A real-world analysis of clinical outcomes in AML with myelodysplasia-related changes: a comparison of ICC and WHO-HAEM5 criteria.

ABSTRACT: The proposed fifth edition of the World Health Organization classification of hematolymphoid tumors (WHO-HAEM5) and International Consensus Classification (ICC) provide different definitions of acute myeloid leukemia with myelodysplasia-related genetics (AML-MR). We conducted a retrospective study which included a cohort of 432 patients, with 354 patients fulfilling WHO-HAEM5 criteria for WHO-AML-MR or 276 patients fulfilling ICC criteria for ICC-AML-MR by gene mutation or cytogenetics (ICC-AML-MR-M/CG). The clinicopathological features were largely similar, irrespective of the classification used, except for higher rates of complex karyotype, monosomy 17, TP53 mutations, and fewer RUNX1 mutations in the WHO-AML-MR group. TP53 mutations were associated with distinct clinicopathological features and dismal outcomes (hazard ratio [HR], 2.98; P < .001). ICC-AML-MR-M/CG group had superior outcome compared with the WHO-AML-MR group (HR, 0.80, P = .032), largely in part due to defining TP53 mutated AML as a standalone entity. In the intensively-treated group, WHO-AML-MR had significantly worse outcomes than AML by differentiation (HR, 1.97; P = .024). Based on ICC criteria, ICC-AML-MR-M/CG had more inferior outcomes compared to AML not otherwise specified (HR, 2.11; P = .048 and HR, 2.55; P = .028; respectively). Furthermore, changing the order of genetic abnormalities defining AML-MR (ie, by gene mutations or cytogenetics) did not significantly affect clinical outcomes. ICC-AML-MR-M/CG showed similar outcomes regardless of the order of assignment. We propose to harmonize the 2 classifications by excluding TP53 mutations from WHO-HAEM5 defined AML-MR group and combining AML-MR defined by gene mutations and cytogenetics to form a unified group.

AML with CEBPA mutations: A comparison of ICC and WHO-HAEM5 criteria in patients with 20% or more blasts.

AML with CEBPA mutation and AML with in-frame bZIP CEBPA mutations define favorable-risk disease entities in the proposed 5th edition of the World Health Organization Classification (WHO-HAEM5) and the International Consensus Classification (ICC), respectively. However, the impact of these new classifications on clinical practice remains unclear. We sought to assess the differences between the ICC and WHO-HAEM5 for AML with CEBPA mutation. 741 AML patients were retrospectively analyzed. Cox proportional-hazard regression was used to identify factors predictive of outcome. A validation cohort from the UK-NCRI clinical trials was used to confirm our findings. 81 (11%) AML patients had CEBPA mutations. 39 (48%) patients met WHO-HAEM5 criteria for AML with CEBPA mutation, among which 30 (77%) had biallelic CEBPA mutations and 9 (23%) had a single bZIP mutation. Among the 39 patients who met WHO-HAEM5 criteria, 25 (64%) also met ICC criteria. Compared to patients only meeting WHO-HAEM5 criteria, patients with in-frame bZIP CEBPA mutations (ie. meeting both WHO-HAEM5 and ICC criteria) were younger, had higher bone marrow blast percentages and CEBPA mutation burden, infrequently harboured 2022 ELN high-risk genetic features and co-mutations in other genes, and had superior outcomes. The associations in clinicopathological features and outcomes between the CEBPA-mutated groups were validated in the UK-NCRI cohort. Our study indicates that in-frame bZIP CEBPA mutations are the critical molecular aberrations associated with favorable outcomes in AML patients treated with curative intent chemotherapy. Compared to WHO-HAEM5, the ICC identifies a more homogenous group of CEBPA-mutated AML patients with favorable outcomes.

Upfront Next Generation Sequencing in Non-Small Cell Lung Cancer.

In advanced non-small cell lung cancer (NSCLC), patients with actionable genomic alterations may derive additional clinical benefit from targeted treatment compared to cytotoxic chemotherapy. Current guidelines recommend extensive testing with next generation sequencing (NGS) panels. We investigated the impact of using a targeted NGS panel (TruSight Tumor 15, Illumina) as reflex testing for NSCLC samples at a single institution. Molecular analysis examined 15 genes for hotspot mutation variants, including AKT1, BRAF, EGFR, ERBB2, FOXL2, GNA11, GNAQ, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, RET and TP53 genes. Between February 2017 and October 2020, 1460 samples from 1395 patients were analyzed. 1201 patients (86.1%) had at least one variant identified, most frequently TP53 (47.5%), KRAS (32.2%) or EGFR (24.2%). Among these, 994 patients (71.3%) had clinically relevant variants eligible for treatment with approved therapies or clinical trial enrollment. The incremental cost of NGS beyond single gene testing (EGFR, ALK) was CAD $233 per case. Reflex upfront NGS identified at least one actionable variant in more than 70% of patients with NSCLC, with minimal increase in testing cost. Implementation of NGS panels remains essential as treatment paradigms continue to evolve.

Measurable residual disease monitoring provides insufficient lead-time to prevent morphologic relapse in the majority of patients with core-binding factor acute myeloid leukemia.

Core-binding factor acute myeloid leukemia is characterized by t(8;21) or inv(16) and the fusion proteins RUNX1-RUNX1T1 and CBFB-MYH11. International guidelines recommend monitoring for measurable residual disease every 3 months for 2 years after treatment. However, it is unknown if serial molecular monitoring can predict and prevent morphologic relapse. We conducted a retrospective single-center study of 114 patients in complete remission who underwent molecular monitoring with RT-qPCR of RUNX1-RUNX1T1 or CBFB-MYH11 transcripts every 3 months. Morphologic relapse was defined as re-emergence of >5% blasts and molecular relapse as ≥1 log increase in transcript level between 2 samples. Over a median follow-up time of 3.7 years (range 0.2-14.3), remission persisted in 71 (62.3%) patients but 43 (37.7%) developed molecular or morphologic relapse. Patients who achieved <3 log reduction in RUNX1-RUNX1T1 or CBFB-MYH11 transcripts at end of chemotherapy had a significantly higher risk of relapse compared to patients who achieved ≥3 log reduction (61.1% vs. 33.7%, p=0.004). The majority of relapses (74.4%, n=32) were not predicted by molecular monitoring and occurred rapidly with <100 days from molecular to morphologic relapse. Molecular monitoring enabled the detection of impending relapse and permitted pre-emptive intervention prior to morphologic relapse in only 11 (25.6%) patients. The current practice of molecular monitoring every 3 months provided insufficient lead-time to identify molecular relapses and prevent morphologic relapse in the majority of patients with core-binding factor acute myeloid leukemia treated at our institution. Further research is necessary to determine the optimal monitoring strategies for these patients.

Radar plots facilitate differential diagnosis of acute promyelocytic leukemia and NPM1+ acute myeloid leukemia by flow cytometry.

BACKGROUND: Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological emergencies and requires a prompt correct diagnosis by cytomorphology and flow cytometry (FCM) with later confirmation by cytogenetics/molecular genetics. However, nucleophosmin 1 muted acute myeloid leukemia (NPM1+ AML) can mimic APL, especially the hypogranular variant of APL. Our study aimed to develop a novel, Radar plot-based FCM strategy to distinguish APLs and NPM1+ AMLs quickly and accurately. METHOD: Diagnostic samples from 52 APL and 32 NPM1+ AMLs patients were analyzed by a 3-tube panel of 10-color FCM. Radar plots combining all markers were constructed for each tube. Percentages of positive leukemic cells and mean fluorescence intensity were calculated for all the markers. RESULTS: APL showed significantly higher expression of CD64, CD2, and CD13, whereas more leukemic cells were positive for CD11b, CD11c, CD15, CD36, and HLA-DR in NPM1+ AMLs. Radar plots featured CD2 expression, a lack of a monocytic component, lack of expression of HLA-DR and CD15, and a lack of a prominent CD11c+ population as recurring characteristics of APL. The presence of blasts with low SSC, presence of at least some monocytes, some expression of HLA-DR and/or CD15, and a prominent CD11c population were recurrent characteristics of NPM1+ AMLs. Radar plot analysis could confidently separate all hypergranular APL cases from any NPM1+ AML and in 90% of cases between variant APL and blastic NPM1+ AML. CONCLUSION: Radar plots can potentially add to differential diagnostics as they exhibit characteristic patterns distinguishing APL and different types of NPM1+ AMLs.

Primary Marginal Zone Lymphoma of the Subcutis Associated With Panniculitis and Fat Necrosis.

OBJECTIVES: Lymphocytic infiltrates in the subcutaneous adipose tissue, often accompanied by fat necrosis, are typically seen in benign panniculitis. Diagnostic considerations include subcutaneous panniculitis-like T-cell lymphoma and cutaneous γδ T-cell lymphoma, whereas a primary subcutaneous B-cell lymphoma in this setting has not been previously described. METHODS: We report the case of a 72-year-old woman with multiple deep cutaneous nodules on the trunk and upper extremities. RESULTS: During 3 years of clinical follow-up, new skin nodules developed, while existing lesions remained stable or regressed. No other organ involvement was detected. Sequential biopsy specimens of the subcutaneous lesions revealed patchy, predominantly septal, lymphocytic infiltrates associated with extensive hyaline fat necrosis. The histologic and immunophenotypic features were consistent with marginal zone lymphoma. Genotyping revealed an identical monoclonal immunoglobulin gene rearrangement across all biopsy specimens. CONCLUSIONS: This case represents, to our knowledge, the first reported case of primary subcutaneous B-cell lymphoma closely associated with panniculitis.

Sample features associated with success rates in population-based EGFR mutation testing.

INTRODUCTION: Epidermal growth factor receptor (EGFR) mutation testing has become critical in the treatment of patients with advanced non-small-cell lung cancer. This study involves a large cohort and epidemiologically unselected series of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer in a North American population to determine sample-related factors that influence success in clinical EGFR testing. METHODS: Data from consecutive cases of Canadian province-wide testing at a centralized diagnostic laboratory for a 24-month period were reviewed. Samples were tested for exon-19 deletion and exon-21 L858R mutations using a validated polymerase chain reaction method with 1% to 5% detection sensitivity. RESULTS: From 2651 samples submitted, 2404 samples were tested with 2293 samples eligible for analysis (1780 histology and 513 cytology specimens). The overall test-failure rate was 5.4% with overall mutation rate of 20.6%. No significant differences in the failure rate, mutation rate, or mutation type were found between histology and cytology samples. Although tumor cellularity was significantly associated with test-success or mutation rates in histology and cytology specimens, respectively, mutations could be detected in all specimen types. Significant rates of EGFR mutation were detected in cases with thyroid transcription factor (TTF)-1-negative immunohistochemistry (6.7%) and mucinous component (9.0%). CONCLUSIONS: EGFR mutation testing should be attempted in any specimen, whether histologic or cytologic. Samples should not be excluded from testing based on TTF-1 status or histologic features. Pathologists should report the amount of available tumor for testing. However, suboptimal samples with a negative EGFR mutation result should be considered for repeat testing with an alternate sample.

Genetic determinants of extracellular magnesium concentration: analysis of multiple candidate genes, and evidence for association with the estrogen receptor alpha (ESR1) locus.

BACKGROUND: Serum magnesium concentration is a quantitative trait with substantial heritability. Although the pool of candidate genes continues to grow, only the histocompatibility locus has been associated with magnesium levels. To explore other possibilities, we targeted 6 candidate genes physiologically relevant to magnesium metabolism. METHODS: We studied a large cohort (n=471) derived from a well-characterized population of healthy Caucasian women 18 to 35 years. Total serum magnesium and calcium were measured by atomic absorption spectrophotometry (aaMg & aaCa). Genomic DNA was amplified and SNPs in candidate genes (CASR, VDR, ESR1, CLDN16, EGF1, TRPM6) genotyped by routine methods. RESULTS: We found a significant association between estrogen receptor alpha (ESR1) polymorphisms, PvuII and XbaI, and magnesium (r=-0.116, p=0.012 and r=-0.126, p=0.006, respectively). Stratifying by PvuII genotype (P/p alleles), the mean adjusted total magnesium (aaMg) concentration was significantly higher (p=0.01) in the pp group (0.823+/-0.005 mmol/l, n=130) than in PP homozygotes (0.805+/-0.006 mmol/l, n=70), and the mean in Pp heterozygotes was intermediate (0.810+/-0.005 mmol/l, n=180). No significant associations were observed with the other candidate genes tested. CONCLUSIONS: The significant association between magnesium and ESR1 polymorphisms supports previous studies linking physiologic changes in serum magnesium to estrogen status.

Lung transplantation complicated by graft-versus-host disease and confounded by incidental transfusion-associated macrochimerism.

BACKGROUND: Passenger lymphocyte-mediated graft-versus-host disease (GVHD) in solid organ transplantation (SOT-GVHD) is considered a rare complication, particularly among recipients of lung allografts. The risk of transfusion-associated GVHD (TA-GVHD) in solid organ transplants is also considered rare. The suspicion of either may be heralded by signs and symptoms of GVHD in the company of a population of passenger lymphocytes in excess of 1 percent (microchimerism). This case report illustrates the challenge of a patient who presented with macrochimerism both from the lung transplant allograft and from transfusions. STUDY DESIGN AND METHODS: Chimerism assessments of the pre- and posttransplant donor lung, and the recipient's aplastic marrow, were made using DNA-based polymerase chain reaction testing. RESULTS: Macrochimerism was observed in both the posttransplant aplastic host marrow and the engrafted donor lung, with the former predominantly consisting of lung donor lymphocytes and the latter a mixture of lung and presumably transfusion source donor lymphocytes. The pretransplant donor lung exhibited no GVHD-like pathology. CONCLUSION: This case demonstrates SOT-GVHD, with the unusual feature of concomitant macrochimerism from transfusions. SOT-GVHD likely predisposed this patient to the observed transfusion-associated macrochimerism. However, the dissociation between transfusion-attributable macrochimerism and attributable pathology is intriguing. Furthermore, the risk spectrum of transfusion-associated macrochimerism and TA-GVHD in solid organ transplant recipients with and without the complication of SOT-GVHD is unknown and warrants further study.

Inter-laboratory comparison of chronic myeloid leukemia minimal residual disease monitoring: summary and recommendations.

In patients with chronic myeloid leukemia, the use of real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for measuring BCR-ABL1 transcripts has become standard methodology for the diagnosis and monitoring of minimal residual disease. In 2004 and 2005, 38 different laboratories from North America participated in three separate sample exchanges using real-time qRT-PCR to measure RNA transcript levels in unknown diluents of a BCR-ABL1-positive cell line, K562. In this study we compared results of quantitative testing for BCR-ABL1 from laboratories using different platforms, internal controls, reagents, and calculation methods. Our data showed that there can be considerable variability of results from laboratory to laboratory, with log reduction calculations varying from 1.6 to 3 log between laboratories at the same dilution. We found that none of the variables tested had a significant impact on the results reported, except for the use of ABL1 as the internal control (P < 0.001). Laboratories that used ABL1 consistently underreported their log reduction values. Regardless of the specific methodology and platform used for real-time qRT-PCR testing, it is important for laboratories to participate in proficiency testing to ensure consistent and acceptable test accuracy and sensitivity. Our study emphasizes the need for optimization of real-time qRT-PCR before offering clinical testing and the need for widely available universal standards that can be used for test calibration.

Recurrent familial hypocalcemia due to germline mosaicism for an activating mutation of the calcium-sensing receptor gene.

De novo activating mutations in the calcium-sensing receptor (CASR) gene are a common cause of sporadic isolated hypoparathyroidism. Here, we describe a family in which two affected siblings were found to be heterozygous for a novel F788L mutation in the fifth transmembrane domain encoded by exon 7 of the CASR. Both parents and the third sibling were clinically unaffected and genotypically normal by direct sequencing of their leukocyte exon 7 PCR amplicons. However, the mother was revealed to be a mosaic for the mutation by sequence analysis of multiple subclones as well as denaturing HPLC of the CASR exon 7 leukocyte PCR product. A functional analysis of the mutation was performed by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. The mutant CASR was expressed at a similar level as the wild type. The F788L mutant produced a significant shift to the left relative to the wild-type CASR in the MAPK response to increasing extracellular calcium concentrations. This is the first report of mosaicism for an activating CASR mutation and suggests that care should be exercised in counseling for risks of recurrence in a situation where a de novo mutation appears likely.