Highlights in Pathology: Hematopathology (September 2021)

Dr. Thomas Jeff Lim Jr. and Dr. Sarah-Jeanne Pilon, Hematopathology Fellows at the University of Toronto

Dr. Rosemarie Tremblay-LeMay, Hematopathologist, University Health Network

VEXAS syndrome: a newly described entity

VEXAS (Vacuoles, E1 Enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a newly described entity characterized by late-onset autoinflammatory manifestations, and may come to the attention of pathologists during the investigation of hematologic manifestations. 

Relevance to pathologists

VEXAS syndrome shows morphological overlap with other hematologic entities such as MDS, raising possible potential pitfall in diagnosis. By increasing recognition of these patients, better treatment approaches could be investigated. 

Summary

VEXAS was first described by Beck et al. in October 2020. This entity is characterized by a somatic mutation of the X-linked UBA1 gene. The disease characteristically affects men older than 45 years of age that present with late onset treatment-refractory inflammatory syndrome. 

Clinical manifestations are non-specific but reflect activated inflammatory response. Common signs and symptoms include fever, fatigue, cutaneous lesions, pulmonary infiltrates, arthritis/arthralgia, chondritis, venous thrombosis. Hematologic manifestations typically consist of macrocytic anemia, followed by thrombocytopenia, or pancytopenia. Lymphopenia is also common. Patients may meet diagnostic criteria for rheumatologic (ex. relapsing polychrondritis, Sweet syndrome) or hematologic diseases (MDS, MGUS or plasma cell neoplasm). There is a high mortality rate due to disease-related issues or treatment complications.

The most striking morphologic finding is the presence of cytoplasmic vacuoles in the myeloid and erythroid lineages. They tend to be more pronounced in precursors than in the more mature forms. Cytoplasmic vacuoles are not specific and can be seen in disorders such as copper deficiency, zinc toxicity, excessive alcohol consumption, MDS. However, the combination of systemic autoinflammatory manifestations as described and vacuolation can raise the differential diagnosis of VEXAS. Lacombe and colleagues suggested that ≥10% myeloid precursor cells with vacuoles may be a significant cut-off for VEXAS syndrome, although this would need further investigation. While most reports describe vacuolation in the myeloid and erythroid lineages, they are also reported to a lesser degree in plasma cells, eosinophils, monocytes and megakaryocytes. As more cases are identified and we learn more about this new entity, our understanding of the range of morphologic features may evolve. Some degree of dysplasia is usually observed and may in some cases meet diagnostic criteria for MDS.

Key points

• VEXAS syndrome should now be considered as part of the differential diagnosis for vacuolation of hematopoietic precursors: the diagnosis could be suggested in an appropriate clinical context (e.g. history of inflammatory manifestations associated with the syndrome) in patients with cytopenia and suggestive morphology, so a sample can be sent for molecular confirmation. 
• Of interest, since the disease has been described a few patients referred to UHN were confirmed to have the syndrome, often after years of refractory rheumatologic manifestations. Identifying patients with VEXAS syndrome could lead to a better understanding of the syndrome and appropriate treatment modalities could be further investigated.

Selected references

Beck, D. B. et al. (2020). "Somatic Mutations in UBA1 and Severe Adult-Onset Autoinflammatory Disease." N Engl J Med 383(27): 2628-2638.

Bourbon, E. et al. (2021). "Therapeutic options in VEXAS syndrome: insights from a retrospective series." Blood 137(26): 3682-3684.

Grayson, P. C., B. A. Patel and N. S. Young (2021). "VEXAS syndrome." Blood 137(26): 3591-3594.

Lacombe, V. et al. (2021). "Vacuoles in neutrophil precursors in VEXAS syndrome: diagnostic performances and threshold." Br J Haematol.

Obiorah, I. E. et al.  (2021). "Benign and malignant hematologic manifestations in patients with VEXAS syndrome due to somatic mutations in UBA1." Blood Advances 5(16): 3203-3215.

CD30 reporting for lymphoma in the era of CD30-targeted therapy 

Relevance to pathologists

With the advent of drugs targeting CD30 for the treatment of lymphoma, it has become important for pathologists to provide quantitative reporting of CD30, as the level of expression may dictate whether patients are eligible to receive such therapies. However, there are many challenges related to CD30 reporting.

Recent publications have highlighted these challenges and provide recommendations to allow harmonization of practices and lead to more standardized reporting of CD30 expression.

Background

CD30 is a surface cell receptor that is typically detected by immunohistochemistry or by flow cytometry. One of its primary uses was in the diagnosis of classic Hodgkin lymphoma and certain types of non-Hodgkin T-cell lymphomas such as anaplastic large cell lymphoma. In recent years, a drug targeting CD30 was developed, highlighting the importance of CD30 as a potential target for treatment.

Herein are two selected papers that complement one another and highlight some of the potential challenges in using CD30 as a predictive biomarker and suggest best practice in reporting of CD30 to harmonize practices across institutions.

1. Xu ML, et al. Practical Approaches on CD30 Detection and Reporting in Lymphoma Diagnosis.  Am J Surg Pathol 2020; 44:e1–e14

This special article highlights challenges in reporting CD30 and addresses 3 major unresolved questions for the predictive use of CD30 expression: 

• “What defines a “POSITIVE” level of CD30 expression?
• How do we evaluate and report CD30 expression?
• What are the caveats in the evaluation of CD30 expression?”

The relevant cut-off of CD30 expression for therapeutic purposes has not yet been clearly established in the literature and is context dependent. Depending on the study or clinical trial, the cut-offs vary from 1% to 75%. The cut-offs also vary between entities. The evaluation of CD30 is complicated by the fact that it is sometimes hard to discriminate between reactive and tumor cells. Therefore, reporting a percentage of tumor cells (or total cells in challenging cases), as opposed to a “positive/negative” result. 

There is also currently no standardized protocol for CD30 testing. There are many caveats in evaluating CD30 that can be separated between pre-analytical, analytical, and post-analytical variables. The article identifies key issues in each of those three categories and what can be done to minimize the occurrence and the effect of these issues. The main issues addressed relate to fixation of tissue, development of IHC assay (referring to NordiQC guidelines), heterogeneous expression within tumors (with special considerations for core biopsies), potential issues with interobserver variability and confidence for reporting CD30 expression below certain thresholds.

Key points

The authors:

• recommend that CD30 be reported as a percentage of tumor cells (or total cells in challenging cases).
• emphasize the impact of pre-analytical and analytical considerations in the evaluation of CD30 for predictive purposes.

2. Gru AA et al.  Best Practices in CD30 Testing, Interpretation and Reporting: An Expert Panel Consensus. Archives of Pathology and Laboratory Medicine, in press. 

The objective of this expert panel consensus is to “develop and propose general best practice recommendations for reporting CD30 expression in lymphoma biopsies to harmonize practices across different institutions and countries and to facilitate assessment of its significance in clinical decision making.” 

The panel is composed of hematopathologists and two clinical hematologists /oncologists from fourteen academic institutions in North America. Recommendations are made on four key areas: indications for testing, how to test, assay readout and test interpretation and reporting. 

Here is a brief summary of the recommendations:

• Indications for testing:  there are two indications for testing; diagnostic and therapeutic. The former includes cases where classic Hodgkin lymphoma and peripheral T-cell lymphomas, amongst other, are in the differential diagnosis. For therapeutic considerations, the recommendation is to test CD30 for all patients diagnosed with T-cell lymphoma, and in selected cases of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) or primary mediastinal large B-cell lymphoma.
• How to test: immunohistochemistry is recommended as the method of choice. Steps should be taken to optimize the immunohistochemistry process and they provide guidance of pre-analytical, analytical and post-analytical considerations. Selection of appropriate controls to optimize the assay is of particular importance; as per guidelines from the Nordic Immunohistochemical Quality Control (NordiQC), using tissues with known descriptive low limit of detection (e.g. CD30-positive centroblasts in germinal centres in benign tonsil) is recommended.
• Assay read-out: Any positivity interpreted as “specific staining” should be reported regardless of intensity, by giving a percentage of tumor cells that are positive whenever possible. This may be difficult if there is a significant contingent of admixed inflammatory cells; in such cases it can be adequate to report out of the total number of lymphocytes, stating the method used. It is recommended to report % positive tumor cells as follows: 0, 1-10%, and in 10% increments or ranges thereafter. A descriptive comment can be provided if non-tumor cells are positive; if possible, estimate the % non-tumor cell positivity. Because of heterogeneity, if one block is tested and is negative, it may be relevant to test additional blocks if available.
• Test interpretation and reporting: guidelines should be followed for interpretation for diagnostic purposes. When CD30 provides potentially therapeutic information, the pathologist should report a percentage of positive cells, distinguishing expression in tumor cells and non-tumors cells as much as feasible. Pathologists are not required to provide interpretation of the clinical significance of the results, the oncologist can interpret the percentage in view of the latest published results in clinical trials

Key points

• For therapeutic considerations, CD30 testing should be included in the work-up of all T-cell lymphomas, and in selected cases of DLBCL.
• Pre-analytical and analytical variables are important to allow standardization of testing across institutions and the article provides a number of recommendations for IHC protocols.
• As there is variability in the expression cut-offs used for treatment in various clinical trials, it is recommended to report % positive tumor cells (or total cells in challenging cases) as follows: 0, 1-10%, and in 10% increments or ranges thereafter. That way clinicians can interpret the result in view of the latest available clinical trials.

Summary

Both articles highlight numerous challenges in evaluating and reporting CD30 expression for therapeutic purposes in lymphomas. They recommend using NordiQC guidelines when developing CD30 assays in order to improve standardization across institutions and advocate for a rigorous approach in testing and reporting CD30 expression for lymphomas where there may be therapeutic implications.