Clinical Indications

Hotspots, copy number variants, and selected fusions evaluated by this assay can aid in the diagnostic and therapeutic assessment of a variety of tumor types, including non-small cell lung cancer, melanoma, colorectal cancer, prostate cancer, breast cancer, glioblastoma, thyroid cancer, and others.

This panel can also provide focused tumor profiling for patients with locally advanced/metastatic disease, who are candidates for anti-cancer therapy, to identify uncommon but targetable alterations.

Interpretation

Variants are classified according to established guidelines, and an interpretation is provided. Reported variants include those of strong or potential clinical significance and variants of unclear clinical significance.

Common population variants are not included in the report.

Methodology & Limitations

Extracted nucleic acid from the specimen, both DNA and RNA, were subjected to separate targeted amplification reactions using AmpliSeq custom primers designed by Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA). Hotspots and selected fusions in gene regions listed below were sequenced using Illumina (San Diego, CA) 2×150 paired-end cycle chemistry. A customized bioinformatics analytical platform was used for read alignment (Genome Build GRCh37/hg19), variant identification, and annotation. Single nucleotide variants (SNVs), insertion, deletion (indels), and copy number gain variants are detected by DNA sequencing. Select fusions and aberrant transcripts (EGFR vIII and MET exon 14 skipping transcripts) are detected by RNA sequencing. Variants are classified according to established guidelines. Reported results include variants of strong or potential clinical significance and variants of unclear clinical significance. Benign population polymorphisms are not included in the report.

Based on validation, the DNA testing delivered an average of >500x coverage, and >99% of targeted regions showed over 100x coverage. A minimum coverage depth of 100 reads is required across the entire region of interest; a list of low-coverage areas is included in the report as applicable. The test demonstrated 100% sensitivity and 100% specificity in identifying SNVs, indels, and copy number gains. The lower limit of detection of this assay is approximately 5% variant allele fraction (VAF) for SNV/indels and 6 copies or greater for copy number gains. Variants below these thresholds may be reported at the discretion of the molecular pathology professional staff if the technical quality of the sequencing is sufficient at that location and the call is unequivocal.

Based on validation, the RNA fusion testing averaged >150,000 total reads. The test demonstrated 93%sensitivity and 100% specificity in gene fusion identification compared to NGS sequencing and 69% sensitivity and 100% specificity compared to fluorescence in situ hybridization (FISH) of fusion drivers (unknown fusion partner). Overall sensitivity is 78%, and accuracy is 99%. The lower limit of detection is approximately 1% of total sequencing reads.

Sequence changes outside the analyzed alterations hotspots, including intronic and noncoding regions, will not be identified by this test. Insertions and deletions larger than 20 and 40 bp, respectively, may not be identified by this test. Negative results from specimens for which the percentage of tumor cells is 10% or less should be interpreted with caution. Although variant allele fraction is provided as a percentage, this is not a quantitative test. RNA fusions involving alternative partners or breakpoints outside of the targeted regions cannot be detected by this test. This test does not distinguish between somatic and inherited variants. Tumor heterogeneity, tumor burden, specimen degradation or other limitations of the technology may affect the sensitivity and limit of detection, either broadly across the regions of interest or for specific regions, and may lead to false negative results. This test is not intended to be used for circulating tumor evaluation.

Genes & Hotspot Regions

Genes & Hotspot Regions Covered for SNV/Indels:

Fusions Detected in RNA

Genes reported for copy number gains: ALK, AR, BRAF, CCND1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, FGFR4, KIT, KRAS, MET, MYC, MYCN, PDGFRA, PIK3CA

References

1. Li MM, et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017 Jan;19(1):4-23. doi: 10.1016/j. jmoldx.2016.10.002. PMID: 27993330; PMCID: PMC5707196.

2. Kumar-Sinha C, Tomlins SA, Chinnaiyan AM. Recurrent gene fusions in prostate cancer. Nat Rev Cancer. 2008 Jul;8(7):497-511. doi: 10.1038/nrc2402. Epub 2008 Jun 19. PMID: 18563191; PMCID: PMC2711688.

3. Baca SC, Garraway LA. The genomic landscape of prostate cancer. Front Endocrinol (Lausanne). 2012 May 16;3:69. doi: 10.3389/fendo.2012.00069. PMID: 22649426; PMCID: PMC3355898.

4. Bennett C, et al. Breast Cancer Genomics: Primary and Most Common Metastases. Cancers (Basel). 2022 Jun 21;14(13):3046. doi: 10.3390/cancers14133046. PMID: 35804819; PMCID: PMC9265113.

5. Natrajan R, Tutt ANJ, Lord CJ. Driver Oncogenes but Not as We Know Them: Targetable Fusion Genes in Breast Cancer. Cancer Discov. 2018 Mar;8(3):272-275. doi: 10.1158/2159-8290.CD-18-0091. PMID: 29500329; PMCID: PMC6372062.

6. Singh A, Ham J, Po JW, Niles N, Roberts T, Lee CS. The Genomic Landscape of Thyroid Cancer Tumourigenesis and Implications for Immunotherapy. Cells. 2021 May 1;10(5):1082. doi: 10.3390/cells10051082. PMID: 34062862; PMCID: PMC8147376.

7. Sepulveda AR, et al. Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology. J Clin Oncol. 2017 May 1;35(13):1453-1486. doi: 10.1200/JCO.2016.71.9807. Epub 2017 Feb 6. PMID: 28165299.

8. Lindeman NI, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. Arch Pathol Lab Med. 2018 Mar;142(3):321-346. doi: 10.5858/arpa.2017-0388-CP. Epub 2018 Jan 22. PMID: 29355391.

9. Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008 Oct 23;455(7216):1061-8. doi: 10.1038/nature07385. Epub 2008 Sep 4. Erratum in: Nature. 2013 Feb 28;494(7438):506. PMID: 18772890; PMCID: PMC2671642.

10. Lombardi MY, Assem M. Glioblastoma Genomics: A Very Complicated Story. In: De Vleeschouwer S, editor. Glioblastoma [Internet]. Brisbane (AU): Codon Publications; 2017 Sep 27. Chapter 1. PMID: 29251861.

11 .Guhan S, Klebanov N, Tsao H. Melanoma genomics: a state-of-the-art review of practical clinical applications. Br J Dermatol. 2021 Aug;185(2):272-281. doi: 10.1111/bjd.20421. Epub 2021 Jun 6. PMID: 34096042.

12. Tornillo L, Terracciano LM. An update on molecular genetics of gastrointestinal stromal tumours. J Clin Pathol. 2006 Jun;59(6):557-63. doi: 10.1136/jcp.2005.031112. PMID: 16731599; PMCID: PMC1860404.

13. Malone ER, Oliva M, Sabatini PJB, Stockley TL, Siu LL. Molecular profiling for precision cancer therapies. Genome Med. 2020 Jan 14;12(1):8. doi: 10.1186/s13073-019-0703-1. PMID: 31937368; PMCID: PMC6961404.

14. Chakravarty D et al. Somatic Genomic Testing in Patients With Metastatic or Advanced Cancer: ASCO Provisional Clinical Opinion. J Clin Oncol. 2022 Apr 10;40(11):1231-1258. doi: 10.1200/JCO.21.02767. Epub 2022 Feb 17. Erratum in: J Clin Oncol. 2022 Jun 20;40(18):2068. PMID: 35175857.

Clinical Indications

According to available guidelines:

Methodology

Targeted variant analysis is performed using LightMix® and LightSNiP® melt curve technology (TIB MOLBIOL) on the LightCycler480 II (Roche) to identify four alleles of the SERPINA1 gene, PI*S, PI*F, PI*I and PI*Z, which combine to create the genotypes listed in Table 1.

Genomic DNA is isolated from the peripheral blood and four regions containing the variants of interest are amplified by PCR. Following PCR, the amplified DNA sequences are subjected to a temperature gradient, allowing fluorescently labeled probes targeted to each variant to bind to the double stranded DNA sequence and fluoresce. As the temperature increases, the probes dissociate, or “melt”, at a specific temperature based on the nucleotide sequence of the probe and fluorescence decreases. Measurement of fluorescence throughout the gradient allows the specific temperature at which melting occurs to be recorded. Mismatches between the probe and the DNA sequence cause the probe/DNA hybrid to be less stable and the probe to melt at a lower temperature, allowing discrimination between variant and normal.

Interpretation

This laboratory-developed test will not detect other mutations that may cause AATD.

Uncommon variants or polymorphisms in the regions of interest may affect the binding of LightMix® or LightSNiP® probes and may result in a false negative, false positive, or indeterminate result.

Absence of the S, Z, F, and I alleles is consistent with (but does not confirm) the normal, aka wild type, PI*MM genotype.

Importantly, there are over 100 known rare variants of SERPINA1 that are not detected in 201306.081 (1.20 rev) by this PCR test. Therefore, the correlation of the genotype with the patient’s serum alpha-1 antitrypsin level and clinical manifestations is strongly recommended.

When discrepancies exist between the enzyme level and targeted genotype results (e.g., the serum AAT level is low but no abnormal allele is identified by PCR), sequencing of the coding regions of the SERPINA1 gene to identify rare mutations should be considered.

References

1. Bornhorst JA, Greene DN, Ashwood ER, Grenache DG. α1-Antitrypsin phenotypes and associated serum protein concentrations in a large clinical population. Chest. 2013;143:1000-8.

2. Dati F, Schumann G, Thomas L, Aguzzi F, et al. Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges for 14 proteins in serum based on the standardization against the IFCC/BCR/CAP reference material (CRM 70), Eur J Clin Chem Biochem. 1996;34:517-520.

3. Rodriguez-Frias F, Miravitlles M, Vidal R, Camos S, Jardi R. Rare alpha-1-antitrypsin variants: are they really so rare? Ther Adv Respir Dis. 2012 Apr;6(2):79-85.

4. Sandhaus RA, Turino G, Brantly ML, Campos M, Cross CE, Goodman K, Hogarth DK, Knight SL, Stocks JM, Stoller JK, Strange C, Teckman J. “The Diagnosis and Management of Alpha-1 Antitrypsin Deficiency in the Adult.” Chronic Obstr Pulm Dis. 2016 Jun 6;3(3):668-682.

5. Silverman EK, Sandhaus RA. Clinical practice. Alpha 1-antitrypsin deficiency. N Engl J Med. 2009;360(26):2749-57.

6. Sinden NJ, Koura F, Stockley RA. The significance of the F variant of alpha-1 antitrypsin and unique case report of a PiFF homozygote. BMC Pulm Med. 2014 Aug 7;14:132.

7. Stoller JK, Aboussouan LS. A review of α1-antitrypsin deficiency. Am J Respir Crit Care Med. 2012;185(3):246-59.

8. Stoller JK, Lacbawan FL, Aboussouan LS. Alpha-1 Antitrypsin Deficiency. 2006 Oct 27 [Updated 2017 January 19]. In Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews. [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2018. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1519/

Methodology

The IMMULITE^® 2000/2000 XPi TSI assay (Siemens) is FDA-approved for in vitro diagnostic purposes.

The test principle is based on automated, random-access, two-cycle chemiluminescent bridging immunoassay. The solid phase (polystyrene beads) are coated, through a monoclonal Ab, with a chimeric human TSHR (Mc4). In Mc4, the TSH binding site is intact, but its membrane-proximal part has been replaced by rat luteinizing hormone-choriogonadotropin receptor to not only stabilize the molecule so it can maintain its native form but also to preclude “other” TSHR-binding (but non-stimulating) Abs of binding to this chimeric molecule. Mc4 is also used by a commercial bioassay. During the second step, the capture TSHR is added, but the latter is conjugated to alkaline phosphatase (ALP), which will generate the chemiluminescent signal upon the addition of the substrate. The generated photons are read as relative light units, or counts per second, for calculations (Figure).

The kit uses two levels (low, high) of “adjustors” in replicates of four (logistical model). These are m22 Abs, as described above, and are used as calibrators based on which the final TSI concentration is calculated in international units per liter (IU/L).

The calibration is based upon a lot-specific master curve generated by the manufacturer. By using such adjustors and the test design, the stimulatory antibodies are typically measured. Furthermore, it has been demonstrated that stimulatory antibodies, such as m22, are able to bind to the concave region of TSHR LRDs where TSH also binds, whereas the blocking antibodies, such as K1-70, bind to the receptor more N-terminally and 155˚ away from where stimulatory antibodies bind.¹² This key difference helps this kit to significantly avoid bridging the receptors by blocking antibodies due to steric hindrance, the latter of which is further accentuated by the upside-down nature of the signal receptor and presence of the conjugated ALP.

According to the manufacturer, this assay is traceable to WHO 2^nd International Standard for thyroid-stimulating antibody, National Institute for  Biological Standards and Control (NIBSC), code: 08/204.

The kit also has three controls (negative, low, and high-positive) using m22 Ab. The reportable range is 0.10-40 IU/L, but the instrument can do further dilutions to calculate the final concentration. The manufacturer recommends 0.55 IU/L as the positivity cut-off based on which the clinical sensitivity and specificity are 98.6% and 98.5%, respectively. This is congruent with the most recent American Thyroid Association (ATA) guideline that states:

In the setting of overt thyrotoxicosis, newer TRAb binding and bioassays have a sensitivity of 96–97% and a specificity of 99% for GD. ⁷

It is noteworthy to mention that ATA did not specifically mention this test, and by “TRAb”, they referred to the two commercial ELISAs (TBI). TSI Immulite received FDA approval after the guideline was finalized. It was also previously demonstrated that TSI Immulite test had higher sensitivity than bioassay in patients with GD on ATDs.⁹ Furthermore, the manufacturer claims that IMMULITE^® 2000/2000 XPi TSI test results are not affected in sera spiked by high concentrations of FSH, LH, TSH, HCG, anti-thyroglobulin, and anti-thyroid peroxidase.

Last but not least, a bioassay only measures cyclic adenosine monophosphate (cAMP); however, other TSHR activation pathways that do not result in cAMP production, such as phospholipase C cascade, are not assessed using current bioassays.  This is even of more importance when, in Grave’s orbitopathy, IGF-1 receptor forms a complex with TSHR to trigger orbital fibroblasts for glycosaminoglycan production and lipogenesis.⁶

Advantages

The IMMULITE^® 2000/2000 XPi TSI assay analytically and clinically maintains high levels of quality and performance characteristics while significantly lowering the reagent cost and technologist’s time. In comparison, a bioassay requires cell culture, multiple reagents, ancillary tests, and manual calculations, all of which predispose this test to analytical and post-analytical errors, QC failures, lack of amenability to automation, and extreme labor intensiveness.  These factors adversely affect the cross-training of new technologists.

This test is performed at Cleveland Clinic on a random-access platform, Monday through Friday.  Tests can be added at any time during working hours without the need for batch-testing, obviating the prolonged bioassay turnaround time, as the former only takes 65 minutes to complete. This walk-away system immensely spares the laboratory technician’s time.

While Cleveland Clinic currently utilizes the IMMULITE^®2000 system, another random-access, commercially-available platform is Elecsys® Anti-TSHR (Roche). However, in addition to the published reports^{9, 10} that show relatively poor performance, the test principle ultimately makes this option a sub-optimal choice: porcine TSH (instead of the Mc4 construct), murine monoclonal antibodies (that make heterophile Ab interference likely), the utilization of biotin (for conjugation of the latter Abs that make the test prone to falsely-elevated results due to dietary biotin), combined together argue against its usefulness for TSI testing.

Upon discussion with several other laboratories in the United States that have adopted the IMMULITE^® 2000/2000 XPi TSI assay, each expressed high satisfaction.

Validation Summary

To verify the manufacturer’s claims, the TSI Immulite MMULITE® 2000/2000 XPi TSI test was validated in-house within the Immunopathology Laboratory on Cleveland Clinic’s main campus.

The linearity and accuracy study used a neat sample and multiple dilutions up to 1:250 in triplicate. The recovery rate, slope, intercept, and observed error were all within acceptable and established limits. For the precision study, the inter- and intra-run simple and complex precision was performed using low, medium, and high-level samples. The %CV values all were well within acceptable limits, confirming high precision, which is ideal for monitoring patients over time.

The method comparison study was performed in two parts:

• One was performed using known positive sera (70% female, age: 24–78 years) received from ARUP Laboratories.  ARUP utilizes an in-house-developed bioassay. Our laboratories received samples within 128-480% (low to high positive); their test uses 123% as the positivity cut-off.
• The second part was performed using sera received from the Ohio State University Laboratories, as they also utilize the IMMULITE® 2000/2000 XPi TSI assay.
• Both panels showed 100% categorical agreement, confirming comparability.

According to our alternate proficiency testing results, our in-house bioassay had 100% concordance with the ARUP in-house-developed bioassay.

A carryover study used three replicates in low, high, low order, and three replicates of a high positive sample with 1:10 dilution. The results confirmed no significant instrument carryover.

The reference range study used apparently healthy subjects, as none tested positive for TSI (their TSI results were all <0.10 IU/L). This verified the manufacturer positivity cut-off of 0.55 IU/L.

References

1. Frank CU, Braeth S, Dietrich JW, Wanjura D, Loos U. Bridge Technology with TSH Receptor Chimera for Sensitive Direct Detection of TSH Receptor Antibodies Causing Graves’ Disease: Analytical and Clinical Evaluation. Horm Metab Res. 2015 Nov;47(12):880-8. doi: 10.1055/s-0035-1554662. Epub 2015 Jun 16.

2. McLachlan SM, Rapoport B. Thyrotropin-blocking autoantibodies and thyroid-stimulating autoantibodies: potential mechanisms involved in the pendulum swinging from hypothyroidism to hyperthyroidism or vice versa.

3. Nguyen CT, Sasso EB2, Barton L, Mestman JH. Graves’ hyperthyroidism in pregnancy: a clinical review. Clin Diabetes Endocrinol. 2018 Mar 1;4:4. doi: 10.1186/ s40842-018-0054-7. eCollection 2018.

4. Bitcon V, Donnelly J, Kiaei D. Sensitivity of assays for TSH-receptor antibodies. J Endocrinol Invest. 2016 Oct;39(10):1195-6. doi: 10.1007/s40618-016-0520-y. Epub 2016 Aug 16.

5. Tozzoli R, D’Aurizio F, Villalta D, Giovanella L. Evaluation of the first fully automated immunoassay method for the measurement of stimulating TSH receptor autoantibodies in Graves’ disease. Clin Chem Lab Med. 2017 Jan 1;55(1):58-64. doi: 10.1515/cclm-2016-0197.

6. Smith TJ, Hegedüs L. Graves’ Disease. N Engl J Med. 2016 Oct 20;375(16):1552-1565.

7. Ross DS, Burch HB, Cooper DS, Greenlee MC, Laurberg P, Maia AL, Rivkees SA, Samuels M, Sosa JA, Stan MN, Walter MA. 2016 American Thyroid Association Guidelines for Diagnosis and Management of Hyperthyroidism and Other Causes of Thyrotoxicosis. Thyroid. 2016 Oct;26(10):1343-1421.

8. Sanders J, Evans M, Premawardhana LD, Depraetere H, Jeffreys J, Richards T, Furmaniak J, Rees Smith B. Human monoclonal thyroid-stimulating autoantibody. Lancet. 2003 Jul 12;362(9378):126-8.

9. David J. Kemble, Tara Jackson, Mike Morrison, Mark A. Cervinski, Robert D. Nerenz. Analytical and Clinical Validation of Two Commercially Available Immunoassays Used in the Detection of TSHR Antibodies. DOI: 10.1373/jalm.2017.024067 Published October 2017.

10. Y Li, J Kim, T Diana, R Klasen, P D Olivo, and G J Kahaly. A novel bioassay for anti-thyrotrophin receptor autoantibodies detects both thyroid-blocking and stimulating activity. Clin Exp Immunol. 2013 Sep; 173(3): 390–397.

11. Diana T, Krause J, Olivo PD, König J, Kanitz M, Decallonne B, Kahaly GJ. Prevalence and clinical relevance of thyroid-stimulating hormone receptor-blocking antibodies in autoimmune thyroid disease. Clin Exp Immunol. 2017 Sep;189(3):304-309. doi: 10.1111/cei.12980.

12. Sanders P1, Young S, Sanders J, Kabelis K, Baker S, Sullivan A, Evans M, Clark J, Wilmot J, Hu X, Roberts E, Powell M, Núñez Miguel R, Furmaniak J, Rees Smith B. Crystal structure of the TSH receptor (TSHR) bound to a blocking-type TSHR autoantibody. J Mol Endocrinol. 2011 Feb 15;46(2):81-99. doi: 10.1530/JME-10-0127. Print 2011 Apr.

Clinical Indications

Beryllium is a lightweight metal with a high melting point, high strength, and good electrical conductivity. As a result, beryllium has become widely used in a variety of industrial applications, such as thermal coating, nuclear reactors, rocket heat shields, micro-circuits, brakes, X-ray tubes, golf clubs, ceramics, and dental plates. Frequently, it is formulated as an alloy or an oxide.

Correlation between the clinical status of CBD (chronic beryllium disease) and in vitro responses to beryllium in an LPT was developed.² An elevated blood Be-LPT result may indicate beryllium sensitization, but a definitive diagnosis of CBD requires lung biopsy in the context of compatible signs and symptoms and radiological findings; therefore, as a follow-up step, individuals may undergo pulmonary evaluation, including bronchoscopy, trans-bronchial biopsy, or BAL collection for BAL Be-LPT. This is important in the differential diagnosis of CBD, as sarcoidosis can clinically mimic CBD. Extra-pulmonary CBD may also be occasionally seen such as cutaneous nodules.

Methodology

The blood lymphocyte proliferation test for beryllium sensitization (Be-LPT) is measured by radioactive ³H-thymidine uptake in a cell culture system on two different days after exposure to a range of beryllium sulfate to re-stimulate effector memory CD4+ T cells in peripheral blood. As a control, a common mitogen, phytohemagglutinin (PHA), and Candida antigen are used to ensure acceptable and normal T cell proliferative responses. Emitted beta rays are counted by a beta counter instrument in count per minute (CPM), the stimulation index (SI) is calculated and reported out along with an interpretive comment. The results are reviewed by staff.

Interpretation

The S.I. for PHA should be ≥50.0.

The S.I. for Candida albicans should be ≥2.0 to indicate normal T-cell function.

If all six beryllium indices are less than 3.0, this signifies a normal result, meaning no evidence of beryllium sensitization.

Ratios of greater-than-or-equal-to 3.0 in two or more beryllium indices constitute an abnormal response that is compatible with beryllium hypersensitivity.

References

1. McCanlies EC, Kreiss K, Andrew M, Weston A. 2003. HLA-DBP1 and chronic beryllium disease: a HuGE review. Am. J Epidemiology. 157:388-398.

2. Deodhar SD, Barna BP, Van Ordstrand HS. 1973. A study of the immunologic aspects of chronic berylliosis. Chest. 63:309-313.

3. Barna BP, Culver DA, Yen-Lieberman B, Dweik RA, Thomassen MJ. Clinical application of Beryllium Lymphocyte Proliferation Testing. Clin and Diag Lab Immunol. 2003;10:990-994.

4. U. S. Department of Labor, Occupational Safety and Health Administration. Directorate of Science, Technology and Medicine Office of Science and Technology Assessment, Safety and Health Information Bulletin, September 2, 1999.

Clinical Indications

The Symptomatic Patient: This test should be ordered when the cause of vesicular or similar lesion is uncertain.

The Asymptomatic Patient: Asymptomatic vaginal shedding of HSV is a risk factor for the baby during delivery. This test is warranted in pregnant women who are near delivery if there is a concern for the possibility of HSV shedding.

Interpretation

Qualitative test results (i.e. Positive or Negative for HSV1, HSV2, or VZV) will be reported.

Methodology

The Solana HSV 1+2/ VZV Assay is an FDA-approved isothermal amplification assay that detects and differentiates HSV1, HSV2, and VZV. Clinical studies performed at Cleveland Clinic (manuscript in preparation) have demonstrated a 94.7% sensitivity and 100% specificity for the detection of HSV with this assay. The formerly-employed assay (i.e. HSV ELVIS Test System) had a sensitivity and specificity of 71.1% and 93.2%, respectively.

Similarly, the detection of VZV with Solana was superior to direct immunofluorescence (DFA). The sensitivity/ specificity of the Solana assay for the detection of VZV in this study was 95.3%/100%, whereas for DFA these were 71.4%/100%, respectively.

Limitations

This test has limited sensitivity. In rare circumstances, false positives may occur if other intracytoplasmic inclusions are present; however, if the peripheral blood smear is negative, and these diseases are suspected, PCR testing and/or serologic studies are recommended to aid in diagnosis.

References

1. Faron M, Mashock M, Connolly J, Ledeboer N, Buchan B. Evaluation of the Solana HSV 1+2 assay for simultaneous detection of herpes simplex virus 1 and 2, and varicella zoster virus from cutaneous lesions. Session P091; P1894. 27th ECCMID. Vienna, Austria. 22-25 April 2017.

2. Granato PA, Degillo MA, Wilson EM. The unexpected detection of varicella-zoster in genital specimens using the Lyra™ Direct HSV1+2/VZV Assay. J Clin Virol 2016; Nov;84:87-89. doi: 10.1016/j.jcv.2016.10.007. Epub 2016 Oct 11.

3. Huppert JS, Batteiger BE, et al. Use of an Immunochromatographic Assay for Rapid Detection of Trichomonas vaginalis in Vaginal Specimens. Journal of Clinical Microbiology. 2005; 684-687.

4. Solana HSV 1+2/VZV Assay product information and package insert, Quidel, San Diego, CA 92130.

Clinical Indications

A patient with feverish symptoms following tick exposure may suspect ehrlichiosis/anaplasmosis. This test is for individuals who are thought to possibly have ehrlichiosis/ anaplasmosis based on symptoms and clinical presentation.

Morulae may be observed in white blood cells with a peripheral blood smear. Although these intracytoplasmic bodies are not frequently seen, their presence is highly-specific if detected by an experienced microscopist.

Interpretation

A positive result indicates the presence of intracytoplasmic morulae. Determining the type of white blood cell that is infected aids in the differentiation between ehrlichiosis and anaplasmosis.

While the specificity of this assay is high, its sensitivity is limited; a negative result does not exclude the possibility of anaplasmosis or ehrlichiosis.

Methodology

Peripheral blood smear (thin film) review following standard Giemsa staining.

Limitations

This test has limited sensitivity. In rare circumstances, false positives may occur if other intracytoplasmic inclusions are present; however, if the peripheral blood smear is negative, and these diseases are suspected, PCR testing and/or serologic studies are recommended to aid in diagnosis.

References

1. National Institute of Allergy and Infectious Disease. Ehrlichiosis and Anaplasmosis. http://www.niaid.nih.gov/topics/ehrlichiosisanaplasmosis/Pages/Default.aspx. Accessed Feb. 1, 2013.

2. Garcia, Lynne S., ed., et. al. Clinical Microbiology Procedures Handbook, 3rd ed., ASM Press, Washington D.C., 2010, Chapters 2.1.17, 3.4.1.17, 11.7.1-11.7.7.

3. Howard, Barbara J., et. al. Clinical and Pathogenic Microbiology, 2nd ed., Mosby Year Book, St. Louis, 1994, pgs. 859-890.

4. Mahon, Connie R., Textbook of Diagnostic Microbiology, 3rd ed., Saunders Elsevier, St. Louis, Mo., 2007, pgs. 1068-1069.

5. Versalovic, James, ed., et al. Manual of Clinical Microbiology, Vol. 1, 10th edition, ASM Press, Washington D.C., 2011, pgs. 240, 1013-1026.

Clinical Indications

Cleveland Clinic Laboratories offers CEBPA mutation analysis for classification and prognostic assessment of new acute myeloid leukemias, especially those with normal cytogenetics. Concurrent NPM1 and FLT3 studies are also recommended.

Interpretation

Mutations in CEBPA include single and dual (usually biallelic) mutations. Initial studies reported that the presence of any CEBPA mutation was associated with a favorable clinical course, while more recent studies have suggested that the favorable clinical course and distinctive clinicopathologic features are limited to acute myeloid leukemia with dual CEBPA mutations.^[2-6]

All identified mutations are reported.  Cases are classified as wild type (no mutations detected), single mutated, or dual mutated.

Methodology

DNA is extracted from peripheral blood (CEBPA) or bone marrow (CEBPAM). The entire CEBPA coding region is amplified by PCR and analyzed by Sanger sequencing.

Limitations

Sanger sequencing is expected to identify >99% of mutations, provided that mutations represent at least 15-20% of total CEBPA alleles.

This test is not intended for the detection of minimal residual disease.

References

1. Arber DA et al. (2008). Acute myeloid leukaemia with recurrent genetic abnormalities. In: Swerdlow SH, Campo E, Harris NL et al., eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: WHO Press. 110-23.

2. Taskesen E, Bullinger L, Corbacioglu A, et al. Prognostic impact, concurrent genetic mutations and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients: further evidence for CEBPA double mutant AML as a distinctive disease entity. Blood. 2011;117:2469-2475.

3. Green CL, Koo KK, Hills RK, et al. Prognostic significance of CEBPA mutations in a large cohort of younger adult patients with acute myeloid leukemia: impact of double CEBPA mutations and the interaction with FLT3 and NPM1 mutations. J Clin Oncol. 2010;28:2739-47.

4. Dufour A, Schneider F, Metzeler KH, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol. 2010;28:570-7.

5. Pabst T, Eyholzer M, Fos J, et al. Heterogeneity within AML with CEBPA mutations: only CEBPA double mutations, but not single CEBPA mutations are associated with favorable prognosis. Br J Cancer. 2009;100:1343-6.

6. Wouters BJ, Lowenberg B, Erpelinck-Verschueren CA, et al. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood. 2009;113:3088-91.

Clinical Indications

Investigations leading to the diagnosis of systemic amyloidosis can be triggered by a variety of symptoms induced by heart or kidney failure, neuropathy, or coagulation abnormalities. Abnormalities suggestive of cardiac amyloidosis have been described by ultrasonography. Amyloid deposits can be identified during workups for low-grade lymphomas, plasma cell myelomas, familial conditions, or renal failure.

There are four major types of systemic amyloidosis: primary amyloidosis, familial amyloidosis, secondary amyloidosis, and senile amyloidosis. The most common form of amyloidosis, primary amyloidosis, is the consequence of deposits of immunoglobulin light chains. Primary amyloidosis is usually diagnosed in patients with plasma cell neoplasms, from monoclonal gammopathy to plasma cell myeloma. Familial amyloidosis is a consequence of inherited mutations, most common in the transthyretin gene. Transthyretin, this time in its wild-type form, is the main component of amyloid in senile amyloidosis, a condition most often diagnosed in the heart. With the decrease in the incidence of chronic inflammatory diseases, mainly infectious, and with better management of autoimmune disorders, the incidence of secondary amyloidosis has markedly decreased and is currently a rare disease. The main amyloidogenic protein in secondary amyloidosis is Amyloid A. Overall, at least 28 proteins have been described to be responsible for the formation of amyloid deposits.

In addition to the proteins that are considered amyloidogenic, amyloid deposits in all types of systemic amyloidosis constantly include other proteins: Serum P component (SAP), Apolipoprotein E (ApoE), Apolipoprotein A-I (ApoA-I), and Apolipoprotein A-IV (ApoA-IV).

Methodology & Interpretation

In histologic sections stained with hematoxylin-eosin, amyloid deposits can be difficult to differentiate from serum or dense collagen. Several stains have been developed to assist in the diagnosis of amyloid, with the Congo Red stain currently considered the gold standard. The microfibrillar nature of the amyloid deposits can be confidently identified by electron microscopy, while the beta-pleated sheet structure of the amyloid can only be demonstrated with x-ray diffraction, a technique exclusively used in research.

While, from a purely morphologic point of view, there are no significant differences between different types of amyloid, the treatment of each type of amyloidosis is different. Immunohistochemical stains show the amyloid deposits to be positive for SAP, but this does not always allow the differentiation of amyloid from serum. In many cases, stains for Transthyretin, Amyloid A, kappa, or lambda immunoglobulin light chains allow further characterization of the components of the amyloid deposits; however, in a significant number of cases, these techniques fail to identify with confidence the amyloidogenic protein. Factors that prevent a confident diagnosis include abnormal protein folding and truncation, as well as the presence of many other endogenous proteins in the amyloid deposits. This results in a significant fraction of cases being inadequately typed and, as a consequence, treated.

Recent studies have shown liquid chromatography-tandem mass spectrometry (LC-MS/MS) to be a reliable method in the identification of amyloidogenic proteins, and is considered by some groups as the current gold standard. This technique was initially used on fresh or frozen tissue, but recently it has been shown that analysis of formalin- fixed, paraffin-embedded tissue (FFPET) can lead to similar results. The analysis usually begins with visual identification of amyloid deposits, followed by their dissection under the microscope (laser microdissection). The specimen is then digested with trypsin, resulting in the generation of peptide fragments. LC-MS/MS is then used to analyze these peptide fragments, resulting in an m/z spectrum. The specific m/z characteristics of the different peaks are compared to those in several databases, leading to the identification of the peptide fragments in the amyloid digested by trypsin. These peptide fragments are quantified and the higher the number of peptide fragments originating from a particular amyloidogenic protein, the higher the degree of confidence that the particular protein is a component of the amyloid. Peptides from the structures of SAP, Transthyretin, ApoE, ApoAI, and ApoA-IV are used as internal controls, indicating the adequate identification of amyloid. When more than one amyloidogenic protein is identified, a comparison of the relative abundance of peptide fragments can indicate which protein is the most abundant in the sample.

This amyloid typing test allows typing of the amyloid deposits with a precision superior to the other available techniques. It incorporates multiple internal controls and allows amyloid typing to be performed in archived specimens. When necessary, in addition to the LC-MS/MS, alternative techniques, such as immunohistochemical stains, can be employed.

Limitations

This test is not a substitute for a surgical pathology consult.

The diagnosis of amyloidosis should not be made by mass spectrometry, as this method has not been developed as a substitute for a detailed morphologic analysis, Congo Red stain, or immunohistochemistry.

Amyloid analysis by tandem mass spectrometry requires that a sufficient amount of amyloid is microdissected. In a few cases, the assay may fail due to the insufficient amyloid available.

In some cases, additional immunohistochemical stains are necessary in order to increase the confidence with which the diagnosis is rendered.

References

1. Sipe JD, Benson MD, Buxbaum JN, et al. Amyloid fibril protein nomenclature: 2012 recommendations from the Nomenclature Committee of the International Society of Amyloidosis. In Amyloid, 2012; 19(4):167-170.

2. Lavatelli F, Vrana JA. Proteomic typing of amyloid deposits in systemic amyloidoses. In Amyloid, 2011; 18(4):177-182.

3. Vrana JA, Gamez JD, Madden BJ, et al. Classification of amyloidosis by laser microdissection and mass spectrometry-based proteomic analysis in clinical biopsy specimens. In Blood, 2009;114(24)4957-4959.

Limitations

PSA as a marker is only prostatic tissue-specific rather than cancer-specific. Increased PSA in serum was not only detected in patients with prostate cancer, but also in benign prostatic disease.^[1] This phenomenon resulted in a significant false positive rate in PSA-based detection of prostate cancer based on biopsy results from those patients.

Additionally, PSA-based detection of prostate cancer often resulted in over-diagnosis of low-grade cancer that may have never become clinically significant. Several approaches to improve the distinction between cancer and benign conditions have been proposed, including the use of age-adjusted PSA reference ranges, PSA density, PSA velocity, and free-to-total PSA ratio.

Clinical Significance

The traditional cutoff for serum PSA is 4.0 ng/ml. PSA values >4.0 ng/mL were considered abnormal, and these patients were further evaluated with invasive diagnostic approaches, such as prostate biopsy or transrectal ultrasonography of the prostate.^[1] However, multiple studies suggest that the risk of prostate cancer in men with PSA levels <4.0 ng/mL is significant (see Table 1). For example, the prostate cancer prevention trial (PCPT) included 2.950 participants who had PSA levels < 4.0 ng/mL and underwent an end-of-study biopsy. Results showed that more than 15% of these men had prostate cancer.^[5]

The detection rate of prostate cancer is about 47% in men with serum PSA in the range of 4-10 ng/mL.^[5] However, the cancer rate is very similar for men with a PSA range of 2.0-3.0 ng/mL(23%) and that with PSA of 3.0-4.0 ng/ml (26%).

Table 1. Likelihood of Cancer, Based on PSA Level

Data from the prostate cancer prevention trial demonstrated that there is no PSA level below which the risk of having prostate cancer is zero, and suggests that there is no “normal” reference range for PSA. The detection rate of prostate cancer is significantly correlated to the serum PSA levels.

Lowering the PSA cutoff of 4.0 ng/ml to 2.6 ng/ml would increase detection of prostate cancer, while also slightly increasing false positive results.^[6-8]

Test Update Information

The new PSA reference range is 0-2.59 ng/ml; the upper limit is cutoff value for further evaluation.

The following comment will be included with each PSA result:

“For an individual patient, the significance of a PSA level should be interpreted in a broad clinical context, including age, race, family history, digital rectal examination, prostate size, results of prior testing (prostate biopsy, free PSA, PCA3), and use of 5-alpha-reductase inhibitors. Considering the high incidence of asymptomatic cancer in the general population that may not pose an ultimate risk to the patient, the decision to recommend urological evaluation or prostate biopsy should be individualized after considering all of these factors.”

A useful tool that incorporates many of these variables for calculating the risk of cancer is available at myprostatecancerrisk.com. This information may assist physicians in deciding whether a prostate biopsy is appropriate.

References

1. Cooner WH, Mosley BR, Rutherford CL, Jr. et al. Prostate cancer detection in a clinical urological practice by ultrasonography, digital rectal examination and prostate specific antigen. J Urol 1990;143:1146-52; discussion 1152-4.

2. Catalona WJ, Smith DS, Ratliff TL et al. Measurement of prostate-specific antigen in serum as a screening test for prostate cancer. N Engl J Med 1991;324:1156-61.

3. Andriole GL, Crawford ED, Grubb RL 3rd, Buys SS, Chia D et al. [PLCO Project Team]. Mortality results from a randomized prostate-cancer screening trial. N Engl J Med. 2009;360(13):1310-9.

4. Schröder FH, Hugosson J, Roobol MJ et al. ERSPC Investigators. Screening and prostate-cancer mortality in a randomized European study. N Engl J Med. 2009;360(13):1320-8.

5. Thompson IM, Pauler DK, Goodman PJ et al. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med. 2004;350:2239-46.

6. Catalona WJ, Smith DS, Ornstein DK. Prostate cancer detection in men with serum PSA concentrations of 2.6 to 4.0 ng/mL and benign prostate examination. Enhancement of specificity with free PSA measurements. JAMA. 1997;277(18):1452-5.

7. Müntener M, Kunz U, Eichler K et al. Lowering the PSA threshold for prostate biopsy from 4 to 2.5 ng/ml: influence on cancer characteristics and number of men needed to biopsy. Urol Int. 2010;84(2):141-6.

8. Punglia RS, D’Amico AV, Catalona WJ, Roehl KA, Kuntz KM. Effect of verification bias on screening for prostate cancer by measurement of prostate-specific antigen. N Engl J Med. 2003 Jul 24;349(4):335-42.

Clinical Indications

Findings suggestive of community-acquired pneumonia (CAP) include productive cough, fever, pleuritic chest pain, hypoxemia, and an imaging study demonstrating a pulmonary infiltrate.

Microbiologic tests to determine the specific cause of community-acquired pneumonia are often negative; however, if the etiology of CAP is determined, empiric therapy can be optimized to decrease morbidity and mortality. De-escalation of therapy can reduce pressure on the emergence of drug resistance and avoid toxic drug effects.

Sputum and blood cultures (prior to initiation of antimicrobial therapy) are recommended for the evaluation of patients with suspected CAP. Recovery of an organism confirms the diagnosis and allows in vitro susceptibility testing to be performed.

Gram stain of sputum is helpful to guide therapy and for correlation with culture results.

Antigen testing is useful when empiric antimicrobial therapy prevents culture confirmation of pneumococcal disease. Urine antigen testing for S. pneumoniae and Legionella pneumophila are recommended for patients with severe CAP.^[1]

The S. pneumoniae antigen test has a specificity of >90% and a sensitivity of 50-80% for the diagnosis of pneumococcal pneumonia in adults.^[1] However, urinary antigen tests are not recommended for the diagnosis of pneumococcal pneumonia in children because false positive tests are common and attributed to nasopharyngeal colonization with S. pneumoniae.^{[2, 3]}

Methodology

Cleveland Clinic Laboratories uses a qualitative immunochromatographic membrane assay (BinaxNOW^® Streptococcus pneumoniae) to detect pneumococcal soluble antigen in human urine. The S. pneumoniae antigen test provides a rapid, simple method for the presumptive diagnosis of pneumococcal pneumonia.

A urine specimen should be collected prior to antimicrobial therapy in a clean, leak-proof container. The specimen may be stored at room temperature if assayed within 24 hours of collection.

Urine is stable for two weeks if refrigerated or frozen. Boric acid may be used as a preservative; however, other preservatives are unacceptable.

Interpretation & Limitations of the Assay

A positive result is presumptive evidence of pneumococcal pneumonia. Correlation of test results with clinical findings is required.

A negative result does not exclude infection by S. pneumoniae since the antigen present in the sample may be below the detection limit of the test.

This test has not been evaluated on patients taking antibiotics for more than one day, or on patients who recently completed a course of antibiotic therapy.^[4]

Cross-reactivity with closely-related bacteria in the Streptococcus mitis group may occur.

Streptococcus pneumoniae vaccine may cause false positive results within two days following vaccination, and is not recommended within five days following pneumococcal vaccination.

This test has only been validated for urine samples.

Antigen testing is not recommended for the diagnosis of pneumococcal pneumonia in children.^{[2, 3]}

References

1. Mandell LA, Wunderink RG, Anzueto A, et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis. 2007 Mar 1; 44 (Suppl 2):S27-72.

2. Dowell SF, Garman RL, Liu G, et al. Evaluation of Binax NOW, an assay for the detection of pneumococcal antigen in urine samples, performed among pediatric patients. Clin Infect Dis. 2001;32:824-825.

3. Bradley JS, Byington CL, Shah SS, et al. The management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the Pediatric Infectious Diseases Society and the Infectious Diseases Society of America. Clin Infect Dis. 2011;53(t):e25-e76.

4. BinaxNOW® Streptococcus pneumoniae, Package Insert, Alere, Ref. IN710050, Rev. 3, 5/7/10.