCG lymphadenitis that is smaller than tuberculous lymphadenitis. Early phase shows follicular hyperplasia and sinus histiocytosis. Later, there is development of micronodules of epithelioid granulomas without necrosis and epithelioid cell granulomas with central caseous necrosis. Langhans giant cells are rare.

Fungal infections: Fungal infections by Histoplasma, Cryptococcus, coccidiodomycosis, pneumocystis may also cause a necrotizing granulomatous inflammation. There are numerous neutrophils, and fungal structures can be seen. GMS and PAS can be used in cases where it is difficult to the find the fungal elements on H&E.

Suppurative

Tularemia: There are three forms of histological changes, Abscess form, showing abscess with central necrosis and mononuclear cells, Abscess-granulomatous form with granulomas with central necrosis, which form large lesions with central abscesses, and granulomatous form with caseating necrosis at the centre of the granulomas.

Cat Scratch disease: Similar to tularemia, there are three phases of histologic presentation, an early phase of follicular hyperplasia, intermediate phase of microabscess, and a late phase of granulomatous inflammation. Monocytoid B cell clusters are observed close to the abscess.

Conclusion

Sarcoidosis is usually diagnosed by excluding other causes of granulomatous inflammation, as we did in this case. Characteristic non-necrotizing, discrete granulomas were seen throughout the lymph node. The age of the patient and female gender epidemiologically support the diagnosis. This case reflects an example work up of a granulomatous lymphadenitis that is a morphologic presentation of myriad diseases.

-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.

Hematology Case Study: A 69 Year Old Female with Breast Implants

Case History

A sixty nine year old female who underwent right breast reconstruction about 13 years ago due to breast cancer presents to the doctor office with right breast pain and right breast enlargement over the last two months. She has lost some weight and does not recall any trauma to this area. She had a textured saline implant. Examination reveals no definite palpable masses. MRI of right breast showed intact saline implant with moderate amount of fluid surrounding the implant within the intact external capsule. No adenopathy was noted. Right breast implant was removed and complete capsulectomy was performed.

Image 1. A. Section of breast capsule with rare atypical hyperchromatic cells (arrow). B. Cytospin preparation of the fluid surrounding the implant with numerous atypical lymphocytes. C. Cell block of the fluid with large atypical lymphocytes. D, E. Lymphocytes are positive for CD30 (image D) and negative for ALK-1 (image E). F. CD30 positive cells in the section of the implant.

Diagnosis

Breast implant-associated anaplastic large cell lymphoma.

Discussion

Breast implant associated anaplastic large cell lymphoma is a provisional entity that is morphologically and immunophenotypically similar to ALK-negative anaplastic large cell lymphoma. It arises primarily in association with a breast implant. It is a very rare entity with an incidence of 1 in 500,000 to 3 million women with implants. Tumor cells may be localized to the seroma cavity or may involve pericapsular fibrous tissue. Sometimes it can form a mass lesion. Locoregional lymph node may be involved. The mean patient age is 50 years. Most patient presents with stage 1 disease, usually with peri-implant effusion. The mean interval from implant placement to lymphoma diagnosis is 10.9 years. There is no association with the type of implant. Histologic examination shows two different types of proliferations. In patients with seroma, the proliferation is confined to the fibrous capsule (“in situ” iALCL). However, the distribution of neoplastic lymphocytes could be heterogeneous with some cellular areas with numerous large pleomorphic cells of varying size and some fibrotic areas with rare atypical lymphocytes. It is beneficial to look at the seroma fluid in addition to capsule sections, because sometimes the neoplastic lymphocytes are predominantly present in fluid (as in our case). Patients presenting with tumor mass show more heterogeneous proliferations infiltrating surrounding tissues (“infiltrative” iALCL). They consists of either sheets are clusters of large neoplastic cells accompanied by a large number of eosinophils. By immunohistochemistry, the tumor cells are strongly positive for CD30. CD2 and CD3 are more often positive than CD5. CD43 is almost always expressed. Most cases are CD4 positive. The prognosis is very good in patients with disease confined to the capsule. The median overall survival is 12 years. However, patients with a tumor mass could have a more aggressive clinical outcome.

References

1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4^th edition). IARC: Lyon 2017.

2. Jaffe, E , Arber, D, et al. Hematopathology (second edition) 2017.

-Junaid Baqai, MD, was born in Chicago, IL but spent most of his life in Karachi, Pakistan. He graduated from DOW Medical College in Pakistan and did his residency in anatomic and clinical pathology at Danbury Hospital, CT followed by hematopathology fellowship from William Beaumont Hospital, Michigan and oncologic-surgical pathology fellowship from Roswell Park Cancer Institute, New York. He currently serves as Medical Director of hematology, coagulation and flow cytometry at Memorial Medical Center and Medical Director of Laboratory at Taylorville Memorial Hospital.

Hematopathology Case Study: A 76 Year Old Man with Lymphadenopathy

Case History

76 year old man with a history of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with new anterior mediastinal mass and increasing lymphadenopathy.

Lymph Node Biopsy

H&E

Diagnosis

Tissue sections show a diffuse atypical lymphoid infiltrate that completely effaces the normal nodal architecture. The infiltrate is composed of numerous small lymphocytes with round to mildly irregular nuclei, clumped chromatin, inconspicuous nucleoli and scant cytoplasm. There are also expanded pale areas that contain intermediate sized cells with more open chromatin and distinct single to multiple nucleoli. These cells are most consistent with prolymphocytes/paraimmunoblasts and form the proliferation centers characteristic of CLL/SLL. Occasional centroblastic-type B-cells are noted within these proliferation centers. In addition, there are scattered single to multinucleated cells that have irregular nuclear membranes with pale, vesicular chromatin and prominent inclusion-like, eosinophilic nucleoli. These cells morphologically resemble Hodgkin cells, Reed-Sternberg cells, mummified forms and other variants. These large cells are more evident in areas with a histiocyte rich background and around foci of necrosis. Occasionally, apoptotic bodies and mitotic figures are seen.

Immunohistochemical studies show that the vast majority of the small-intermediate lymphocytes express B-cell markers CD20 (dim) and PAX5 and co-express CD5 and CD23 (subset). This is consistent with a background of CLL/SLL. The large atypical cells are positive for CD30, PAX5 and CD20 (variable). CD3 highlights numerous scattered background small T-cells, which are increased in the areas with the large cells. In situ hybridization for Epstein Barr viral RNA (EBER ISH) is mainly staining the large atypical cells. By Ki-67, the proliferation fraction is overall increased (40%) with increased uptake by the large atypical cells.

The morphologic and immunophenotypic findings are consistent with involvement by the patient’s known small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) with aggressive morphological features. The aggressive features include expanded proliferation centers and an elevated Ki-67 proliferative index (40%). Additionally there are histiocyte/T-cell rich areas composed of multiple EBV positive large atypical cells with morphologic and immunophenotypic features compatible with Hodgkin/ Reed-Sternberg cells. These areas are most in keeping with evolving classic Hodgkin lymphoma. Sheets of large cells indicative of large cell transformation are not seen, although increased scattered large centroblastic-type B cells are present.

Discussion

Lymph node involvement by CLL/SLL will typically show a diffuse proliferation of small lymphocytes with effacement of the normal nodal architecture. The small lymphocytes have round nuclei, clumped chromatin and scant cytoplasm. Scattered paler areas known as proliferation centers are characteristic of this entity. The proliferation centers are composed of a mixture of cell types including small lymphocytes, prolymphocytes and paraimmunoblasts. Prolymphocytes are small to medium in size with relatively clumped chromatin, whereas paraimmunoblasts are larger cells with round to oval nuclei, dispersed chromatin, eosinophilic nucleoli and slightly basophilic cytoplasm. Some cases show increased and enlarged proliferation centers with a higher proliferation rate. This must be distinguished from large cell transformation.¹

Aggressive features of CLL/SLL include proliferation centers that are broader than a 20x field or becoming confluent. An increased Ki-67 proliferation >40% or >2.4 mitoses in the proliferation centers can also portend a more aggressive course. These cases tend to have worse outcomes than typical CLL/SLL and better outcomes than cases that have undergone Richter transformation to diffuse large B-cell lymphoma (DLBCL). Transformation to DLBCL occurs in 2-8% of patients with CLL/SLL. Less than 1% of patients with CLL/SLL develop classic Hodgkin lymphoma (CHL). In order to diagnose CHL in the setting of CLL/SLL, classic Reed-Sternberg cells need to be found in a background appropriate for CHL, which includes a mixed inflammatory background. The majority of these CHL cases will be positive for EBV.¹

Richter’s transformation is defined as an aggressive evolution of CLL. While the most common type of transformation is to a high-grade B-cell Non-Hodgkin lymphoma, other histological transformations have been described. This includes CHL, lymphoblastic lymphoma, hairy cell leukemia and high-grade T-cell lymphomas. The prognosis for patients who present with transformation to CHL is poor compared to de novo CHL.² A large study from the M.D. Anderson Cancer Center described 4121 patients with CLL/SLL and found that only 18 patients or 0.4% developed CHL. The median time from CLL to CHL diagnosis was 4.6 years. Fourteen of the patients received chemotherapy. The overall response rate was 44% with a complete response rate of 19%. The median overall survival was 0.8 years and all patients eventually died from disease recurrence or progressive disease.³ This dismal prognosis is similar to patients with Richter transformation to DLBCL and much worse than patients with de novo CHL, which is curable in >85% of cases.¹

References

Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4^th edition). IARC: Lyon 2017.
Janjetovic S, Bernd HW, Bokemeyer C, Fiedler W. Hodgkin’s lymphoma as a rare variant of Richter’s transformation in chronic lymphocytic leukemia: A case report and review of the literature. Mol Clin Oncol. 2016;4(3):390–392.doi:10.3892/mco.2016.727.
Tsimberidou, AM, O’Brien, S and Kantarjian, HM, et. al. Hodgkin transformation of chronic lymphocytic leukemia. Cancer. 2006;107(6).doi.org/10.1002/cncr.22121.

–Chelsea Marcus, MD is a Hematopathology Fellow at Beth Israel Deaconess Medical Center in Boston, MA. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Hematopathology Case Study: A 77 Year Old Man with Rash

Case History

The patient is a 77 year old man with a longstanding history of increased white blood cell (WBC) count who presented with a new rash and increasing absolute lymphocytosis.

Labs

Peripheral Blood Smear

Peripheral blood smear shows small to medium-sized lymphocytes with basophilic cytoplasm, cytoplasmic protrusions or blebs, round to oval nuclei with indented nuclear contours and some cells with prominent nucleoli.

Bone Marrow Biopsy

Bone marrow aspirate (top left) shows increased lymphocytes with similar features to those seen in the peripheral blood. The core biopsy (top right) shows an abnormal lymphocytic infiltrate. By immunohistochemistry, CD3 highlights markedly increased interstitial T-lymphocytes (30-40%) that predominantly express CD4. CD8 highlights only few scattered T-cells.

Flow Cytometry

Concurrent flow cytometry identifies an expanded population of lymphocytes comprising 73% of the total cellularity. Of the lymphocytes, 98% are T-cells. The T-cell population is almost entirely composed of CD4 positive cells (CD4/8 ratio = 301). The T-cells show expression of TCR (a/b), normal T-cell antigens CD3, CD2, CD5 and CD7 and express CD52 (bright).

Cytogenetics

Concurrent chromosome analysis shows that 90% of the metaphase bone marrow cells examined have a complex abnormal karyotype with a paracentric inversion of chromosome 14 that results in the TRA/D/TCL1 gene rearrangement. There is also a rearrangement resulting in three copies of 8q with partial loss of 8p as well as other chromosome aberrations.

Diagnosis

Altogether, the presence of an abnormal CD4 positive and CD52 (bright) lymphocyte population with the characteristic cytogenetic finding of inv(14), is diagnostic of T-cell prolymphocytic leukemia (T-PLL). This patient’s course is unusual in that he initially presented with indolent disease that ultimately progressed. The lymphocyte morphology was also somewhat atypical in that only occasional cells had prominent nucleoli. This is consistent with the “small cell variant” of T-PLL.

Discussion

T-PLL is generally an aggressive disorder characterized by small to medium sized mature T-cells that are found in the peripheral blood, bone marrow, lymph nodes, spleen, liver and sometimes skin. T-PLL is rare and occurs in adults usually over 30 years old. The clinical presentation includes a lymphocytosis, often >100 x 10^9/L, hepatosplenomegaly and lymphadenopathy. Serous effusions and skin infiltration can be seen in a subset of cases. On microscopy, the cells are usually small to medium in size with basophilic cytoplasm, round to irregular nuclei and visible nucleoli. Characteristic cytoplasmic blebs or protrusions are a common feature. The immunophenotype is of a mature T-cell and cells are positive for CD2, CD3, CD5 and CD7. They are negative for TdT and CD1a. Another characteristic feature is bright expression of CD52. Sixty percent of cases are positive for CD4, while 25% show double expression of CD4 and CD8. The most frequent chromosome abnormality is inversion of chromosome 14 at q11 and q32, which is seen in 80% of patients. Translocations involving chromosome X and 14 are also seen, as well as abnormalities of chromosome 8. The overall prognosis is generally poor with a median survival of 1-2 years. Patients with expression of CD52 may respond well to the monoclonal anti-CD52 antibody alemtuzumab, but other treatment options are limited.¹

The small cell variant (SV) of T-cell prolymphocytic leukemia was once referred to as T-cell chronic lymphocytic leukemia due to a predominant population of small lymphocytes with condensed chromatin and lack of conspicuous nucleoli. In addition, unlike the aggressive course seen in most patients with T-PLL, patients with this morphology tended to have an indolent or more chronic disease course. Eventually, it became clear that this was merely a variant of T-PLL due to similar immunophenotypic and cytogenetic findings. Ultimately, the term T-cell CLL was retired from use.²

In a comparison of patients with SV T-PLL to three large studies of classic T-PLL patients, the SV patients were found to have a higher frequency of a normal karyotype and increased double negative (CD4-/CD8) immunophenotype. Interestingly, 38% of the SV patients did not receive treatment for the entire duration of follow-up, while 19% required treatment after initially just being observed. This time period ranged between 2 months to 3 years. The remaining patients were treated at diagnosis. Most of the patients ultimately progressed and the cause of death was disease progression in 86% of the patients who died during follow-up. Overall, SV T-PLL tended to show less aggressive clinical behavior than classic T-PLL, however many aggressive cases of patients with the small cell variant have been seen. Likewise, more indolent cases of classic T-PLL featuring cells with larger nuclei with prominent nucleoli have also been described.²

While cases of SV T-PLL may initially present with more indolent disease, they almost always progress to a similarly aggressive disease course as seen in classic T-PLL. T-PLL is generally resistant to most conventional chemotherapies. As mentioned earlier, cases of T-PLL tend to express bright CD52, which is a glycoprotein present on the surface of mature lymphocytes. CAMPATH-1H is an anti-CD52 monoclonal antibody that may result in complement-mediated lysis and antibody-dependent cellular cytotoxicity. In a study by Dearden et. al., thirty-nine patients with T-PLL received CAMPATH-1H treatments. The overall response rate was 76% with 60% achieving complete remission. These rates are significantly higher than those reported for conventional therapies like CHOP. Unfortunately, almost all of the patients ultimately progressed and all but 2 had relapsed following 1 year of therapy. This indicates that CAMPATH-1H is good for first line therapy, but is not a curative treatment for this aggressive and most often deadly disease. ³

References

Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4^th edition). IARC: Lyon 2017.
A. Rashidi and S. Fisher. T-cell chronic lymphocytic leukemia or small-cell variant of T-cell prolymphocytic leukemia: a historical perspective and search for consensus. European Journal of Haematology. 2015(Vol 95).
C. Dearden, E. Matutes and B. Cazin, et. al. High remission rate in T-cell prolymphocytic leukemia with CAMPATH-1H. Blood. 2001(98)1721-1726.