Morphological Clues of Acute Monocytic Leukemia in COVID-19-Induced Transient Leukoerythroblastic Reaction with Monocytosis.

Viral infections, including those caused by COVID-19, can produce striking morphologic changes in peripheral blood. Distinguishing between reactive changes and abnormal morphology of monocytes remains particularly difficult, with low consensus rates reported amongst hematopathologists. Here, we report a patient who developed transient monocytosis of 11.06 × 109/L with 32% promonocytes and 1% blasts during hospitalization that was secondary to severe COVID-19 infection. Three days later, the clinical status of the patient improved and the WBC had decreased to 8.47 × 109/L with 2.2 × 109/L monocytes. Flow cytometry studies did not reveal immunophenotypic findings specific for an overt malignant population. At no time during admission did the patient develop cytopenia(s), and she was discharged upon clinical improvement. However, the peripheral blood sample containing promonocytes was sent for molecular testing with an extended next-generation sequencing myeloid panel and was positive for pathogenic NPM1 Type A and DNMT3A R882H mutations. Subsequently, despite an essentially normal complete blood count, the patient underwent a bone marrow assessment that showed acute myeloid leukemia with 77% promonocytes. This case emphasizes the critical importance of a full work up to exclude acute leukemia when classical promonocyte morphology is encountered in the peripheral blood. Promonocytes are not a part of the reactive changes associated with COVID-19 and remain specific to myeloid neoplasia.

Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer, and in this process, some "old" biomarkers are reutilized and given a new purpose. It is not always recognized that by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory-developed tests (LDTs) of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays with 32 cases and performed their in-house Ki-67 assays. The results were assessed using QuPath, an open-source software application for bioimage analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20%, and 30% cutoffs. Overall, PPA and NPA varied depending on the selected cutoff; participants were more successful with 5% and 10%, than with 20% and 30% cutoffs. Only 4 of 16 laboratories had robust IHC protocols with acceptable PPA for all cutoffs. The lowest PPA for the 5% cutoff was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cutoff was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cutoffs. The poor agreement was not due to the readout but rather due to IHC protocol conditions. International Ki-67 in Breast Cancer Working Group (IKWG) recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Identification of Potential Therapeutic Targets for Plasmablastic Lymphoma Through Gene Expression Analysis: Insights into RAS and Wnt Signaling Pathways.

Plasmablastic lymphoma (PBL) is a rare and aggressive B-cell lymphoma with overlapping characteristics with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. Hyperactive Wnt signaling derails homeostasis and promotes oncogenesis and chemoresistance in DLBCL and multiple myeloma. Evidence suggests active cross-talk between the Wnt and RAS pathways impacting metastasis in solid cancers in which combined targeted therapies show effective results. Recent genomic studies in PBL demonstrated a high frequency of mutations linked with the RAS signaling pathway. However, the role of RAS and Wnt signaling pathway molecule expression in PBL remained unknown. We examined the expression of Wnt and RAS pathway-related genes in a well-curated cohort of PBL. Because activated B cells are considered immediate precursors of plasmablasts in B cell development, we compared this data with activated B-cell type DLBCL (ABC-DLBCL) patients, employing NanoString transcriptome analysis (770 genes). Hierarchical clustering revealed distinctive differential gene expression between PBL and ABC-DLBCL. Gene set enrichment analysis labeled the RAS signaling pathway as the most enriched (37 genes) in PBL, including upregulating critical genes, such as NRAS, RAF1, SHC1, and SOS1. Wnt pathway genes were also enriched (n = 22) by gene set enrichment analysis. Molecules linked with Wnt signaling activation, such as ligands or targets (FZD3, FZD7, c-MYC, WNT5A, WNT5B, and WNT10B), were elevated in PBL. Our data also showed that, unlike ABC-DLBCL, the deranged Wnt signaling activity in PBL was not linked with hyperactive nuclear factor κB and B-cell receptor signaling. In divergence, Wnt signaling inhibitors (CXXC4, SFRP2, and DKK1) also showed overexpression in PBL. The high expression of RAS signaling molecules reported may indicate linkage with gain-in-function RAS mutations. In addition, high expression of Wnt and RAS signaling molecules may pave pathways to explore benefiting from combined targeted therapies, as reported in solid cancer, to improve prognosis in PBL patients.

Reinventing Nuclear Histo-score Utilizing Inherent Morphologic Cutoffs: Blue-brown Color H-score (BBC-HS).

Immunohistochemistry (IHC) is a testing methodology that is widely used for large number of diagnostic, prognostic, and predictive biomarkers. Although IHC is a qualitative methodology, in addition to threshold-based stratification (positive vs. negative), the increasing levels of expression of some of these biomarkers often lead to more intense staining, which published evidence linked to specific diagnosis, prognosis, and responses to therapy. It is essential that the descriptive thresholds between positive and negative staining, as well as between frequently used graded categories of staining intensity (eg, 1+, 2+, 3+) are standardized and reproducible. Histo-score (H-score) is a frequently used scoring system that utilizes these categories. Our study introduces categorization of the cutoff points between positive and negative results and graded categories of staining intensity for nuclear IHC biomarker assays based on color interaction between hematoxylin and diaminobenzidine (DAB); the Blue-brown Color H-score (BBC-HS). Six cases of diffuse large B-cell lymphoma were stained for a nuclear marker MUM1. The staining was assessed by H-score by 12 readers. Short tutorial and illustrated instructions were provided to readers. The novel scoring system in this study uses the interaction between DAB (DAB, brown stain) and hematoxylin (blue counterstain) to set thresholds between "0" (negative nuclei), "1+" (weakly positive nuclei), "2+" (moderately positive nuclei), and "3+" (strongly positive nuclei). The readers recorded scores for 300 cells. Krippendorff alpha (K-alpha) and intraclass correlation coefficient (ICC) were calculated. We have also assessed if reliability improved when counting the first 100 cells, first 200 cells, and for the total 300 cells using K-alpha and ICC. To assess the performance of each individual reader, the mean H-score and percent positive score (PPS) for each case was calculated, and the bias was calculated between each reader's score and the mean. K-alpha was 0.86 for H-score and 0.76 for PPS. ICC was 0.96 for H-score and 0.92 for PPS. The biases for H-score ranged from -58 to 41, whereas for PPS it ranged from -27% to 33%. Overall, most readers showed very low bias. Two readers were consistently underscoring and 2 were consistently overscoring compared with the mean. For nuclear IHC biomarker assays, our newly proposed cutoffs provide highly reliable/reproducible results between readers for positive and negative results and graded categories of staining intensity using existing morphologic parameters. BBC-HS is easy to teach and is applicable to both human eye and image analysis. BBC-HS application should facilitate the development of new reliable/reproducible scoring schemes for IHC biomarkers.

Dual Expression of Immunoglobulin Light Chains in Plasma Cell Myeloma: A Case Report and Literature Review.

Typically, myeloma cells express a monoclonal immunoglobulin (Ig), either heavy or light chain. Here, we present a case of multiple myeloma with clonal dual expression of kappa and lambda light chain in a 74-year-old woman. Awareness of rare biphenotypic myeloma is important for proper diagnostic workup. A 74-year-old woman underwent hip replacement with an incidental finding of 20% plasma cells in the femoral head. Subsequent bone marrow biopsy also showed about 30% of plasma cells negative for CD20, CD56, and CD117. Immunohistochemistry (IHC) and in situ hybridization studies showed a mixture of kappa and lambda plasma cells. Flow cytometry showed ambiguous results for cytoplasmic Ig light chains kappa and lambda. However, cyclin D1 was highly expressed by plasma cells, and increased free kappa light chains were identified in serum. Further investigation by double IHC demonstrated co-expression of kappa and lambda light chains in the same cells. Fluoresces in situ hybridization studies were positive for t(11;14)(q13;q32) and the deletion 13q. Since its first description by Taylor and Burns in 1974, the demonstration of restricted cytoplasmic Ig light chain expression by immunohistochemistry is 1 of the basic tools for corroborating clonality of the plasma cells in tissue biopsy. IHC results in myeloma with dual expression of Ig light chains may suggest polyclonal plasma cell population, especially when plasma cells do not form sheets in the bone marrow. In an appropriate clinical setting, other investigations are needed to exclude plasma cell neoplasm, even with seemingly "polytypic" results by IHC.

A Consortium for Analytic Standardization in Immunohistochemistry.

CONTEXT.­: The authors announce the launch of the Consortium for Analytic Standardization in Immunohistochemistry, funded with a grant from the National Cancer Institute. As with other laboratory testing, analytic standards are important for many different stakeholders: commercial vendors of instruments and reagents, biopharmaceutical firms, pathologists, scientists, clinical laboratories, external quality assurance organizations, and regulatory bodies. Analytic standards are customarily central to assay development, validation, and method transfer into routine assays and are critical quality assurance tools. OBJECTIVE.­: To improve immunohistochemistry (IHC) test accuracy and reproducibility by integrating analytic standards into routine practice. To accomplish this mission, the consortium has 2 mandates: (1) to experimentally determine analytic sensitivity thresholds (lower and upper limits of detection) for selected IHC assays, and (2) to inform IHC stakeholders of what analytic standards are, why they are important, and how and for what purpose they are used. The consortium will then publish the data and offer analytic sensitivity recommendations where appropriate. These mandates will be conducted in collaboration and coordination with clinical laboratories, external quality assurance programs, and pathology organizations. DATA SOURCES.­: Literature review and published external quality assurance data. CONCLUSIONS.­: Integration of analytic standards is expected to (1) harmonize and standardize IHC assays; (2) improve IHC test accuracy and reproducibility, both within and between laboratories; and (3) dramatically simplify and improve methodology transfer for new IHC protocols from published literature or clinical trials to clinical IHC laboratories.

Simultaneous Imaging and Therapy Using Epitope-Specific Anti-Epidermal Growth Factor Receptor (EGFR) Antibody Conjugates.

Matuzumab and nimotuzumab are anti-EGFR monoclonal antibodies that bind to different epitopes of domain III of EGFR. We developed 89Zr-matuzumab as a PET probe for diagnosis/monitoring of response to treatment of a noncompeting anti-EGFR nimotuzumab antibody drug conjugate (ADC) using mouse colorectal cancer (CRC) xenografts. We developed 89Zr-matuzumab and performed quality control in EGFR-positive DLD-1 cells. The KD of matuzumab, DFO-matuzumab and 89Zr-matuzumab in DLD-1 cells was 5.9, 6.2 and 3 nM, respectively. A competitive radioligand binding assay showed that 89Zr-matuzumab and nimotuzumab bound to noncompeting epitopes of EGFR. MicroPET/CT imaging and biodistribution of 89Zr-matuzumab in mice bearing EGFR-positive xenografts (HT29, DLD-1 and MDA-MB-231) showed high uptake that was blocked with pre-dosing with matuzumab but not with the noncompeting binder nimotuzumab. We evaluated nimotuzumab-PEG6-DM1 ADC in CRC cells. IC50 of nimotuzumab-PEG6-DM1 in SNU-C2B, DLD-1 and SW620 cells was dependent on EGFR density and was up to five-fold lower than that of naked nimotuzumab. Mice bearing the SNU-C2B xenograft were treated using three 15 mg/kg doses of nimotuzumab-PEG6-DM1, and 89Zr-matuzumab microPET/CT was used to monitor the response to treatment. Treatment resulted in complete remission of the SNU-C2B tumor in 2/3 mice. Matuzumab and nimotuzumab are noncompeting and can be used simultaneously.