Investigating Erythrocytosis: Changing Practice Patterns in the Era of Molecular Diagnostics

Abstract Background: Since the identification of JAK2 mutations in polycythemia vera (PV) in 2005 (Kralovics et al., NEJM 2005), molecular testing of JAK2 in patients with erythrocytosis has become part of routine clinical practice. We hypothesized that changes in the World Health Organization (WHO) diagnostic criteria for PV in 2016, which lowered the hemoglobin threshold to >165 g/L for men and >160 g/L for women, may have resulted in increased molecular testing. This study examines changing patterns of utilization of molecular diagnostics in patients referred for erythrocytosis at a tertiary care center. Methods: We examined all patients with erythrocytosis who underwent JAK2 testing, which included testing for JAK2 V617F with PCR between 2015 and 2017, and JAK2 V617F and exon 12 mutations with Next-Generation Sequencing (NGS) between 2018 and 2020 at London Health Sciences Centre in Ontario, Canada. We performed a retrospective chart review to extract laboratory and clinical data, including information on medical comorbidities and medications, with a focus on known secondary causes of erythrocytosis. Results: A total of 668 patients with erythrocytosis underwent JAK2 testing at our institution between August 1, 2015 and December 31, 2020. There was an overall increase in testing over the five-year study period, with a decline in the positive detection rate: 8/29 (28%) in 2015, 15/94 (16%) in 2016, 15/100 (15%) in 2017, 19/136 (14%) in 2018, 17/162 (10%) in 2019, and 14/147 (10%) in 2020 (Figure 1). The average hemoglobin levels in patients with erythrocytosis who underwent testing remained similar across all years (range 170-173 g/L for women, 179-181 g/L for men). In our cohort, there was a high proportion of patients with known or suspected secondary causes of erythrocytosis who underwent molecular testing. Between 2018 and 2020, 324/445 (73%) of patients who underwent molecular testing had either chronic obstructive pulmonary disease, obstructive sleep apnea, other hypoxic lung disease, smoking history, erythropoietin-secreting tumor, or potential drug-induced erythrocytosis. Specifically, we observed an increase in proportion of patients who underwent molecular testing on sodium-glucose cotransporter-2 (SGLT-2) inhibitors, a known secondary cause of erythrocytosis, with 15/136 (11%) in 2018, 17/162 (10%) in 2019, and 25/147 (17%) in 2020. In contrast, the proportion of patients on testosterone was relatively constant at 15/136 (11%) in 2018, 11/162 (6.8%) in 2019, and 11/147 (7.5%) in 2020. Conclusion: This study revealed that a high proportion of patients with known or suspected secondary causes of erythrocytosis underwent JAK2 testing, resulting in increase in molecular testing over time and a decline in positive detection rate. In particular, we observed a number of patients on SGLT-2 inhibitors who had investigation, suggesting that this class of medications may be an underrecognized cause of drug-induced erythrocytosis (Chin-Yee et al., CMAJ 2020). Our findings underscore the importance of careful medical history and medication review to support more judicious use of molecular testing. Similarity in average hemoglobin levels across the five-year study period suggests that other factors, such as increased availability of 'routine' molecular testing, rather than changes in the WHO diagnostic criteria may explain increases in JAK2 testing. Our study indicates a need to develop an effective clinical prediction rule for JAK2 positivity to better risk stratify patients with suspected PV based on clinical and laboratory parameters to optimize utilization of molecular diagnostics. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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A Case of Erythrocytosis in a Patient Treated with an Aromatase Inhibitor for Breast Cancer

Case Reports in Hematology ◽

10.1155/2013/615189 ◽

2013 ◽

Vol 2013 ◽

pp. 1-3 ◽

Cited By ~ 1

Author(s):

Abhinav Iyengar ◽

Dawn Sheppard

Keyword(s):

Breast Cancer ◽

Ductal Carcinoma ◽

Jak2 V617f ◽

Myeloproliferative Disorder ◽

Pulmonary Diseases ◽

Molecular Testing ◽

Carcinoma Of The Breast ◽

Obstructive Sleep ◽

Serum Erythropoietin ◽

Secondary Causes

A previously healthy 79-year-old female was referred to hematology for further evaluation of erythrocytosis. Two years earlier she had been diagnosed with ER/PR-positive ductal carcinoma of the breast and was receiving hormonal therapy with exemestane. No secondary cause of erythrocytosis was identified. Serum erythropoietin (EPO) level was normal, and molecular testing for the JAK2 V617F and exon 12 mutations was negative. A bone marrow biopsy showed a mild increase in erythropoiesis, and no spontaneous erythroid colonies were demonstrated. Erythrocytosis is common reason for referral to a hematologist. The myeloproliferative disorder, polycythemia vera, and the rare congenital polycythemias represent primary erythrocytosis. Common secondary causes include smoking, obstructive sleep apnea, and other pulmonary diseases. Erythrocytosis is well described with certain classes of drugs, including androgens. We hypothesize that exemestane contributed to the development of erythrocytosis in our patient. To our knowledge, erythrocytosis has not been previously described in association with aromatase inhibitors. These drugs prevent the conversion of androstenedione and testosterone to estrogen; thus the physiologic mechanisms may be similar to those responsible for erythrocytosis seen with exogenous androgens. These mechanisms are not well understood, but may include altered iron metabolism by a reduction in hepcidin levels.

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Problem in diagnostic criteria preparation of drug induced hepatopathy.

Kanzo ◽

10.2957/kanzo.45.332 ◽

2004 ◽

Vol 45 (6) ◽

pp. 332-336 ◽

Cited By ~ 1

Author(s):

NAOKI YOROZUYA

Keyword(s):

Diagnostic Criteria ◽

Drug Induced

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Toward harmonization of clinical molecular diagnostic reports: findings of an international survey

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2017-1080 ◽

2018 ◽

Vol 0 (0) ◽

Author(s):

Deborah A. Payne ◽

Katarina Baluchova ◽

Graciela Russomando ◽

Parviz Ahmad-Nejad ◽

Cyril Mamotte ◽

...

Keyword(s):

Molecular Diagnostics ◽

Clinical Decision Making ◽

Clinical Chemistry ◽

Clinical Decision ◽

Molecular Diagnostic ◽

Molecular Testing ◽

End User ◽

Iso 15189 ◽

Reporting Practices ◽

Reporting Phase

Abstract Background: The International Organization for Standardization (ISO) 15189 standard provides recommendations for the postexamination reporting phase to enhance quality in clinical laboratories. The purpose of this study was to encourage a broad discussion on current reporting practices for molecular diagnostic tests by conducting a global survey of such practices. Methods: The International Federation of Clinical Chemistry and Laboratory Medicine’s Committee for Molecular Diagnostics (IFCC C-MD) surveyed laboratories on selected ISO 15189 recommendations and topics. The survey addressed the following aspects: (1) laboratory demographics, (2) report format, (3) result reporting/layout, (4) comments in report and (5) interpretation and clinical decision-making information. Additionally, participants indicated categories needing standardization. Results: Sixteen responses from laboratories located in Asia, Europe, the Middle East, North America and South America were received. Several categories yielded 100% agreement between laboratories, whereas other categories had less than or equal to 50% concordance. Participants scored “nomenclature” and “description of methodologies” as the two most frequently cited aspects needing standardization. Conclusions: The postexamination phase requires extensive and consistent communication between the laboratory, the healthcare provider and the end user. Surveyed laboratories were most likely to follow explicit ISO 15189 recommendations vs. recommendations when the term(s) “where appropriate or where applicable” was used. Interpretation and reporting of critical values varied among participants. Although the outcome of this study may not fully represent the practices of all molecular testing laboratories in countries around the world, the survey identified and specified several recommendations that are requirements for harmonized reporting in molecular diagnostics.

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Proposal of diagnostic criteria of drug induced hepatic injury in DDW-J2004 workshop.

Kanzo ◽

10.2957/kanzo.46.85 ◽

2005 ◽

Vol 46 (2) ◽

pp. 85-90 ◽

Cited By ~ 49

Author(s):

H Takikawa ◽

M Onji ◽

Y Takamori ◽

Y Murata ◽

H Taniguchi ◽

...

Keyword(s):

Diagnostic Criteria ◽

Hepatic Injury ◽

Drug Induced

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Overlapping DRESS and Stevens-Johnson Syndrome: Case Report and Review of the Literature

Case Reports in Dermatology ◽

10.1159/000475802 ◽

2017 ◽

Vol 9 (2) ◽

pp. 1-7 ◽

Cited By ~ 4

Author(s):

Aneline Casagranda ◽

Mariano Suppa ◽

Florence Dehavay ◽

Véronique del Marmol

Keyword(s):

Diagnostic Criteria ◽

Adverse Reactions ◽

Stevens Johnson Syndrome ◽

Drug Induced ◽

Johnson Syndrome ◽

Systemic Symptoms ◽

The Right ◽

Exanthematous Pustulosis ◽

Cutaneous Adverse Reactions

Drug-induced severe cutaneous adverse reactions (SCARs) include acute generalized exanthematous pustulosis, drug reaction with eosinophilia and systemic symptoms (DRESS), and epidermal necrolysis (Stevens-Johnson syndrome [SJS], toxic epidermal necrolysis). The identification of the causal drug is crucial in order to avoid further exposure, but making the right differential diagnosis of the type of SCAR is equally important since treatment, follow-up, and prognosis of different SCARs are not the same. These syndromes are distinct entities with different clinical, biological, and histological patterns, but sometimes the early distinction between 2 SCARs can be extremely challenging, and overlapping conditions could therefore be taken into consideration, although true overlapping SCARs are very rare when using strict diagnostic criteria (described by the RegiSCAR group). Only a better understanding of the physiopathology of the SCARs could possibly explain these ambiguities and overlaps. We report a case of SCAR in an 86-year-old patient probably induced by allopurinol and simultaneously fulfilling the diagnostic criteria for DRESS and SJS, thus considered as an overlapping case of SCARs.

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Brief Synopsis: Review of Renal Tubule and Interstitial Anatomy and Physiology and Renal INHAND, SEND, and DIKI Nomenclature

Toxicologic Pathology ◽

10.1177/0192623318798758 ◽

2018 ◽

Vol 46 (8) ◽

pp. 920-924

Author(s):

Torrie A. Crabbs ◽

Kevin S. McDorman

Keyword(s):

Diagnostic Criteria ◽

Renal Tubule ◽

Kidney Injury ◽

Scientific Session ◽

Laboratory Animals ◽

Drug Induced ◽

Diagnostic Features ◽

Collaborative Project ◽

Anatomy And Physiology ◽

Historical Control Data

This article provides a synopsis of the first two presentations from the second scientific session of the 37th Annual Symposium of the Society of Toxicologic Pathology in Indianapolis, Indiana, on June 18, 2018; the session focused on acute kidney injury. The first presentation, given by Dr. Kevin McDorman, focused on “Fundamentals of Renal Tubule and Interstitial Anatomy and Physiology.” Several common background findings from toxicity studies were additionally discussed. Lastly, factors that impact the relevance and usefulness of historical control data, such as quality and consistency of histopathology, were discussed. The second presentation, given by Dr. Torrie Crabbs, provided a review of International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), Standard for Exchange of Nonclinical Data (SEND), and drug-induced kidney injury (DIKI) nomenclature. INHAND is a global collaborative project that provides internationally accepted standardized nomenclature and diagnostic criteria for proliferative and nonproliferative changes in laboratory animals in toxicity and carcinogenicity studies. SEND is currently a required standard for data submission to the Food and Drug Administration (FDA). Since the FDA has indicated its preference for INHAND nomenclature, SEND will predominately use INHAND terminology; thus, familiarity with INHAND terminology is critical for toxicologic pathologists. The diagnostic features of three common DIKI findings, in addition to several complicated INHAND terminologies, were reviewed.

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A Rare Case of JAK2 V617F Positive Polycythemia Vera in a Child with Neurofibromatosis Type I.

Blood ◽

10.1182/blood.v110.11.4661.4661 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4661-4661

Author(s):

Jason N. Berman ◽

Wenda L. Greer ◽

Mignon Loh ◽

Christie Riddell ◽

Barbara Morash ◽

...

Keyword(s):

Polycythemia Vera ◽

Diagnostic Criteria ◽

Peripheral Blood ◽

Jak2 V617f ◽

Neurofibromatosis Type ◽

Juvenile Myelomonocytic Leukemia ◽

Type I ◽

Jak2 Mutation ◽

Genetic Event ◽

The Common

Abstract Polycythemia vera (PV), is a myeloproliferative disease (MPD) originating in a hematopoietic stem cell resulting in clonal expansion of erythroid progenitors. It is associated with thromboses and malignant transformation. Recently the V617F alteration arising from a mutation in JAK2 has been identified in greater than 90% of cases of PV in adults. PV is rare in children and the frequency of the common JAK2 mutation is significantly lower than in adult patients, indicating that alternative genetic events are involved in the pathogenesis of this disease. We have identified a child diagnosed with PV at the age of 15 months, the youngest described in the literature to date. Initial laboratory values demonstrated a WBC of 33 x109/L, hemoglobin 181 g/L, and platelet count of 579 x109/L. Bone marrow cytogenetics were normal. Erythroid colony forming units demonstrated erythropoietin-independent growth. Peripheral blood, buccal swab and saliva analysis revealed the presence of the common JAK2 V617F mutation, but a B lymphocyte cell line and skin-fibroblast-culture from this patient were negative, indicating that the JAK2 mutation was somatic. Peripheral blood from her parents and older brother demonstrated normal blood counts and wild type JAK2 status. This child was also diagnosed with Neurofibromatosis type 1 (NF1) based on meeting NIH consensus diagnostic criteria diagnostic criteria, having the requisite number of appropriately sized café-au-lait macules and Lisch nodules. NF1 and PV have no previously known association, however NF1 is associated with another MPD, juvenile myelomonocytic leukemia (JMML). Patients with NF1 and JMML demonstrate loss of heterozygosity (LOH) at the NF1 locus while 60% of JMML patients without NF1 alternatively demonstrate somatic mutations in NRAS, KRAS2 or PTPN11. Taken together, these genetic lesions result in hyperactivation of the RAS/MAPK pathway. Low density single nucleotide polymorphism arrays performed on peripheral blood from this patient failed to demonstrate obvious LOH at the NF1 locus. NF1 gene sequencing failed to identify the cause of the NF1 phenotype. Mutations were not identified in the commonly mutated regions of NRAS, KRAS2 or PTPN11. This case reveals the presence of the most commonly acquired somatic JAK2 mutation in a young child with PV and indicates that buccal swabs and saliva are unreliable sources of unaffected tissue for assessing the presence of germline mutations in PV patients. Moreover, it suggests that some patients with clinical NF1 are at risk for developing other MPDs besides JMML. For this patient, a novel unidentified genetic abnormality resulting in the clinical phenotype of NF1 may serve as a predisposing genetic event accounting for the unusually young age of presentation. The investigation of rare children with PV has the potential to provide valuable insight into the molecular interactions underlying the pathogenesis of MPDs.

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Impact of Molecular Testing for JAK2V617F in the Diagnosis of Polycythemia in An Integrated Community Based Hematology/Oncology Group Practice

Blood ◽

10.1182/blood.v118.21.846.846 ◽

2011 ◽

Vol 118 (21) ◽

pp. 846-846

Author(s):

Paul I. Roda ◽

Erlich Porat

Keyword(s):

Bone Marrow ◽

Diagnostic Criteria ◽

Molecular Testing ◽

Red Cell ◽

Community Based ◽

Progress Notes ◽

Serum Erythropoietin Level ◽

Additional Testing ◽

Minor Criteria ◽

Serum Erythropoietin

Abstract Abstract 846 Background: In 2005, a specific mutation in the gene for JAK2 (JAK2V617F) was documented in virtually all patients with Polycythemia vera (PV). In 2009, the WHO issued new diagnostic criteria for PV, listing erythrocytosis and a positive test for JAK2V617F as major diagnostic criteria; and listing either a low serum erythropoietin level or characteristic bone marrow histopathological abnormalities as minor criteria for the diagnosis of PV. The importance of testing for JAK2 V617F in making the diagnosis of PV was documented by Seaman et al Cancer Epidemiol Biomarkers Prev 2009;18(2). February 2009. In Seaman's study of PV patients in Northeast Pa., molecular testing confirmed that only 33 of 62 patients thought to have PV were correctly diagnosed. This study was initiated at the request of the ATSDR with goals of determining if similar discrepancies existed in another, larger population; as well as determining whether community hematologists were integrating molecular testing into their diagnostic paradigms. Methodology: The Geisinger Medical Center is an integrated health provider, with facilities throughout much of north central and east Pennsylvania. A single EMR has been utilized at outpatient locations since 2004. After obtaining approval from the Institutional Review Board, the records of all patients being followed between 2004 and 2009 for PV by a Geisinger hematologist/oncologist were identified. A total of 279 records were located. These records were reviewed for the physician's narrative diagnosis, the use of molecular testing, serum erythropoietin levels, bone marrow histology, as well as other tests historically used to assess polycythemia (red cell volume, spleen size by imaging, etc.). Of these records 252 contained adequate physician documentation to be evaluable. Results: While all progress notes listed PV as the final diagnosis, the progress notes documented sufficient data to make a diagnosis of PV in only 139 patients. 89 were diagnosed with secondary polycythemia, while twenty one were ultimately found to have spurious polycythemia. Once the patient was diagnosed with PV, it was rarely changed when further testing indicated either secondary or spurious polycythemia. JAK2V617F mutation testing was performed in 132 of these patients, with 94 of these tests obtained in 2008 or later. 80 of the 132 patients tested for this mutation were JAK2V617F positive. Seventy eight of the JAK2V617F positive. patients underwent additional testing. Serum erythropoietin levels were measured in 39, while bone marrow samples were obtained in 20 patients. Only 41 JAK2V617F positive patients met the 2009 WHO “minor” criteria by having either a serum erythropoietin level near or below the lower limit of normal, and/or a bone marrow characteristic of PV. In 14 patients, JAK2V617F testing led to a change in diagnosis, excluding PV in eleven, while three patients initially diagnosed with secondary polycythemia were found to have PV. Of patients who did not undergo molecular testing, only six had low serum erythropoietin levels and abnormal bone marrow findings. Determinations of the red cell mass with, or without plasma volume was rarely performed after 2004. Conclusions: From these data we conclude that JAK2V617F testing has made a major impact in facilitating the successful delineation of the cause of polycythemia in patients evaluated in a large, community – based Hematology/Oncology practice. In contrast the complete WHO diagnostic criteria do not appear to be widely utilized by these physicians, nor does it appear that these additional tests add to the diagnostic evaluation in most patients. The necessity of utilizing the minor criteria while assessing JAK2V617F positive patients requires further evaluation. Furthermore this study demonstrates that physicians do not change their initial diagnosis to reflect the impact of the additional testing. This has implications for those utilizing ICD – 9 codes to study myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

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Smoking history and frequency of somatic KRAS mutations in adenocarcinoma of the lung

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.7573 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 7573-7573

Author(s):

V. A. Miller ◽

G. J. Riely ◽

M. G. Kris ◽

D. Rosenbaum ◽

J. Marks ◽

...

Keyword(s):

Cigarette Smoking ◽

Mutational Analysis ◽

Smoking History ◽

Egfr Mutations ◽

Molecular Testing ◽

Kras Mutations ◽

Exon 2 ◽

Significant Difference ◽

Never Smokers ◽

Lung Adenocarcinomas

7573 Background: Somatic mutations in the epidermal growth factor receptor (EGFR) gene are more common in patients with adenocarcinoma, especially those who smoked < 15 pack years (py). KRAS mutations are found in ∼25% of lung adenocarcinomas, most commonly in codons 12 and 13 of exon 2 (∼85%) and have been associated with poor prognosis in resected disease [Winton NEJM 2005] and resistance to EGFR tyrosine kinase inhibitors [Pao PLoS Med 2005]. KRAS mutations are uncommon in non-small cell lung cancer histologies other than adenocarcinoma. We sought to determine the association between quantitative measures of cigarette smoking and presence of KRAS mutations in lung adenocarcinomas. Methods: Standard direct sequencing techniques were used to identify KRAS codon 12 and 13 mutations in lung adenocarcinoma specimens from surgical resections between 2001 and 2006 and tumor specimens sent for KRAS molecular analysis in 2006. Surgical specimens were obtained from an institutional tumor bank. Detailed smoking history (age at first cigarette, packs per day, years smoked, years since quitting smoking) was obtained from the medical record and a patient-completed smoking questionnaire. Results: KRAS mutational analysis was performed on 408 lung adenocarcinomas from 242 women and 166 men. Median age was 68 (range 33–89). KRAS mutations were present in 19% (78/408, 95% CI 15 to 23%). The frequency of KRAS mutation was not associated with age or gender. The presence of KRAS mutations was not related to smoking history with 15% (9/61) of never smokers having KRAS mutations compared with 19% (51/275) of former smokers. When compared with never smokers, there was no significant difference in frequency of KRAS mutations for tumors from patients with 1–5 py (5%, p=0.44), 6- 10 py (12%, p=0.99), 11–15 py (25%, p=0.45), 16–25 py (16%, p=0.99), 26–50 py (25%, p=0.129), 51–75 py (20%, p=0.48), >75 py (20%, p=0.47) history of cigarette smoking. Conclusions: While the incidence of EGFR mutations has a strong inverse relationship with the amount of cigarettes smoked, allowing the selective molecular testing for EGFR mutations, the frequency of KRAS mutations cannot be predicted by age, gender, or smoking history. KRAS mutational analysis of all adenocarcinomas is required to reliably identify patients with KRAS mutations. No significant financial relationships to disclose.

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Detection of BRAF mutations in melanoma: Rate of mutation detection at codon 600 using Sanger sequencing as compared to the cobas 4800 method.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.8596 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 8596-8596

Author(s):

Kevin Z. Qu ◽

Qiulu Pan ◽

Xi Zhang ◽

Luis Rodriguez ◽

Jennifer Uyeji ◽

...

Keyword(s):

Molecular Diagnostics ◽

Sanger Sequencing ◽

Detection Rate ◽

Mutation Detection ◽

Mutation Detection Rate ◽

Braf Mutations ◽

Roche Molecular Diagnostics ◽

Ffpe Tissues ◽

Sanger Method ◽

Braf V600 Mutation

8596 Background: Detection of BRAF V600 mutations is currently a prerequisite for approved use of vemurafenib in patients with metastatic melanoma. The cobas 4800 BRAF V600 Mutation Test (Roche Molecular Diagnostics), a PCR-based assay approved to aid in selecting patients for vemurafenib therapy, primarily detects V600E. It is also reported to detect V600K, which has been associated with vemurafenib response as well. We compared the mutation detection rate of the cobas assay with that of Sanger sequencing. Methods: 125 de-identified FFPE tissues submitted for BRAF mutation analysis that all showed histologically-confirmed melanoma were tested. BRAF mutations were detected using both the cobas kit and bidirectional Sanger sequencing using BigDye kits (Applied Biosystems). DNA was extracted from 5-um sections without macrodissection using the cobas DNA extraction kit (for the cobas test) or from 5-10-um sections using Agencourt extraction kits (Beckman Coulter) following macrodissection. Results: The two methods showed agreement in 104/125 (83.2%) of cases (Table). Sanger sequencing detected V600 dinucleotide mutations in 9 samples that were negative by the cobas assay. Sanger sequencing produced no results in 10 cases owing to suboptimal PCR, including 2 that were positive by the cobas assay. The cobas assay produced 2 invalid results, including 1 that was positive for V600E by Sanger.The cobas assay detected 7/11 V600K mutations. Conclusions: Overall agreement between cobas and Sanger sequencing was 83.2%. The Sanger method had higher analytic sensitivity, resulting in nine additional V600 mutations not called by cobas compared to the two seen by cobas but not Sanger sequencing. Thus, 16% (9/57) more patients would be identified as candidates for vemurafenib therapy using the Sanger method. [Table: see text]

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