Cytogenetics, FISH, and PCR Testing for Hematopathology Samples

UT-Medical School at Houston, Pathology/ Last Revision on: 7/14/2020



 

NOTES: for patient already in remission, we will just order tests to cover known positive abnormalities from previous diagnostic bone marrow (translocations, mutations, etc). Only for patients still refractory or with relapse we would order all tests as for new cases to cover any genomic evolution that yield new abnormalities.

 

1. AML

 (a) Sample in green-top tube for

-Oncology Chromosome Analysis

-Rapid AML Therapeutic Panel (under Molecular Genetics):

(i)Sanger sequencing (6 genes): FLT3 (ITD and TKD), NPM1, CEBPA, IDH1/IDH2, and TP53

(ii)FISH probes: 5q-, -5 (5p15, 5q31, 5q33) | 7q-, -7 (Cen 7, 7q22, 7q31) | RUNX1/RUNX1T1 (ETO/AML1) t(8;21) | MLL (11q23) | PML/RARA t(15;17) | CBFB inv(16), t(16;16) | 17p- (TP53 17p13.1, NF1 17q11.2)

 

(b) Sample in lavender-top tube for quantitative PCR testing:

-BCR/ABL1 Standard p210,p190

-PML-RARA Translocation, t(15;17) (Quantitative PML/RARA) for diagnostic and follow-up bone marrows with APL cases

-For AML cases with known t(8;21) or inv(16), typically by FISH from baseline BM, we will send for Quantitative PCR with f/u BMs; i.e. RUNX1-RUNX1T1 (AML1-ETO) Translocation, t(8;21). Send specimen (purple-top tube) for this.

 

-Note: for difficult APL cases, call Neogenomics to request for a STAT FISH testing for the PML/RARA component. It can be done on the same day of Neogenomics receiving the sample in their lab. The other FISH tests in the AML panel can be completed later. Note STAT along with MD contact name and phone number to receive STAT results.

 

 

2. ALL

(a) Sample in green-top tube for

-Oncology Chromosome Analysis

-For adult: ALL, Adult FISH Panel [TCF3/PBX1 (E2A/PBX1) t(1;19) | Trisomy or Tetrasomy 4, 6, 10, 17 (Cen 4, Cen 6, Cen 10, Cen 17) | MYC (8q24) | BCR/ABL1/ASS1 t(9;22) | MLL (11q23) | IgH (14q32) |]

-For pediatric: ALL, Pediatric FISH Panel [TCF3/PBX1 (E2A/PBX1) t(1;19) | Trisomy or Tetrasomy 4, 6, 10, 17 (Cen 4, Cen 6, Cen 10, Cen 17) | MYC (8q24) | BCR/ABL1/ASS1 t(9;22) | MLL (11q23) | ETV6/RUNX1 (TEL/AML1) t(12;21) | IgH (14q32)]. Note that we need to write down name of this test in request slip.

 

-For bcr-abl1-like ALL: ALL, Ph-like  [PDGFRb (5q32), BCR/ABL1-ASS1 t(9;22), JAK2 (9p24.1), EPOR (19p13.2) and CRLF2 (Xp22.33/Yp11.32)]

 

Note: STAT processing is available by request for BCR-ABL1. Note STAT along with MD contact name and phone number to receive STAT results.

 

b) Sample in lavender-top tube for quantitative PCR testing:

-BCR/ABL1 Standard p210,p190

 

3. CML

(a) Sample in green-top tube for

-Oncology Chromosome Analysis

 

(b) Sample in lavender-top tube for quantitative PCR testing:

-BCR/ABL1 Standard p210,p190 (diagnostic and follow-up bone marrows)

 

(c) For detection of resistance to Imatinib Mesylate if clinically indicated: Abl1 Kinase Domain (Gleevec Resistance), sample in purple-top tube for PCR

 

4. PV

(a) Sample in green-top tube for

-Oncology Chromosome Analysis

 

(b) Sample in lavender tube for PCR: JAK2 V617F qualitative (check box “if negative reflex to JAK2 Exon 12-14 mutation”)

 

5. ET, PM, MPN-NOS, MPN/MDS

(a) Sample in green-top tube for

-Oncology Chromosome Analysis

-MPN FISH panel: PDGFRa, CHIC2, FIP1L1 (4q12) | PDGFRb (5q33) | FGFR1 (8p11) | BCR/ABL1 t(9;22) including ASS1 (9q34)

(b) Sample in lavender tube for MPN Extended Reflex Panel:

JAK2 V617F, JAK2 Exon 12-14, CALR exon 9, and MPL exon 10.

 

6. Myeloid and Lymphoid Neoplasms with Eosinophilia

Sample in green-top tube for

-Oncology Chromosome Analysis

-Eosinophilia FISH profile: PDGFRa, CHIC2, FIP1L1 (4q12) | PDGFRb (5q33) | FGFR1 (8p11) | CBFB inv(16), t(16;16) |

 

7. PLASMA CELL MYELOMA

Sample in green top tube for

-Oncology Chromosome Analysis

 

-Plasma cell myeloma FISH panel [1p-, 1q+, iso(1q): CDKN2C (1p32), CKS1B (1q21) | +5, hyperdiploidy (5p15) | +9, hyperdiploidy (9q22) | +15, hyperdiploidy (15q22) | 13q- (13q14, 13q34) | IgH (14q32) | 17p- (TP53 17p13.1, NF1 17q11.2)]; check “with reflex to IgH Complex if positive [FGFR3/IgH t(4;14) | CCND1/IgH t(11;14) | IgH/MAF t(14;16) | IgH/MAFB t(14;20)],

 

8. CLL

(a) Sample in green top tube for

-Oncology Chromosome Analysis

-CLL FISH Profile: 6q- [SEC63 (6q21), MYB (6q23)] | ATM (11q22.3) | p53 (17p13.1) | Trisomy 12 (Cen 12) | 13q-/-13 (13q14, 13q34) | CCND1/IgH t(11;14)

 

(b) Sample in lavender-top (baseline bone marrow only) for

-IgVH Hypermutation analysis

 

9. MDS

Sample in green top tube for

-Oncology Chromosome Analysis

-MDS FISH standard panel: 5q-, -5 (5p15, 5q31, 5q33) | 7q-, -7 (Cen 7, 7q22, 7q31) | Trisomy 8 (Cen 8) | MLL (11q23) | 20q- (20q12, 20qter)

 

10. B cell Non-Hodgkin Lymphoma FISH Panel, if needed for diagnosis

(bone marrow aspirate or unstained slides of bone marrow clot)

-Low grade B cell: Low grade/small B cell lymphoma FISH panel: BCL6 (3q27) | CCND1/IgH t(11;14) IgH/BCL2 t(14;18) | MALT1 (18q21)

-High grade B cell: High grade/large B cell lymphoma FISH panel:  BCL6 (3q27) | MYC (8q24) | BCL2 (18q21) |. Note that this B-NHL High Grade FISH profile is needed for large B cell lymphoma that meets one or more of the following criteria:

(a)           Ki67 ≥70%

(b)           Germinal center type (based on Hans algorithm with IHCs)

(c)           Positive cMyc by IHC (≥40% of cells)

These cases also need to have the following IHCs: bcl2, bcl6, cMyc to report double/triple expressors

11. (Neotype) Myeloid Profile, if needed for diagnosis of MDS; not universally adopted

Sample in lavender-top tube for Next-Gen Sequencing testing:

ABL1, ASXL1, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CEBPA, CSF3R, CUX1, DNMT3A, DDX41, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, GNB1, HRAS, IDH1, IDH2, IKZF1, JAK2 including V617F and Exons 12+14, JAK3, KDM6A, KIT, KRAS, MLL, MPL, MYD88, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PML, PPM1D, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG2, STAT3, STAT5B, TET2, TP53, U2AF1, WT1, ZRSR2.

12. LPL

Sample in lavender-top tube for PCR testing: MYD88

 

13. HCL

(a) Sample in green top tube for

-Oncology Chromosome Analysis

 

(b) Sample in lavender-top for PCR

-BRAF

 

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SOLID TISSUE (lymph node, bone marrow clot)

Note that decalcified bone marrow biopsy is not accepted

 

(A) FISH sample for solid tissue:

-Unstained slides at 5 micron thickness

-Minimum 2 slides for each probe; add 2 extra slides

 

(B) PCR sample for solid tissue:

-Unstained slides at 10 micron thickness

-Minimum 2 slides for each probe; add 2 extra slides

 

(C) EBER sample for solid tissue (technical only, to be read by us):

-3 unstained slides at 5 micron thickness

 

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NEOGENOMICS

Account Number: 17336

·       Account Name: Memorial Hermann – Texas Medical Center

·       Client Service Phone: 866.776.5907, option 3 and email: client.services@neogenomics.com

·       Client Service Fax: 239.690.4237

·       NeoLink Online Portal Access:  https://neolink.neogenomics.com/APvX/security/login.aspx 

·       Test Menu Link: www.neogenomics.com/test-menu

·       Requisition Forms:

o   Heme: https://www.neogenomics.com/sites/default/files/Requisition/Neo-Hematopathology-Requisition.pdf

o   Solid Tumor: https://www.neogenomics.com/sites/default/files/2020-06/Neo_Solid-Tumor-Req_062620_Final_FF.pdf

o   IHC: https://www.neogenomics.com/sites/default/files/2019-07/Neo-IHC-Req.PDF