The utility of disc space and vertebral body specimens cell count differential for the diagnosis of native vertebral osteomyelitis: a prospective cohort study.

BACKGROUND: Diagnostic methods for native vertebral osteomyelitis (NVO) often yield inconclusive results. Image-guided spine biopsies for culture are specific but diagnose NVO in only 50% of cases. Pre-exposure to antimicrobials further reduces diagnostic yield. Our study assesses the value of neutrophil percentage in disc space fluid and vertebral body (DS/VB) samples for diagnosing NVO. METHODS: Adults referred for spine biopsy at Mayo Clinic from August 2022 to September 2023 were consented and enrolled at the time of biopsy. Following routine specimen collection, the biopsy needle was rinsed in saline into an EDTA tube for cell analysis. NVO diagnosis required organism identification in spine tissue or blood and/or positive histopathology, and consistent symptoms and imaging. RESULTS: Sixty-eight patients were prospectively enrolled, comprising 14 with NVO and 54 with alternative diagnoses. The median biopsy sample polymorphonuclear (PMN) percentage for NVO patients was 80.5% (IQR 72.5-85.2), compared to 64.5% (IQR 54.0-69.0) for those without NVO (p < 0.001). Nine (64.3%) NVO patients received antibiotics within 10 days prior to spine biopsy. As a continuous measure, PMN differential showed a moderately strong ability in classifying NVO status with an area under ROC curve of 0.795; an optimal point on the curve of 71.5% corresponded to a sensitivity of 78.6%, specificity of 79.6%, negative predictive value of 93.5% and positive predictive value of 50.0%. CONCLUSION: PMN differential in DS/VB biopsies may serve as an effective diagnostic tool in the evaluation of patients with NVO particularly in ambiguous cases with an initially negative spine biopsy. Future efforts will aim to implement these findings within routine clinical practice.

Salivary gland amyloidosis: Proteomic identification and clinicopathologic characterization of 57 cases.

Salivary gland amyloidosis is an uncommon diagnosis. Most studies have focused on minor salivary gland biopsies as a surrogate site for diagnosing systemic amyloidosis, while only few studies have investigated major salivary gland amyloidosis. We retrospectively identified 57 major and minor salivary gland amyloidosis cases typed using a proteomics-based method between 2010 and 2022. Frequency of amyloid types, clinicopathologic features, and distribution patterns of amyloid deposits were assessed. The indication for salivary gland biopsy/resection (known in 34 cases) included suspected amyloidosis (N = 14; 41.2%), lesion/mass (N = 12; 35.3%), swelling/enlargement (N = 5; 14.7%), and rule out Sjogren syndrome (N = 3; 8.8%). Concurrent pathology was reported in 16 cases, and included chronic sialadenitis (N = 11), extranodal marginal zone lymphoma (N = 3), plasma cell neoplasm (N = 1), and pleomorphic adenoma (N = 1). We identified 3 types of amyloidosis: immunoglobulin light chain/AL (N = 47; 82.5%); immunoglobulin heavy chain/AH (N = 1; 1.8%), and transthyretin/ATTR (N = 9; 15.8%). The patterns of amyloid deposits (assessed in 35 cases) included: 1) Perivascular and/or periductal distribution (N = 18; 51.4%); 2) Mass formation (N = 9; 25.7%); 3) Stromal micronodule formation (N = 7; 20.0%); and 4) Diffuse interstitial involvement (N = 1; 2.9%). We also identified one case of AL amyloidosis localized to the major salivary gland, where only 6 other cases with adequate staging workup to exclude systemic amyloidosis were previously reported. In conclusion, salivary gland amyloidosis is an uncommon diagnosis but may be underrecognized due to low index of suspicion. Most cases of salivary gland amyloidosis are AL type, but a minority are ATTR. Therefore, proteomics-based typing remains essential for treatment and prognosis.

Identification of amyloidosis of the urinary tract and prostate: Opportunities for early diagnosis & intervention in systemic disease.

OBJECTIVES: To determine the prevalence of different amyloid types and frequency of associated systemic amyloidosis in the urinary tract/prostate. METHODS: We studied Congo red-positive prostate (n = 150) and urinary tract (n = 767) specimens typed by a proteomics-based method between 2008 and 2020. Clinical follow up was available for a subset (urinary tract, n = 111; prostate, n = 17). Amyloid types were correlated with various clinicopathologic features. For patients with clinical follow up, chart review was performed to establish localized versus systemic disease, frequency of initial diagnosis of amyloidosis on urinary tract/prostate specimens, presence of cardiac disease, and death from disease-related complications. RESULTS: The most common amyloid types were AL/AH in urinary tract (479/767, 62 %) and localized ASem1 in prostate (64/150, 43 %). Urinary tract AL/AH amyloid was usually localized, but systemic AL amyloidosis occurred in both sites (urinary tract: 5/71, 7 %; prostate: 2/2, 100 %). ATTR amyloidosis was seen in over a third of cases (urinary tract: 286/767, 37 %; prostate: 55/150, 37 %). Urinary tract/prostate was the site of the initial ATTR amyloidosis diagnosis in 44/48 patients (92 %), and 38/48 (79 %) were subsequently found to have cardiac involvement. Seminal vesicle/ejaculatory duct involvement was pathognomonic for ASem1-type amyloidosis (39/39, 100 %). CONCLUSIONS: Over 40 % of patients had systemic amyloidosis, with urinary tract/prostate often the first site in which amyloid was identified. Since early recognition of systemic amyloidosis is critical for optimal patient outcomes, there should be a low threshold to perform Congo red stain. Proteomics-based amyloid typing is recommended since treatment depends on correctly identifying the amyloid type.

Gastrointestinal amyloidosis: an often unexpected finding with systemic implications.

The gastrointestinal (GI) tract is a common site of amyloidosis, but the incidence, clinicopathologic features, and systemic implications of different types of GI amyloidosis are not well understood. GI amyloid specimens (N = 2511) typed using a proteomics-based method between 2008 and 2021 were identified. Clinical and morphologic features were reviewed in a subset of cases. Twelve amyloid types were identified, including AL (77.9%), ATTR (11.3%), AA (6.6%), AH (1.1%), AApoAIV (1.1%), AEFEMP1 (0.7%), ALys (0.4%), AApoAI (0.4%), ALECT2 (0.2%), Aß2M (0.1%), AGel (0.1%), and AFib (<0.1%). Amino acid abnormalities indicative of known amyloidogenic mutations were detected in 24.4% ATTR cases. AL, ATTR, and AA types all commonly involved submucosal vessels. They also showed some characteristic patterns of involvement of more superficial anatomic compartments, although there was significant overlap. Common indications for biopsy were diarrhea, GI bleed, abdominal pain, or weight loss. Amyloidosis was usually an unexpected finding, but most AL and ATTR patients were ultimately found to have cardiac involvement (83.5% of AL; 100% of ATTR). Although most GI amyloid is of AL type, over 10% are ATTR, over 5% are AA, and twelve different types were identified in total. GI amyloid is often unexpected but usually signals systemic amyloidosis, thus there should be a low threshold to perform biopsy with Congo red stain in patients with unexplained GI symptoms. Clinical and histologic features are nonspecific, and typing should be performed via a robust method such as proteomics as treatment hinges on correctly identifying the amyloid type.

Proteomic and Clinicopathologic Assessment of Penile Amyloidosis: A Single Institutional Review of 12 Cases.

OBJECTIVES: There is a paucity of data on penile amyloidosis. We aimed to assess the frequency of different amyloid types in surgical specimens from the penis involved by amyloidosis and correlate relevant clinicopathologic parameters with proteomic findings. METHODS: Since 2008, our reference laboratory has performed liquid chromatography/tandem mass spectrometry (LC-MS/MS) for amyloid typing. The institutional pathology archive and reference laboratory database were queried to retrospectively identify all penile surgical pathology specimens with LC-MS/MS results between January 1, 2008, and November 23, 2022. Archived H&E-stained and Congo red-stained sections were re-reviewed. RESULTS: Twelve cases of penile amyloidosis were identified, which represented 0.35% (n = 3,456) of penile surgical specimens. AL-type amyloid was most frequent (n = 7), followed by keratin-type amyloid (n = 3) and ATTR (transthyretin)-type amyloid (n = 2). AL-type amyloid cases often showed diffuse dermal/lamina propria deposition, whereas all keratin-type amyloid cases were localized to the superficial dermis. Two cases with keratin-type amyloid had concomitant cutaneous findings (penile intraepithelial neoplasia and condyloma). CONCLUSIONS: This series, the largest to date, demonstrates that penile amyloidosis has a heterogeneous proteomic landscape. To the best of our knowledge, this is the first study describing ATTR (transthyretin)-type penile amyloid.

Proteomic Identification and Clinicopathologic Characterization of Splenic Amyloidosis.

The spleen is a commonly encountered specimen in surgical pathology. However, little is known about the incidence, morphologic pattern, and clinical features of spleens involved by amyloidosis. We retrospectively identified 69 spleen amyloid cases typed using a proteomics-based method between 2008 and 2020. The frequency of amyloid types, clinicopathologic features, and distribution of amyloid deposits were assessed. Four amyloid types were detected: immunoglobulin light chain (AL) (N=30; 43.5%); leukocyte chemotactic factor 2 amyloidosis (ALECT2) (N=30; 43.5%); amyloid A (AA) (N=8; 11.6%); and fibrinogen alpha (AFib) (N=1; 1.4%). The splenic amyloid showed 5 distinct distribution patterns: (1) diffuse pattern, exhibited by most AL cases; (2) red pulp pattern, exhibited by most ALECT2 cases; (3) multinodular pattern, seen in subsets of AA and AL-kappa cases; (4) mass-forming pattern, seen in the AFib case; and (5) vascular only, seen in a subset of AA cases. Atraumatic splenic rupture was the most common reason for splenectomy in AL cases, while most ALECT2 spleens were removed incidentally during an unrelated abdominal surgery. Splenomegaly was significantly more common in AA spleens than in AL or ALECT2 spleens and was often the reason for splenectomy in this group. In conclusion, splenic amyloid may be underrecognized as it is often an incidental finding. Although, as expected, many of the spleens were involved by AL amyloidosis, ALECT2 emerged as another common spleen amyloid type. Although the spleen amyloid types exhibited characteristic distribution patterns, proteomics-based typing is warranted as some morphologic overlap still exists. Awareness of ALECT2 as a major spleen amyloid type is important for appropriate diagnostic workup and patient management.

Bone marrow amyloid: a comprehensive analysis of 1,469 samples, including amyloid type, clinical features, and morphologic distribution.

BACKGROUND: Bone marrow biopsy is common in patients suspected of having systemic AL amyloidosis. However, little is known about the incidence, morphology and clinical phenotype of non-AL amyloid types in bone marrow. METHODS: We retrospectively identified N = 1469 bone marrow amyloid biopsies typed using a proteomics-based method between 2008-2020. Frequency of amyloid types (N = 1469), distribution of amyloid deposits (N = 139), and clinical phenotypes (N = 355), with particular emphasis on cardiac involvement, were assessed. RESULTS: The amyloid types were: AL (N = 1172; 79.8%), ATTR (N = 240; 16.3%), AH (N = 38; 2.6%), AA (N = 17; 1.2%), and Aß2M (N = 2; 0.1%). Although there were characteristic morphologic features, including periosteal soft tissue and/or vascular involvement in ATTR, interstitial vascular involvement in AA, and variable anatomic compartment involvement in AL, none were pathognomonic. Most patients with both an M-spike and cardiac involvement had AL amyloid in their BM, but in over 10% the amyloid type was ATTR. Compared to AL patients, ATTR patients had higher stage cardiac amyloidosis and lower overall survival, which was mainly due to advanced cardiac stage. CONCLUSIONS: ATTR amyloid is common in bone marrow and its morphologic distribution overlaps with AL. Amyloid typing is critical as over 10% of patients with bone marrow amyloid, cardiac amyloidosis, and an M-spike have ATTR amyloidosis.

Chronic lymphocytic leukemia (CLL) with Reed-Sternberg-like cells vs Classic Hodgkin lymphoma transformation of CLL: does this distinction matter?

The distinction between chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with isolated Hodgkin/Reed-Sternberg cells (CLL-HRS; background milieu with a paucity of inflammatory cells) and overt transformation to classic Hodgkin lymphoma (CLL-HL; mixed inflammatory background) is incompletely understood. This retrospective study examined the clinicopathologic features of CLL-HRS (n = 15) and CLL-HL (n = 31) patients seen over the past three decades from a single institution. The phenotypic features of Reed-Sternberg cells in both groups were similar, including expression of CD30, CD15, and PAX5, as well as EBV status. However, a spectrum of background CLL/SLL infiltration amongst the HRS cells was noted on pathologic review, and four patients had both diagnoses, either concurrently or in succession. The median overall survival (OS) of patients with CLL-HRS was 17.5 months compared to 33.5 months for patients with CLL-HL (P = 0.24). Among patients with CLL-HRS, those who received Hodgkin-directed therapy had a significantly longer median OS (57 months) compared to those who received CLL-directed therapy (8.4 months, P = 0.02). Our clinical and pathologic findings suggest a biologic continuum between CLL-HRS and CLL-HL and indicate that CLL-HRS patients may benefit from Hodgkin-directed therapy.

De novo isolated myeloid sarcoma: comparative analysis of survival in 19 consecutive cases.

Institutional database search (1999-2020) for acute myeloid leukaemia (AML) identified 109 cases of myeloid sarcoma (MS), of which 19 were isolated and presented de novo. The latter displayed longer survival (median 78 months), compared to MS with synchronous intramedullary AML (n = 32; median 16 months) and de novo AML without MS (n = 729; median 22 months; P = 0·13). However, the difference in survival was no longer apparent after accounting for bone marrow cytogenetic risk status (P = 0·67). Treatment-induced MS tumour resolution was not affected by the presence of intramedullary disease (P = 0·61). The current study clarifies the prognosis of de novo isolated MS, in the context of AML.

RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis.

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Novel t(1;8)(p31.3;q21.3) NFIA-RUNX1T1 Translocation in an Infant Erythroblastic Sarcoma.

OBJECTIVES: Pure erythroid leukemia (PEL) is exceptionally rare in the pediatric setting. Four pediatric PEL cases with t(1;16)(p31;q24) NFIA-CBFA2T3 were reported previously. We present a case of an infant with PEL presenting with erythroblastic sarcoma and harboring a novel t(1;8)(p31.3;q21.3) NFIA-RUNX1T1 fusion detected by RNA sequencing and conventional karyotype. METHODS: Bone marrow (BM) and abdominal mass biopsies from the patient were evaluated with extensive immunohistochemical, flow cytometric, cytogenetic, and molecular studies. RESULTS: The patient was a female infant who presented between 2 and 5 months of age with cytopenias and an enlarging abdominal mass. Blasts in the BM and abdominal mass expressed CD71 and CD117 with focal expression of CD43, E-cadherin, epithelial membrane antigen, and hemoglobin A. They were negative for additional myeloid, lymphoid, and nonhematolymphoid markers. These findings were most consistent with PEL and erythroblastic sarcoma. RNA sequencing revealed the novel NFIA-RUNX1T1 fusion. CONCLUSIONS: Along with the previously reported PELs with NFIA-CBFA2T3 fusions, we describe a subset of PELs that occur in children, that frequently display extramedullary disease, and that harbor rearrangements of NFIA with core binding factor genes. We hypothesize that, together, these cases represent a rare but distinct clinicopathologic group of pediatric PELs with recurrent genetic abnormality.

Amyloid Typing by Mass Spectrometry in Clinical Practice: a Comprehensive Review of 16,175 Samples.

OBJECTIVE: To map the occurrence of amyloid types in a large clinical cohort using mass spectrometry-based shotgun proteomics, an unbiased method that unambiguously identifies all amyloid types in a single assay. METHODS: A mass spectrometry-based shotgun proteomics assay was implemented in a central reference laboratory. We documented our experience of typing 16,175 amyloidosis specimens over an 11-year period from January 1, 2008, to December 31, 2018. RESULTS: We identified 21 established amyloid types, including AL (n=9542; 59.0%), ATTR (n=4600; 28.4%), ALECT2 (n=511; 3.2%), AA (n=463; 2.9%), AH (n=367; 2.3%), AIns (n=182; 1.2%), KRT5-14 (n=94; <1%), AFib (n=71; <1%), AApoAIV (n=57; <1%), AApoA1 (n=56; <1%), AANF (n=47; <1%), Aß2M (n=38; <1%), ASem1 (n=34; <1%), AGel (n=29; <1%), TGFB1 (n=29; <1%), ALys (n=15; <1%), AIAPP (n=13; <1%), AApoCII (n=11; <1%), APro (n=8; <1%), AEnf (n=6; <1%), and ACal (n=2; <1%). We developed the first comprehensive organ-by-type map showing the relative frequency of 21 amyloid types in 31 different organs, and the first type-by-organ map showing organ tropism of 18 rare types. Using a modified bioinformatics pipeline, we detected amino acid substitutions in cases of hereditary amyloidosis with 100% specificity. CONCLUSION: Amyloid typing by proteomics, which effectively recognizes all amyloid types in a single assay, optimally supports the diagnosis and treatment of amyloidosis patients in routine clinical practice.

Evaluation of automated synovial fluid total cell count and percent polymorphonuclear leukocytes on a Sysmex XN-1000 analyzer for identifying patients at risk of septic arthritis.

BACKGROUND: Total cell counts (TC-BF) and percent polymorphonuclear cells (%PMN) of synovial fluid (SF) aspirates provide important cues for the timely diagnosis and management of septic arthritis. To facilitate faster turnaround time, we compared automated to manual TC-BF and differential counts in order to identify reporting cut-offs for automated TC-BF and %PMN that would allow release of automated results concordant with manual counts and differentials. METHODS: Automated TC-BF and %PMN counts of a non-validated analyzer (Analyzer-B in STAT laboratory) were compared to a validated analyzer (Analyzer-A) and manual TC-BF counts and cytospin differentials. Concordance and %differences of Analyzer-B versus Analyzer-A and manual counts were assessed by linear regression analysis and Bland-Altman comparison. RESULTS: Overall, automated and manual counts displayed good correlation. A majority of samples demonstrated unacceptable (>20%) differences between automated and manual counts at lower TC-BF (<10,000 cells/µl) and %PMN (<60%). CONCLUSIONS: Based on good overall correlation and fewer samples with unacceptable (>20%) differences between automated and manual counts, we adopted TC-BF > 10,000 cells/µl and %PMN > 60% as cutoffs for reporting automated counts. These cutoffs minimize differences between automated and manual cell counts and differentials and would allow rapid automated reporting in the vast majority of septic arthritis cases.

Biology and prognostic impact of clonal plasmacytoid dendritic cells in chronic myelomonocytic leukemia.

Islands of CD123high cells have been commonly described in the bone marrow of patients with chronic myelomonocytic leukemia (CMML). Using a multiparameter flow cytometry assay, we detected an excess of CD123+ mononucleated cells that are lineage-negative, CD45+, CD11c-, CD33-, HLA-DR+, BDCA-2+, BDCA-4+ in the bone marrow of 32/159 (20%) patients. Conventional and electron microscopy, flow cytometry detection of cell surface markers, gene expression analyses, and the ability to synthesize interferon alpha in response to Toll-like receptor agonists identified these cells as bona fide plasmacytoid dendritic cells (pDCs). Whole-exome sequencing of sorted monocytes and pDCs identified somatic mutations in genes of the oncogenic RAS pathway in the two cell types of every patient. CD34+ cells could generate high amount of pDCs in the absence of FMS-like tyrosine kinase 3-ligand (FLT3L). Finally, an excess of pDCs correlates with regulatory T cell accumulation and an increased risk of acute leukemia transformation. These results demonstrate the FLT3L-independent accumulation of clonal pDCs in the bone marrow of CMML patients with mutations affecting the RAS pathway, which is associated with a higher risk of disease progression.

Bone Marrow Biopsy Operator Experience and Impact on Aspirate, Biopsy, and Ancillary Testing Quality.

OBJECTIVE: To assess the relationship between bone marrow (BM) biopsy operator experience and both specimen quality and ancillary testing utilization. PATIENTS AND METHODS: We evaluated all referred and in-house (IH) BM biopsy specimens obtained over a contiguous 6-week period from April 3, 2017, to May 19, 2017. The BM specimens were assessed for the length of interpretable marrow, and aspirates were assessed for the presence of spicules. Subgroup comparisons included IH BM obtained by a trained team of nurses within our institution, patients clinically referred (CR) to our institution with outside-obtained BM specimens, and outside pathologist-referred (PR) consultation cases. Ancillary study usage was compared between the first 100 cases of each group. RESULTS: A total of 1191 BM specimens were analyzed, including 600 IH, 288 CR, and 303 PR cases with biopsies and/or aspirates. The average interpretable biopsy lengths of IH, CR, and PR cases were 16.0 mm, 10.0 mm, and 7.0 mm, respectively (P<.001). World Health Organization-recommended length of 15 mm or more was achieved in 61.4%, 26.6%, and 19.1%, respectively (P<.001). Of the aspirates analyzed among IH, CR, and PR cases, 93%, 71.3%, and 73.5% contained spicules, respectively (P<.001). Use of immunohistochemistry, flow cytometry, karyotype, and fluorescence in situ hybridization was higher in CR and PR cases than in IH cases (all P<.05). The IH, CR, and PR cases used on average 1.5, 2.8, and 4.8 immunohistochemistry stains per case (P<.001). CONCLUSION: Having a dedicated team of BM biopsy operators is likely one factor contributing to improved BM biopsy quality and a reduced need for ancillary testing.

A Test Utilization Approach to the Diagnostic Workup of Isolated Eosinophilia in Otherwise Morphologically Unremarkable Bone Marrow: A Single Institutional Experience.

OBJECTIVES: Determine ancillary test utilization for the workup of isolated eosinophilia in otherwise morphologically unremarkable bone marrow (BM). METHODS: We evaluated BM ancillary testing performed in cases with isolated eosinophilia and otherwise morphologically unremarkable BM. Cases with abnormal morphology (eg, dysplasia, basophilia) and/or findings suggestive of a disorder (eg, unexplained thromboses, lymphoma) are specifically excluded. RESULTS: A total of 132 cases met inclusion criteria. Ten cases had an ancillary testing abnormality that warranted a more specific hematologic diagnosis: four cases of lymphocytic variant of hypereosinophilic syndrome, three cases of myeloid neoplasm with PDGFRA rearrangement, and one case each of myeloid neoplasm with PDGFRB rearrangement, chronic eosinophilic leukemia, and morphologically occult systemic mastocytosis. No cases revealed a cryptic PDGFRB or BCR/ABL1 rearrangement or JAK2 V617F mutation. CONCLUSIONS: Findings from our institutional experience support initial testing in isolated eosinophilia with otherwise unremarkable BM to include PDGFRA rearrangement, tryptase/CD25 immunohistochemistry, cytogenetics, and T-cell flow cytometry/receptor gene rearrangement. This approach achieves diagnostic quality and test utilization efficiency in our clinical practice.