Services
Molecular Analysis
1. Molecular Diagnostic Test Menu
Turn-Around-Time: 24 - 48 hrs
| Test | Clinical Application | Methodology | CPT Codes |
|---|---|---|---|
| B-cell Gene Rearrangement (IgH)+ | Monoclonality detection and confirmation in B-cell malignancies. Monoclonality profiling for disease monitoring. | PCR | 83891, 83896, 83900, 83901, 83909, 83912 |
| B-cell Gene Rearrangement (IgK)+ | For 10 % of B-cell malignancies not detected by IgH assay (germinal and post-germinal center with somatic hypermutations). | PCR | 83891, 83896, 83900, 83901, 83909, 83912 |
| T-cell Gene Rearrangement+ | Monoclonality detection and confirmation in T-cell malignancies. Monoclonality profiling for disease monitoring. | PCR | 83891, 83896, 83900, 83901, 83909, 83912 |
| BCL-2 t(14;18)+ | Quantitative monitoring of t(14;18) for Follicular Lymphoma. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83912 |
| BCL-1 t(11;14)+ | Quantitative monitoring of t(11;14) for Mantle Cell Lymphoma. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83912 |
| ALL Panel * | Detection of BCR-ABL, E2A-PBX1, MLL-AF4 and TEL-AML1 | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| E2A-PBX1 t(1;19) * | Minimal residual disease (MRD) detection of t(1;19) for ALL. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| MLL-AF4 t(4;11) * | MRD detection of t(4;11)and prognostic indicator for ALL. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| TEL-AML1 t(12;21) * | MRD detection of t(12;21)and prognostic indicator for ALL. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| BCR-ABL t(9;22) * | Detection and quantitative monitoring of t(9;22) for CML and ALL. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| AML Panel * | Detection of PML-RARA, AML1-ETO and CBFB-MYH11 | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| PML-RARA t(15;17) * | Detection and quantitative monitoring of t(15;17) for APL. STAT same day analysis available for diagnostic specimens. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| AML 1-ETO t(8;21) * | Detection and monitoring of t(8;21). Favorable prognosis in AML. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| CBFB-MYH11 inv(16) * | Detection and monitoring of inv(16). Favorable prognosis in AML. | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| FIPILI-PDGFRA del(4q12) * | Detection and monitoring of del(4q12) in HES (hypereosinophilic syndrome) and CEL (chronic eosinophilic leukemia). | Real-Time Quantitative PCR | 83891, 83896, 83900, 83901, 83902, 83912, 83913 |
| JAK2+ | V617F JAK2 (Janus kinase 2) point mutation detection for diagnosis of myeloproliferative disorders. High detection sensitivity (0.1 %). Heterozygous versus Homozygous profile detection. | Activating Point Mutation Assay | 83891, 83892, 83896, 83898, 83909, 83912 |
| c-KIT D816V+ | Detection of the D816V c-KIT point mutation as prognostic indicator for core binding factor (CBF) AML, mastocytosis, and systemic mast cell disease (SMCD). | Activating Point Mutation Assay | 83891, 83892, 83896, 83900, 83901, 83909, 83912 |
| MSI+ | Microsatellite Instability for Hereditary non-polyposis colorectal cancer (HNPCC) and sporadic colorectal cancers. | 88380, 83907 83891, 83900, 83901, 83896, 83909, 83912 | |
| CLL IGHV Mutation Analysis |
The lack of hypermutations in the IGHV gene detected by sequence analysis is predictive of poor prognosis. |
* RNA-based assays = please provide minimum of 5 mL peripheral blood or 3 mL of bone marrow in lavender top tube [EDTA]. Overnight shipping and processing within 24 h required for accurate results.
+DNA-based assays = Fresh or paraffin-embedded specimens. FFPE bone marrow biopsies can result in inadequate DNA due to decalcification treatment [clot sections preferable].
For MSI analysis, please submit normal and tumor tissue
2. B- and T-cell gene rearrangement PCR
HematoLogics Inc. offers B-Cell (IGH & IGK) and T-Cell Receptor (TCRG) Gene rearrangement assays to detect monoclonal cell populations in peripheral blood, bone marrow, body fluids and tissues (fresh or paraffin-embedded).
- Unsurpassed 24 - 48 h turn-around time.
- Highly sensitive fluorescence based capillary electrophoresis fragment analysis allows monitoring of individual tumors.
- Optimized and standardized multiplex PCR protocols for clonal gene rearrangment detection by the BIOMED-2 Concerted Action [van Dongen et al. Leukemia, 17:2257-2317, 2003] with unprecedentedly high detection rate.
- Immunoglobulin Light Chain Kappa (IGK) available in addition to the Immunoglobulin Heavy Chain (IGH) gene rearrangement assay. IGK analysis can detect B-cell proliferations in which somatic hypermutations [germinal center and postgerminal center B-cell malignancies] may hamper amplification of the IGH loci.
- Only one specimen for High Resolution Flow Cytometry™ and Molecular Analysis.
- Integrated results for High Resolution Flow Cytometry™ and Molecular Analysis.
- Cell Sorting combined with Molecular Analysis can be used for Minimal Residual Disease detection and for confirmatory testing in difficult cases.
Monoclonal Peaks detected with Immunoglobulin Heavy Chain conserved framework (FR) primers at 340 bp for FR1 and 273 bp for FR2. [blue=FR1; black=FR2; green=FR3; red=size standard] in lymph node specimen suspicious for lymphoma.
BCL-2, BCL-1, ALL Panel, BCR-ABL, AML panel, PML-RARA, and FIPILI-PDGFRA
3. Quantitative RQ-PCR assays
HematoLogics Inc. offers a comprehensive test menu of quantitative PCR assays for detection and monitoring of chromosomal translocations.
BCL-2 t(14;18) (monitoring, follicular lymphoma)
Quantitative real-time polymerase chain reaction (PCR) assays with primers for the BCL2 MBR-JH and the BCL2 mcr-JH rearrangement regions are used for specific amplification of the t(14;18) genomic translocations. In approximately 70 % of follicular lymphoma cases the t(14;18) translocation can be detected by PCR and consequently this assay can be used to monitor minimal residual disease.
BCL-1 t(11;14) (monitoring, mantle cell lymphoma)
A quantitative real-time polymerase chain reaction (PCR) assay with primers for the BCL1 MTC-JH rearrangement region are used for specific amplification of the t(11;14) genomic translocation. In approximately 50 % of mantle cell lymphoma cases the t(11;14) translocation can be detected by PCR and consequently this assay can be used to monitor minimal residual disease.
ALL Panel
- Bcr-Abl t(9;22)
- E2A-PBX1 t(1;19)
- MLL-AF4 t(4;11)
- TEL-AML1 t(12;21)
Quantitative molecular analyses of acute lymphoblastic leukemia can identify chromosomal translocations useful for sensitive disease monitoring and to provide independent prognostic information for treatment strategies. HematoLogics utilizes real-time quantitative polymerase chain reaction (RQ-PCR) assays to detect fusion transcripts which are associated with the presence of the t(9;22), t(1;19), t(4;11) and t(12;21) translocations. All translocation assays can be used individually.
Bcr-Abl t(9;22)
Utilizes real-time quantitative polymerase chain reaction (RQ-PCR) to detect BCR/ABL fusion transcripts which are associated with the presence of t(9;22) Philadelphia (Ph) translocation resulting in a small derivative chromosome 22 known as Ph associated with CML, ALL and/or AML.
This test can detect the m-bcr (minor breakpoint cluster region) e1-a2 transcript encoding the 190 kDA (p190) protein and the M-bcr (major breakpoint cluster region) b2a2 (e13a2) and b3a2 (e14a2) transcripts encoding the 210 kDA (p210) chimeric tyrosine kinase protein with a sensitivity level of approximately > 1 in 10e5 transcripts (0.001 %). Quantitative assay units are reported according to the ‘Europe Against Cancer Program’ (EAC) standardized protocol [Gabert J et al. Leukemia 2003 (17): 2318-2357] and can be used for treatment monitoring.
 |
Quantitative Analysis of BCR/ABL expression levels of different K562 cell dilutions. 10e-1 (red) |
AML Panel
- PML-RARA t(15;17)
- AML1-ETO t(8;21)
- CBFB-MYH11 inv(16)
HematoLogics offers quantitative molecular analyses of acute myeloid leukemia to identify chromosomal translocations useful for sensitive disease monitoring and to provide independent prognostic information for treatment strategies. Our laboratory utilizes real-time quantitative polymerase chain reaction (RQ-PCR) assays to detect fusion transcripts which are associated with the presence of the t(15;17) + t(8;21) + inv(16) translocations. All translocation assays can be used individually.
PML-RARA t(15;17)
Utilizes real-time quantitative polymerase chain reaction (RQ-PCR) to detect PML/RARA fusion transcripts which are the molecular result of the t(15;17) translocation associated with the majority of APL cases, a distinct AML subset with M3 cytomorphology. This test can detect all three possible PML-RARA isoforms, referred to as long (L, or bcr1), variant (V, or bcr2) and short (S, or bcr3) with a sensitivity of at least 1 in 10e4 transcripts (0.01 %). Quantitative assay units are reported according to the ‘Europe Against Cancer Program’ (EAC) standardized protocol [Gabert J et al. Leukemia 2003 (17): 2318-2357] and can be used for treatment monitoring.
FIP1L1-PDGFRA del(4q12)
Utilizes real-time quantitative polymerase chain reaction (RQ-PCR) to detect FIP1L1-PDGFRA fusion transcript, which is associated with the presence of an interstitial deletion on chromosome 4q12.
The identification of the FIP1L1-PDGFRA fusion transcript may assist diagnosis, classification and monitoring of hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL).
4. ABL Kinase Domain Mutation Analysis in CML
Detection and Monitoring
TAT: 7-12 days
BACKGROUND AND CLINICAL SIGNIFICANCE:
The hallmark genomic abnormality of chronic myelogenous leukemia (CML) is caused by a translocation between the long arms of chromosomes 9 and 22 (Philadelphia Chromosome), resulting in a hybrid gene, BCR/ABL. Gleevec® (imatinib), a tyrosine kinase inhibitor, is the current first choice therapy for patients suffering from CML. However, a sub-set of patients who initially respond to Gleevec relapse after a period of time. The emergence of resistant leukemia clones bearing mutations in the BCR-ABL kinase domain (KD) represents a major mechanism of disease recurrence that can be treated by changing therapy. As alternative therapies are available for imatinib resistance, early identification of mutations may prevent disease progression. ABL kinase domain sequence analysis has been previously recommended for patients with significant BCR-ABL RNA increase (> 3-fold) in order to identify imatinib mesylate (Gleevec) resistance mutations [Press et al. Blood 2009 p.2598-2605]. To date, more than 50 different KD mutations, conferring variable degrees of imatinib resistance, have been described and can be detected by this analysis.
METHODOLOGY:
This assay analyzes the patient-specific BCR/ABL fusion transcript sequence for the presence of resistance mutations. Polymerase chain reaction (PCR) of the BCR/ABL kinase domain followed by DNA sequencing using overlapping primer sets is used and mutations are identified by comparison analysis to the un-mutated kinase domain sequence. A mutation must be present in approximately 20% of the cell population to be detected by this assay.
Specimen requirements:
- Peripheral blood or bone marrow sent for flow cytometry analysis (green top, heparin)
- 2 mL of peripheral blood or 1 mL of bone marrow (lavender top, EDTA) for molecular analysis only.
References:
Jones, D. et al, Laboratory Practice Guidelines for Detecting and Reporting BCR-ABL Drug Resistance Mutations in
Chronic Myelogenous Leukemia and Acute Lymphoblastic Leukemia, Journal of Molecular Diagnostics, Vol. 11, No. 1,
January 2009
Branford et al., Detection of BCR-ABL mutations in patients with CMLtreated with imatinib is virtually always accompanied by
clinical resistance, and mutations in the ATPphosphate-binding loop (P-loop) are associated with a poor prognosis, Blood, Vol.
102, No. 1, July 2003
5. Activating Point Mutation Assays
Jak2 (MPD)
The V617F mutation of the JAK2 (Janus kinase 2) gene has been described in 74 – 97 % of polycythemia vera (PV), in 33 – 57 % of essential thrombocythemia (ET) and in 35 – 50 % of idiopathic myelofibrosis (IMF) cases [Baxter et al. The Lancet 2005: 1054 – 1061] [Levine et al. Cancer Cell 2005: 387-397]. The identification of the V617F JAK2 point mutation in myeloproliferative disorders (MPD) is useful to assist diagnosis, classification and monitoring.
c-KIT (Mastocytosis, AML)
The D816V c-Kit point mutation has been associated as a prognostic indicator with shorter event-free survival in core binding factor (CBF) acute myeloid leukemia (AML) [Boissel N et al. Leukemia 2006]. In mastocytosis the c-Kit mutation has been associated with both aggressive systemic disease and increased bone marrow mast cell content [Pardanani et al. Leukemia Research 27 2003: 739-742]. In addition, it has been shown that the D816V c-kit activation loop mutation is highly resistant to Imatinib (Gleevec). Therefore identification of this mutation might be informative for therapeutic decisions in systemic mast cell disease (SMCD) and acute myeloid leukemia (AML) [Krystal GW, Leuk Res 2004: S53-S59; Growney JD et al. Blood 2005 106(2): 721-4].
6. Microsatellite Instability (MSI)
MSI detection can be helpful in identifying patients with hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome). In addition, microsatellite instability has also been reported for approximately 15 % of sporadic colorectal cancers. Determination of MSI status in sporadic cancers might be useful for establishing prognosis and may predict the benefit from certain chemotherapeutic regimens [Benatti et al. Clin Cancer Res 2005,11(23):-8332-40; Ribic et al. N Engl J Med 2003,349(3):247-57].
7. CLL IGHV Mutation Analysis
The determination of the mutational status of rearranged immunoglobulin heavy chain variable (IGHV) genes in patients with chronic lymphocytic leukemia (CLL) has shown strong and independent prognostic value. The lack of hypermutations in the IGHV gene detected by sequence analysis is predictive of a poor prognosis.
8. Retrospect™ DNA Archiving Service
HematoLogics will archive DNA for future Molecular Analysis purposes for all diagnostic B- and T- cell tumor specimens sent for Flow Cytometry analysis at no extra cost.
Tumor clonality fingerprints can be identified by gene rearrangement analysis from diagnostic DNA specimens. Clonality fingerprints can be used for patient specific analysis of follow-up specimens during treatment monitoring or in the case of a suspected relapse. In addition, patient specific tumor clonality fingerprints allow monitoring at sensitivity levels below 0.01 % by combining flow cytometry cell sorting and gene rearrangement analysis.
9. FISH Probes / Panels by Disease Application
| FISH Panels | ||
| Panel | Probes | Validated for use in Paraffin |
| AML Panel | AML1/ETO, PML/RARA, CBFB, MLL, D7S522,D8Z2 | |
| B-Cell Lymphoma Panel | BCL6, MALT, BCL1 (CCND1), BCL2 | x |
| Burkitt's Lymphoma Panel | MYC/IGH | x |
| CML Panel | BCR/ABL ASS | |
| CLL Panel | D13S319/LAMP1, CEP12, ATM, TP53 | |
| Standard ALL Panel | MLL, TEL/AML1 (ETV6/RUNX1), BCR/ABL1, p16/CEP9, IGH/MYC/CEP8 | |
| MDS Panel | EGR1/D5S23, D7S522/CEP7, D20S108/CEP8, TP53/CEP17 | |
| MM/MGUS Panel | TP53/CEP17, FGFR3/IGH, D13S319/13q34, MLL | |
| Hypereosinophilia/Eosinophilia | PDGFRA/CHIC2 | |
| MPD/CMML (PDGFRB)* | PDGFRB | |
| ALL Panel Pediatric Add On | Cen4/Cen10/Cen17, PBX1/TCF3 | |
| Single FISH Probes | |||
| Gene Rearrangement Translocation | Probes Location | Disease Purpose | Validated for use in Paraffin |
| 1p/19q | Glioma, astrocytoma | x |
|
| 258B3*/MYB/252P19* | 6q13/6q23/6q27 | Lymphoma, Myeloma, Waldenstrom's | |
| ALK/NPM | t(2;5) | Anaplastic Large Cell Lymphoma | x |
| AML1/ETO (CBFA2/MTG8) | t(8;21) | AML M2 | |
| BCL6 | 3q27 | DLBCL | x |
| BCL2/IGH | t(14;18) | Follicular or DLBCL | x |
| BCR/ABL1 | t(9;22) | CML, AML or ALL | |
| CBFB | 16q22 | AMLM2/M4 | |
| CCND1/IGH(BCL1/IGH) | t(11;14) | Mantle Cell Lymphoma, Myeloma | x |
| CHOP | t(12;16) or t(12;22) | Liposarcoma | x |
| D13S25/D13S319 | 13q14 | CLL, MM, MPD deletion | |
| D20S108 | 20q12 | Myeloid Disorders Del 20q | |
| D7S486/CEP7 | 7q31 | Myeloid Disorders Del 7q/monosomy 7 | |
| DEK/CAN* | t(6;9) | AML | |
| CHIC2 * | 4q12 deletion | Hypereosinophilia/mast cell disease | |
| EGFR | 7p11-p12 | Non-small cell lung cancer, glioma aplification status | x |
| EGR1/D5S23 | 5q31 | Myeloid disorders deletion 5q/monosomy 5 | |
| ELL/ENL* | t(11;19) | ALL or AML Identify gene on 19p involved in t(11;19) | |
| EVI1 | t(3q26) | Myeloid Leukemia | |
| ETV6/RUNX1 (TEL/AML1) | t(12;21) (cryptic) | Pediatric precursor B-ALL | |
| EWSR 1 | t(11;22) or varient | Ewing Sarcoma | x |
| FGFR3/IGH | t(4:14) | Multiple Myeloma | |
| FKHR | t(2;13) or varient | Rhabdomyosarcoma | x |
| IGH break-apart | IGH gene rearrangement | Lymphoma, MM, HCL | x |
| MAF/IGH | t(14;16) | Multiple Myeloma | |
| MALT1 | t(11;18) or varient | MALT Lymphoma | x |
| MLL | t(11q23/_) | Mixed lineage, AML, ALL, deletion, rearrangment or amplification | |
| MYC break apart | 8q24 | lymphoma, leukemias: amplification, rearrangement | |
| MYC/IGH | t(8;14) | Burkitt's lymphoma | x |
| MYCN | 2p23-24 | Neuroblastoma amplification status | x |
| NUP98 | 11p15 | AML | |
| PAX5* | 9p13 | ALL, Lymphoma | x |
| PDGFRB* | 5q33 | Myeloproliferative disease | |
| PML/RARA | t(15;17) | AML M3 | |
| PTEN | 10q | Glioma | x |
| RB1 | 13q14 | Retinoblastoma deletion | |
| SIL-TAL 1 | 1p32 | T-cell ALL | |
| SHPRH*/MYB/CEP6 | 6q24/6q23 | Myeloma, Waldenstrom's | |
| SYT | t(X;18) | Synovial Sarcoma | x |
| TCRTM* | 14q11.2 | T-cell ALL, PLL | |
| TP53 | 17p13 | Solid tumors, lymphoid or myeloid leukemias, MM | |
| TP58 | 1p36 | Neuroblastoma, glioblastoma deletion 1p36 | x |
| TP16 | 9p21 | T-cell lymphoblastic leukemia, pediatric ALL | |
| WT1* | 11p15 | Wilms tumor | x |
| CEP 12 | 12q10 | CLL | |
| ATM | 11q22.3 | CLL | |
| CEP 4 | 4q10 | Pediatric precursor B-ALL | |
| CEP 10 | 10q10 | Pediatric precursor B-ALL | |
| CEP 17 | 17q10 | Pediatric precursor B-ALL | |
| CEP9 | 9q10 | lymphoid or myeloid leukemia, MDS | |
| CEP8 | 8q10 | Myeloid leukemia or MDS | |
| CEP7 | 7q10 | Myeloid or lymphoid leukemia, NHL | |
| LSI21 | 21q22 | Myeloid or lymphoid leukemia, NHL | |
| CEP X and Y | Xq10, Yq10 | post gender mis-matched BMT or SCT | |
Chromosone Enumeration Probes: Centromere probe for chormosomes 7, 8, 9, 12, 21, X and Y are the most useful for assessment of clinically relevant chromosome gain/loss of these chromosomes in hematologic malignancies and for following patients with sex-mismatched BMT.
* ASR Probes