DNA-FISH Probe Catalog Empowering Personalized Cancer Treatment.

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1 Hematologic Diseases ALCL ALL AML CLL CML MM MDS NHL Solid Tumors Empowering Personalized Cancer Treatment Breast Colon/Rectal Cervical Lung DNA-FISH Probe Catalog

2 Visit Visit our website and keep abreast of our latest product releases, access complete information about our product portfolio, and learn about our R&D pipeline.

3 About CGI Italia Cancer Genetics Italia S.r.l. (Italia) was founded in 2009 and is a wholly owned subsidiary of Cancer Genetics Inc. (CGI), which is located in Rutherford, New Jersey (U.S.A.). CGI was founded in 1999 by worldrenowned human geneticist R.S.K. Chaganti, Ph.D., who is currently a senior faculty member at New York City s Memorial Sloan-Kettering Cancer Center. CGI s affiliation with major cancer centers, such as the Memorial Sloan-Kettering Cancer Center, the National Cancer Institute, and the Mayo Clinic, along with the firm s foundation in world-class scientific knowledge has enabled CGI to develop strong intellectual properties (IP) in solid and blood-borne cancers. It is from this foundation that CGI Italia draws its ability to design and manufacture proprietary DNA- FISH Probes. Our DNA-FISH Probes detect a range of genetic aberrations such as translocations and copy number changes and are protected by the following IPs: U. S. Patent (#7,585,964, Publication: pending) entitled Methods of Analyzing Chromosomal Translocations using Fluorescence in situ Hybridization (FISH). U. S. Patent (#11/932,422, Publication: 30 May 2009) entitled Panel for the Detection and Differentiation of Renal Cortical Neoplasms. EU Patent (# , Publication: 30 May 2009) entitled Panel for the Detection and Differentiation of Renal Cortical Neoplasms. U. S. Patent (#61/380,894, Publication: pending) entitled Panel for the Detection and Classification of HPV Associated Carcinoma Cells. Our products are marketed globally, except within the United States, and are provided in a ready-to-use format as Research Use Only (RUO) or CE marked. Mission Statement Our mission is to provide professionals with robustly designed DNA-FISH Probes that are highly specific, easy to interpret, and possess strong signal quality, for the clinical management of cancer patients.

4 What is FISH? Fluorescence in situ hybridization (FISH) is a sensitive and accurate technique that enables the detection of chromosomal aberrations and is complementary to conventional cytogenetic analysis. The method entails hybridization of a single-stranded fluorescently labeled nucleic acid sequence (probe), which is complementary to a target genomic sequence that is present in metaphase chromosomes as well as interphase nuclei and is able to detect the presence or absence of a given abnormality. The primary advantage of the FISH technique is its applicability to non-dividing cells and a variety of specimen types. FISH is a preferred method for diagnosis, prognosis, treatment response, and minimal residual disease detection in a variety of hematopoietic neoplasms and solid tumors. Overview of FISH Procedure Specimen DNA-FISH Probe (Fluorescently labeled DNA, Two Color) Step1: Heat denaturation Co-denaturation of the DNA-FISH Probe and DNA from the specimen. Step 2: Hybridization Addition of target DNA to specimen. Step 3: Washing Removal of excess DNA-FISH probe. Target DNA Step 4: Analysis Analysis of specimen using an epi-fluorescence microscope. Page 4

5 Cancer Genetics Italia DNA-FISH Probe Catalog Table of Contents 6 Hematologic DNA-FISH Probe Panels 9 Solid Tumor DNA-FISH Probe Panels 11 DNA-FISH Probe Portfolio 32 Scoring Guidance and Signal Interpretation 33 Filter Specifications Troubleshooting 35 Ordering Products 37 DNA-FISH Probe Index Page 5

6 Hematologic DNA-FISH Probe Panels Anaplastic Large Cell Lymphoma ALCL Anaplastic large cell lymphoma (ALCL) is a type of Non-Hodgkin s Lymphoma (NHL) that affects T-cells. A common genetic rearrangement that is observed in ALCL is the translocation of the anaplastic lymphoma kinase (ALK) gene and the nucleophosmin (NPM1) gene, which results in the expression of a novel fusion protein, NPM-ALK. ALCL can present itself as a systemic disease or as a localized cutaneous disease. Compared to systemic ALCL, cutaneous ALCL is ALCL Panel less aggressive and varies in terms of prognosis and clinical management. Overall, ALCL patients with ALK translocations, as determined by FISH, have a better prognosis. ALK Break Apart, t(2p23) ALK/NPM1, t(2;5) Hematologic Probes Acute Lymphoblastic Leukemia ALL Acute lymphoblastic leukemia (ALL), also known as acute lymphocytic leukemia, is a rapidly progressive disease marked by the presence of abnormal levels of lymphoblasts within the bone marrow. The disease is more prevalent amongst children versus adults, with children between the ages of 2 and 5 at greater risks of developing the disease. ALL ALL Panel accounts for 80% of pediatric leukemias and 20% of adult acute leukemias. In children, the cure rate of the disease is greater while amongst adults the disease has a poorer prognosis. A20/PRDM1/SHGC-79576, del(6q23),del(6q21)/6p12 ABL1/BCR, t(9;22) IGH/BCL2, t(14;18) IGH Break Apart, t(14q32) MLL Break Apart, t(11q23) MYB/SHGC-79576, del(6q23) MYC Break Apart, t(8q24) MYC/IGH, t(8;14) PBX1/E2A, t(1;19) Acute Myeloid Leukemia AML Acute Myeloid Leukemia (AML) is the most common acute leukemia affecting adults and results from damage to the DNA of granulocytes and monocytes cells, which prevents the cells from maturing. As the cells divide they begin to overcrowd the number of normal red and white blood cells along with platelets produced within the bone marrow. The incidence AML Panel rate of AML increases with age with ~6% occurring in children and young adults under the age of 20 years and more than 50% occurring in patients over the age of 65 years. AML1/ETO, t(8;21) D7S486/Cen7, del(7q31)/cen7 D20S108/8q11, del(20q12) & trisomy 8 EGR1/5p15, del(5q31)/5p15 MLL Break Apart, t(11q23) MYH11/CBFB, inv(16) PML/RARA, t(15;17) RB1/D13S1009, del(13q14)/13q34 Page 6

7 Cancer Genetics Italia DNA-FISH Probe Catalog Chronic Lymphocytic Leukemia CLL Chronic Lymphocytic Leukemia (CLL) is a type of mature B-cell neoplasm. In CLL, abnormal lymphocytes accumulate and crowd out the healthy cells in the bone marrow and blood. CLL primarily affects the elderly and is considered the most common type of leukemia in adults (25 30% of all forms of leukemia); it is also twice as common in men as in women. Because the clinical course of CLL is heterogeneous, risk stratification based on clinical stage, fitness and CLL Panel comorbidities, and molecular prognostic markers is highly recommended at diagnosis and throughout the course of the disease. Certain chromosome abnormalities detected by FISH have been identified as key prognostic markers and can be used at diagnosis, prognosis and during disease monitoring. ATM/D11S1251, del(11q22)/11p15 D13S25/D13S1009, del(13q14)/13q34 MDM2/D12S1837, del(12q15)/12p11 MYB/SHGC-79576, del(6q23) MYC Break Apart, t(8q24) TP53/RARA, del(17p13)/17q21 Chronic Myeloid Leukemia CML Chronic Myeloid Leukemia (CML) is a type of cancer that affects blood-forming cells within the bone marrow and causes an overproduction of granulocytes. Although CML can occur at any age, it has a higher incidence rate amongst older people with a median age of 65 years at the time of diagnosis. According to the World Health Organization, of the ~290,000 new cases of leukemia diagnosed globally each year, CML accounts for ~15-20% of the cases. The de- CML Panel velopment of CML has been attributed to the translocation between the ABL1 and BCR genes. The reciprocal translocation of the genes is referred to as the Philadelphia chromosome (Ph), which is the cytogenetic hallmark of CML. ABL1/BCR, t(9;22) MYH11/CBFB, inv(16) PML/RARA, t(15;17) RB1/D13S1009, del(13q14)/13q34 TP53/RARA, del(17p13)/17q21 Hematologic Probes Multiple Myeloma MM Multiple Myeloma (MM) is a rare cancer that arises from malignant plasma cells that invade and overcrowd healthy cells in the bone marrow. As the disease progresses the number of red blood cells and platelets begin to decrease, which affects the body s ability to make new bone. The incidence rate of MM is slightly greater in men versus women and is more preva- MM Panel lent amongst African Americans. The disease is not found in children and does not often affect adults younger than 35 years. After the age of 35, the incidence rate significantly increases with ~90% of cases occuring in adults over the age of 50 years. 5p15/9q34/15q24, Hyperdiploidy 5, 9, 15 CCND1/IGH, t(11;14) D13S25/D13S1009, del(13q14)/13q34 FGFR3/IGH, t(4;14) IGH Break Apart, t(14q32) IGH/MAF, t(14;16) RB1/D13S1009, del(13q14)/13q34 Page 7

8 Myelodysplastic Syndrome MDS Myelodysplastic Syndrome (MDS) is a group of blood disorders associated with cytopenias, which can affect the production of one or all three types of the following: red blood cells, white blood cells, or platelets. MDS is more common in men, except the MDS subset, 5q-syndrome, which is more prevalent in elderly women. The incidence rate of MDS in- MDS Panel creases significantly with age and over 85% of patients are over the age of 60 years. Research has shown that 10 to 15% of MDS cases are chemotherapy or radiation therapy related and that 10% to 40% of MDS cases progress to AML. Additionally, research has shown that recurring chromosomal abnormalities are present in 40-70% of primary MDS (de novo) cases and in 95% of therapy related MDS (t-mds) cases. D7S486/Cen7, del(7q31)/cen7 D20S108/8q11, del(20q12) & trisomy 8 EGR1/5p15, del(5q31)/5p15 MYH11/CBFB, inv(16) RB1/D13S1009, del(13q14)/13q34 TP53/RARA, del(17p13)/17q21 Hematologic Probes NHL Non-Hodgkin s Lymphoma Non-Hodgkin s Lymphoma (NHL) is a form of cancer that affects the immune system and is indicative of an overproduction of white blood (lymphocytes) cells in the blood, bone marrow, spleen, and/or lymph nodes. Globally, there was ~355,900 new NHL cases reported and ~191,400 deaths due to NHL in Classification of NHL is based upon the type of lymphocytes affected (B-cell vs. T-cell) and the pattern of growth progression (indolent/low-grade vs. aggressive/ high-grade). There are over 30 types of NHL cases, with B- cell lymphomas accounting for ~85% of cases and T-cell lymphomas accounting for approximately 15% of cases. Diagnosis of NHL subtypes through conventional cytogenetics is ineffective because the subtypes exhibit similar characteristics. However, known genetic rearrangements, as detected by FISH, have been identified for a number of NHL subtypes, which enables effective diagnosis of NHL subtypes. NHL Panel A20/PRDM1/SHGC-79576, del(6q23),del(6q21)/6p12 API2/MALT1, t(11;18) ATM/D11S1251, del(11q22)/11p15 BCL6 Break Apart, t(3q27) CCND1/IGH, t(11;14) D13S25/D13S1009, del(13q14)/13q34 IGH/BCL2, t(14;18) IGH Break Apart, t(14q32) IGH/MALT1, t(14;18) MDM2/D12S1837, del(12q15) MYC Break Apart, t(8q24) MYC/IGH, t(8;14) Page 8

9 Cancer Genetics Italia DNA-FISH Probe Catalog Solid Tumor DNA-FISH Probe Panels Breast Cancer Breast Amongst women, breast cancer is the leading cause of cancer related deaths and is the most frequently diagnosed worldwide. In 2008, breast cancer accounted for 23% of the total new cancer cases reported globally and for 14% of the total deaths from cancer worldwide. Approximately 5% to 10% of breast cancer cases are a result of inherited gene mutations and women who exhibit such mutations have an 80% chance of developing the disease. The five major types of breast cancer are lobular carcinoma in situ (LCIS), ductal carcinoma in situ (DCIS), invasive (infiltrating) lobular carcinoma (ILC), invasive (infiltrating) ductal carcinoma (IDC), and inflammatory breast cancer (IBC). DCIS is the most common form of non-invasive breast cancer, while LCIS, which is not a cancer, is more a marker of an increased risk for developing breast cancer. IDC is the most common breast cancer and accounts for ~8 out of 10 invasive breast cancer cases, while ILC accounts for ~1 out 10 invasive breast cancer cases. IBC is an uncommon type of breast cancer with an incidence rate of 1% to 3% of all breast cancer cases. Lung Cancer Breast Panel EGFR/Cen7, EGFR Amplification ERBB2/Cen17, ERBB2 Amplification TP53/RARA, del(17p13)/17q21 Lung Globally, smoking is attributed to 80% of the lung cancer cases reported in men and 50% of the lung cancer cases reported in women. Amongst men, lung cancer was the leading cause of death associated with cancer and was the most commonly diagnosed form of cancer globally in Amongst women, it was the fourth most commonly diagnosed cancer and the second leading cause of cancer associated deaths in Early detection of the disease is limited due to the late onset of symptoms, which often appears once the disease has advanced. Lung cancer can be categorized into two major forms, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC); both forms affect different cells within the lung and possess varying growth and progression patterns. NSCLC is the most common form of lung cancer, accounting for 85% Lung Panel to 90% lung cancer cases, and is often associated with a history of smoking. SCLC, which is also known as oat cell lung cancer, accounts for ~10% to 15% of lung cancer cases and is always associated with a history of smoking. ALK Break Apart, t(2p23) EGFR/Cen7, EGFR Amplification TP53/RARA, del(17p13)/17q21 Solid Tumor Probes Colorectal Cancer Colon/Rectal Colorectal cancer (CRC) is the third most diagnosed form of cancer amongst men and the second amongst women worldwide, with over 1.2 million newly reported cases and 608,700 reported deaths in Although there are a number colorectal cancer types, the most common is adenocarcinomas, which accounts for more than 95% of CRC cases. Other rare types of the disease include carcinoid tumors, gastrointestinal stromal tumors (GISTs), lymphomas, and sarcomas. Studies have found that ~5% to 10% of colorectal cancer cases are a result of genetic mutations that are passed from parents to children. Two well-established forms of CRC are familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC), which is also known as Lynch syndrome. FAP accounts for ~1% of all colorectal cancer cases, while HNPCC accounts for ~3% to 5%. Colon/Rectal Panel EGFR/Cen7, EGFR Amplification TP53/RARA, del(17p13)/17q21 Page 9

10 Cervical Cancer Cervical Globally, cervical cancer is the third most diagnosed cancer and the fourth most leading cause of cancer deaths amongst women. In 2008, cervical cancer accounted for 529,800 new cases and 275,100 deaths were reported. The success of screening programs for the detection of for the detection of precancerous cervical lesions based on the appearance of abnormal cells in cytology specimens (Pap smear) is well known. It is estimated that 10% of abnormal specimens will evolve into cancer. The strong correlation between the development of cervical cancer and infection with the human papillomavirus (HPV) is well known. Genomic abnormalities observed in HPV-associated cancers serve as Cervical Panel biomarkers of cancer detection and progression and can be detected by FISH. FHACT TM, 3q26/5p15/20q13/Cen7 Solid Tumor Probes Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). Jaffe, E.S., et al. Hematopathology, 2011 (1st Edition). Jemal, A., et al. A Cancer J for Clinicians, :p Page 10

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13 Cancer Genetics Italia DNA-FISH Probe Catalog Table of Contents p15, 9q34, 15q24 Lesion: Hyperdiploidy 5, 9, 15/Ref: MM A20/PRDM1/SHGC Lesion: del(6q23),del(6q21)/6p12/ref: ALL NHL ABL1/BCR Lesion: t(9;22)/ref: ALL CML ALK Break Apart Lesion: t(2p23)/ref: ALCL Solid Tumors 16 ALK/NPM1 Lesion: t(2;5)/ref: AML1/ETO Lesion: t(8;21)/ref: ALCL AML API2/MALT1 Lesion: t(11;18)/ref: ATM/D11S1251 Lesion: del(11q22)/11p15/ref: CLL NHL BCL6 Break Apart Lesion: t(3q27)/ref: CCND1/IGH Lesion: t(11;14)/ref: MM NHL D7S486/Cen7 Lesion: del(7q31)/cen7/ref: NHL NHL AML MDS D13S25/D13S1009 Lesion: del(13q14)/13q34/ref: CLL MM NHL D20S108/8q11 Lesion: del(20q12) & trisomy 8/Ref: EGFR/Cen7 Lesion: EGFR Amplification/Ref: EGR1/5p15 Lesion: del(5q31)/5p15/ref: ERBB2/Cen17 Lesion: ERBB2 Amplification/Ref: FGFR3/IGH Lesion: t(4;14)/ref: FHACT TM Lesion: 3q26/5p15/20q13/Cen7/Ref: AML MDS Solid Tumors AML MDS Solid Tumors MM Solid Tumors 23 IGH/BCL2 Lesion: t(14;18)/ref: ALL NHL IGH Break Apart Lesion: t(14q32)/ref: ALL MM NHL 24 IGH/MAF Lesion: t(14;16)/ref: IGH/MALT1 Lesion: t(14;18)/ref: MM NHL MDM2/D12S1837 Lesion: 12q15/12p11/Ref: CLL NHL MLL Break Apart Lesion: t(11q23)/ref: ALL AML MYB/SHGC Lesion: 6q23/6p12/Ref: ALL CLL MYC Break Apart Lesion: t(8q24)/ref: ALL CLL NHL MYC/IGH Lesion: t(8;14)/ref: ALL NHL MYH11/CBFB Lesion: inv(16)/ref: AML CML MDS PBX1/E2A Lesion: t(1;19)/ref: PML/RARA Lesion: t(15;17)/ref: AML CML RB1/D13S1009 Lesion: del(13q14)/13q34/ref: AML CML MDS MM TP53/RARA Lesion: del(17p13)/17q21/ref: CLL CML MDS Solid Tumors ALL Page 13

14 5p15, 9q34, 15q24 5p15, 9q34, 15q24 Three Color, Enumeration Probe Ref: The 5p15, 9q34, 15q24 DNA-FISH Probe is designed to detect changes in copy number of chromosome(s) 5, 9, and 15 by fluorescence in situ hybridization (FISH). Hyperdiploidy is characterized by increased copy numbers of chromosomes such that the modal chromosomal number is [1] It is observed in 30-50% of multiple myeloma (MM) cases where the most common trisomies involve chromosomes 3, 5, 7, 9, 15, 19, and 21. [2-4] In a diagnostic setting, gain of at least two of the three chromosomes 5, 9, or 15 in MM is considered defining for hyperdiploidy. [5] When assessed by G-banded karyotype and in the absence of other aberrations such as IGH translocations or deletion of chromosome 13, hyperdiploidy in MM is associated with a good prognosis. [6,7] 5p15 5 AFMA055ZD9 ~554kb 15q ~269kb 9q34 D15S169 D9S64 ~468kb MM 1. Shaffer, L. G., et al. ISCN 2005 (1st Edition). 2. Liebisch, P., et al. Eur J Cancer, (11): p Terpos, E., et al. Leuk Lymphoma, (5): p Neben, K., et al. Haematologica, (7): p Wuilleme, S., et al. Leukemia: Offic J of Leuk Society of America, (2): p Chen, L., et al. Exp Oncol, (2): p Chng, W. J., et al. Leukemia, (5): p A20/PRDM1/SHGC A20/PRDM1/SHGC Three Color, Enumeration Probe Ref: The A20/PRDM1/SHGC DNA-FISH Probe is designed to detect deletion of the A20 gene located on 6q23 and the PRDM1 gene located on 6q21 relative to the control locus SHGC on 6p12, using fluorescence in situ hybridization (FISH). [1] Deletion of 6q is observed in B-cell malignancies and childhood acute lymphoblastic leukemia (ALL) cases, where two commonly deleted regions map to the A20 and PRDM1 gene region. [2] Loss of the A20 gene is observed most frequently in mantle cell lymphomas (MCLs) and diffuse large B-cell lymphomas (DLBCLs) cases with a frequency rate of 31% and 38%, respectively. [3] However, the A20 loss is also observed in ~20% of non-hodgkin lymphoma (NHL) cases, ~20% of mucosa-associated lymphoid tissue (MALT) lymphoma cases [5] and in nonsplenic marginal zone lymphomas (MZLs) cases. [4] In DLBCL cases, inactivation of A20 either by somatic mutation or deletion, is seen more frequently in the activated B- cell (ABC) subtype of DLBCL (50%) versus the germinal center B-cell (GCB) subtype (22%). [3,5,6] DLBCL cases exhibit a variety of complex 6q deletions, which encompasse either A20 or PRDM1 alone or together as part of a larger deletion. [2] Additionally, the deletion of the PRDM1 gene has been observed in 53% of primary central nervous system lymphomas (PCNSLs). [7] 6 ALL NHL 1. Huret, J.L., et al Thelander, E. F., et al. Leuk Lymphoma, (3): p Honma, K., et al. Blood, (12): p Novak, U., et al. Blood, (20): p Kato, M., et al. Nature, (7247): p Lenz, G., et al. PNAS, (36): p Schwindt, H., et al. Leukemia, (10): p SHGC PRDM1 5 3 A ~494kb ~774kb ~772kb Page 14

15 Cancer Genetics Italia DNA-FISH Probe Catalog ABL1/BCR Two Color, Two Fusion Translocation Probe Ref: The ABL1/BCR DNA-FISH Probe is designed to detect the translocation between the ABL1 gene on chromosome 9q34 and the BCR gene on chromosome 22q11 using fluorescence in situ hybridization (FISH). This reciprocal translocation results in the Philadelphia chromosome (Ph), the der(22), and is the hallmark of chronic myeloid leukemia (CML). Approximately 90-95% of CML cases along with up to 5% of pediatric and 20% of adult acute lymphocytic leukemia (ALL) cases are Ph positive. [1-3] A subset of CML (~10%) and ALL (~5%) cases exhibit large deletions adjacent to the breakpoints on chromosomes der(9) and der(22). [4-6] Such submicroscopic losses carry a poor prognosis and can be detected by the Cancer Genetics Italia DNA-FISH Probe. 9q34 22q11 9 ASS ~285kb 22 ABL1 5 3 BCR 5 3 ~390kb ALL CML 1. Huret, J. L., et al. t(9;22)(q34;q11) in CML, Dec Huret, J. L., et al. t(9;22)(q34;q11) in ALL, Sep Nashed, A. L., et al. J Mol Diagn, : Bacher, U., et al. Haematologica, : Douet-Guilbert, N. et al., Cancer Genet Cytogenet, : Gorusu, M., et al. Cancer Genet Cytogenet, : ABL1/BCR ~350kb ~390kb ALK Break Apart Two Color, Break Apart Probe Ref: The ALK Break Apart DNA-FISH Probe is designed to detect the translocation between the ALK gene located at 2p23 and one of at least 14 known translocation partner loci using fluorescence in situ hybridization (FISH). [1] Translocation of the ALK gene occurs in ~50% of anaplastic large cell lymphoma (ALCL) cases with a form of t(2;5)(p23;q35), as determined by conventional cytogenetics; in such cases, the presence of the t(2;5)(p23;q35) form carries a better overall prognoses for ALCL patients. [2] ALK translocation has been observed in vesical inflammatory myofibroblastic tumors (IMT) of the bladder (>66%) [3,4] and serves as a diagnostic biomarker for differential diagnosis of IMT from sarcomatous lesions. [3] ALK translocation is observed in ~5% - 16% of non-small cell lung cancer (NSCLC) cases [5] in the form of inv(2)(p21p23), as determined by FISH, [5] and serves as a biomarker for therapy response. [5,6] The presence of the ALK translocation in NSCLC patients is correlated with a marked sensitivity to pemetrexed and crizotinib treatment. [7] 2p23 ~541kb 2 ALK 3 5 ~556kb ALCL Lung 1. Huret, J. L., et al Drexler, H. G., et al. Leukemia, (9):p Freeman, A., et al. Mod Pathol, (7):p Sukov, W. R., et al. Mod Pathol, (5):p Kim, H. R., et al. Cancer, 2011 June Gerber, D. E., et al. Cancer Cell, (6):p Camidge, D. R., et al. J Thorac Oncol, (4):p ALK Break Apart Page 15

16 ALK/NPM1 ALK/NPM1 Two Color, Two Fusion Translocaton Probe Ref: The ALK/NPM1 DNA-FISH Probe is designed to detect the translocation between the ALK gene located at 2p23 and the NPM1 gene located at 5q35, using fluorescence in situ hybridization (FISH); [1] the translocation between the ALK and NPM1 gene is designated as t(2;5)(p23;q35). By conventional cytogenetics, the translocation occurs in up to 50% of anaplastic large cell lymphoma (ALCL) cases. [2] As assessed by immunohistochemistry, expression of the fusion protein ALK/NPM1 that is generated by t(2;5), occurs more frequently in childhood ALCL with an occurance rate of 83% versus adult ALCL which has an occurance rate of 31%. [2,3] The presence of t(2;5)(p23;q35) carries a better overall prognosis for ALCL patients. [2] 2p23 ~541kb 2 ALK ~556kb ALCL 1. Huret, J.L., et al Drexler, H.G., et al. Leukemia, (9):p Weitzman, S., K., et al. Curr Oncol Rep, (2):p q35 NPM1 5 3 ~552kb ~502kb AML1/ETO AML1/ETO Two Color, Two Fusion Translocation Probe Ref: The AML1/ETO DNA-FISH Probe is designed to detect the translocation between the AML1 (RUNX1) gene on chromosome 21q22 and the ETO (RUNX1T1/ MTG8) gene on chromosome 8q22 using fluorescence in situ hybridization (FISH). [1] This reciprocal nonrandom translocation generates an AML1-ETO fusion protein and is detected in 12% of de novo acute myeloid leukemia (AML) cases and in up to 46% of AML subtype M2 cases. [2,3,4] The detection of t(8;21) on der(8) is clinically relevant because it is generally associated with a favorable prognosis, particularly with high-dose cytorabine-based consolidation chemotherapy. [5] 8q21 21q22 8 ETO 3 5 ~635kb 21 ~472kb AML1 3 5 AML 1. Huret, J.L., et al Nucifora, G., et al. Blood, (1): Peterson, L. F., et al. Oncogene, (24): Cox, M. C., et al. Hematol J., (4): Grimwade, D., et al. Blood, (7): ~570kb ~630kb Page 16

17 Cancer Genetics Italia DNA-FISH Probe Catalog API2/MALT1 Two Color, Two Fusion Translocation Probe Ref: The API2/MALT1 DNA-FISH probe is designed to detect the translocation between the API2 gene located at 11q21 and the MALT1 gene located at 18q21 using fluorescence in situ hybridization (FISH). [1] The translocation between the API2 and MALT1 gene, designated as t(11;18)(q21;q21), can be detected in around 15% of mucosa-associated lymphoid tissue (MALT) lymphomas, but varies in frequency based on primary tumor site. [2] In pulmonal and gastric MALT, t(11;18) is found more frequently (38-53% and 22-24%, respectively) and in these cases is almost always the only detected chromosomal abnormality. [2] When observed in gastric MALT lymphoma, t(11;18) is highly associated with a lack of response to antibiotic H pylori eradication treatment. [3,4] q22 ~498kb API2 5 3 ~645kb NHL 1. Huret, J. L., et al Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). 3. Nakamura, T., et al. J Gastroenterol, (10): p Toracchio, S., et al. Cancer Sci, (5): p API2/MALT1 18q21 ~641kb MALT1 5 3 ~738kb ATM/D11S1251 Two Color, Enumeration Probe Ref: The ATM/D11S1251 DNA-FISH Probe is designed to detect the deletion of the ATM gene located on 11q22 relative to the control locus D11S1251 located on 11p15 by fluorescence in situ hybridization (FISH). [1] The deletion of the ATM gene is detected in ~65% of T-cell prolymphocytic leukemia (T-PLL) cases [2], ~50% of mantle cell lymphoma (MCL) cases [2,3], and ~20% of chronic lymphocytic leukemia (CLL) cases. [3] Deletion of 11q in CLL patients is associated with extensive lymphadenopathy, disease progression, and shorter median survival. [4,5] Significantly improved clinical outcomes in previously untreated CLL patients with ATM loss have been observed using alkylating agent-based chemo-immunotherapy regimens. [6] 11 D11S1251 ~423kb ATM 5 3 ~399kb CLL NHL 1. NCBI/Gene database Monni, O., et al. Leuk Lymphoma, (3-4): p Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). 4. Dohner, H., et al. N Engl J Med, (26): p Neilson, J.R., et al. Leukemia, (11): p Tsimberidou, A.M., et al. Cancer, (2): p ATM/D11S1251 Page 17

18 BCL6 Break Apart BCL6 Break Apart Two Color, Break Apart Probe Ref: The BCL6 Break Apart DNA-FISH Probe is designed to detect the translocations between the BCL6 gene located on 3q27 and one of at least 20 known translocation partner loci as detected by fluorescence in situ hybridization (FISH). [1] Translocation of the BCL6 gene occurs in 6-26% of follicular lymphoma (FL) [2] with higher incidence (44%) in grade 3 cases negative for t(14;18)(q32;q21). [3] Rearrangement of the BCL6 gene is observed at a frequency of 15% to 40% in diffuse large B-cell lymphomas (DLBCL). [2,3] 3 3q27 ~471kb BCL6 3 5 ~756kb NHL 1. NCBI/Gene database Chaganti, R.S., et al. Semin Hematol, (4): p Gu, K., et al. Mod Pathol, (9): p CCND1/IGH CCND1/IGH Two Color, Two Fusion Translocation Probe Ref: The CCND1/IGH DNA-FISH Probe is designed to detect the translocation between the CCND1 gene located on 11q13 and the IGH gene located on 14q32 by fluorescence in situ hybridization (FISH). [1] The translocation between the CCND1 and IGH gene is designated as t(11;14)(q13;q32) and is the cytogenetic hallmark of mantle cell lymphoma (MCL), which distinguishes it from other non-hodgkin lymphomas. [2,3] The t(11;14) rearrangement is also detected in 35-50% of light chain amyloidosis, 15-30% of monoclonal gammopathy of undetermined significance (MGUS), and 15-20% of multiple myeloma (MM) cases. [4,5] The presence of IGH translocations is considered a high-risk feature in MM patients although it has been reported that t(11;14)(q13;q32) is associated with an improved survival. [6] 11q CCND1 5 3 ~513kb ~511kb MM NHL 1. Huret, J. L., et al Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). 3. Bentz, J.S., et al. Cancer, (2):p Barille-Nion, S., et al. Hematology Am Soc Hematol Educ Program, p Liebisch, P., et al. Eur J Cancer, (11):p Fonseca, R., et al. Cancer Res, (4):p q32 3 C segments J segments D segments IGH V segments 5 ~561kb ~420kb Page 18

19 Cancer Genetics Italia DNA-FISH Probe Catalog D7S486/Cen7 Two Color, Enumeration Probe Ref: The D7S486/Cen7 DNA-FISH Probe is designed to detect the deletion of the D7S486 locus located on 7q31 relative to the control locus chromosome 7, using fluorescence in situ hybridization (FISH). [1] Loss of the D7S486 locus is detected in ~5% of adults with de novo myelodysplastic syndrome (MDS) and in ~50% of children with de novo MDS. [2] Additionally, loss of the D7S486 locus is observed in 5% of de novo acute myeloid leukemia (AML) patients and in 30-40% of therapy related MDS (t-mds)/therapy related AML (t-aml) patients. [2-4] Deletion of the D7S486 locus or monosomy of chromosome 7 is associated with a poor prognosis in adults and children diagnosed with MDS and/or AML. [2,3,5] 7 7q31 Cen 7 ~230kb D7S486 AML MDS 1. NCBI/Gene database Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). 3. Mantadakis, E., et al. Cancer, (12):p Cuneo, A., et al. Leukemia, (9):p Haase, D. Ann Hematol, (7):p D7S486/Cen7 D13S25/D13S1009 Two Color, Enumeration Probe Ref: The D13S25/D13S1009 DNA-FISH Probe is designed to detect loss of the D13S25 locus on chromosome 13q14 relative to the control marker D13S1009 on chromosome 13q34 using fluorescence in situ hybridization (FISH). The deletion of D13S25, a locus distal to the RB1 gene, has been detected in B-cell chronic lymphocytic leukemia (B-CLL) cases, [1] in multiple myeloma (MM) cases, [2,3] rarely in a variety of non-hodgkin s lymphoma s (NHL) cases, [4] and several myeloid disorders. [5] Deletion of 13q14 has a strong prognostic value correlating with slower disease progression and better prognosis in B-CLL patients, [5] while in MM patients it is associated with a higher stage of disease and shorter survival. [6] 13 D13S25 D13S1009 ~186 kb ~321 kb CLL MM NHL 1. Nelson, B. P., et al. Am J Clin Pathol, (2): Chen, L., et al. Exp Oncol, (2): Terpos, E., et al. Leuk Lymphoma, (5): Dierlamm, J., et al. Cancer Genet Cytogenet, ;120(1): Ma, ESK, et al. del(13q). org. 6. Reddy, K. S. Br J Haematol, (6): D13S25/D13S1009 Page 19

20 D20S108/8q11 D20S108/8q11 Two Color, Enumeration Probe Ref: The D20S108/8q11 DNA-FISH Probe is designed to detect the deletion of the D20S108 locus on 20q12 and the gain of chromosome 8 using fluorescence in situ hybridization (FISH). [1] Genomic aberrations related to copy number changes are found in myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). [2-4] Deletion of the D20S108 locus is observed in 0.6-5% of de novo MDS patients [2] and in less than 2% of de novo AML patients. [3] In MDS patients, deletion of the D20S108 locus is associated with a good prognosis [4] whereas in AML patients it is a marker of either an intermediate or an unfavorable outcome. [3] The trisomy of chromosome 8 is observed as a sole abnormality in ~5% of MDS patients or as part of a complex karyotype in >15% of such patients [5] and is generally associated with an intermediate prognosis. [2] Additionally, trisomy of chromosome 8 can be observed as a sole aberration in 5% of de novo AML patients or simultaneously with other aberrations in 15% in such patients. [3] However, trisomy 8 is more common in de novo AML cases than in therapy related AML (t-aml) with an occurance of 7.4% vs. 3.3%, respectively [6,7] and has been associated with an intermediate prognosis. [3] q11 AML MDS 1. Huret, J. L., et al Sole, F., et al. Haematologica, (9):p Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). 4. Haase, D. Ann Hematol, (7):p Disperati, P., et al. Leuk Res, (2):p Qian, Z., et al. Chem Biol Interact, (1-2):p Mauritzson, N., et al. Leukemia, (12):p q12 D20S108 ~302kb EGFR & Cen7 EGFR/Cen7 Two Color, Enumeration Probe Ref: The EGFR/Cen7 DNA-FISH Probe is designed to detect an increased copy number of the EGFR gene on 7p11 (previously assigned to band 7p12) relative to the control Cen7 probe using fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded (FPPE) tissues. The EGFR gene encodes a transmembrane protein involved in cell proliferation. [1,2] Increased copy numbers of the EGFR gene has been reported in non-small cell lung cancer (NSCLC) cases and some other carcinomas such as colorectal, head and neck, and breast cancer cases. [2,3,4,5] An increased EGFR copy number is predictive of response to anti-egfr therapies. [1,3,4,5,6] 7p11 7 Cen7 EGFR 5 3 ~ 495 kb Breast Colon/Rectal Lung 1. Toschi and Cappuzzo. The Oncologist, : Bhargava, et al. Modern Patholy, : Hirsch, et al. J Clin Oncol, : Personeni, et al. Clin Cancer Res, (18): Zimmermann, at al. Radiation Oncology, : Varella-Garcia, et al. J Clin Pathol, : Page 20

21 Cancer Genetics Italia DNA-FISH Probe Catalog EGR1/5p15 Two Color, Two Fusion Translocation Probe Ref: AML MDS The EGR1/5p15 DNA-FISH Probe is designed to detect the deletion of the EGR1 gene located on 5q31 relative to the control locus 5p15 by fluorescence in situ hybridization (FISH). [1] The deletion of the EGR1 gene is detected in 10-15% of de novo myelodyplastic syndrome (MDS) and acute myeloid leukemia (AML) patients, and in 35-42% of therapy-related MDS (t-mds) and therapy-related AML (t-aml) patients. [2, 3] When observed as the sole chromosomal aberration in cases of MDS (also called 5q-syndrome), deletion of the EGR1 gene is associated with a favorable prognosis and good response to lenalidomide treatment. [4] In cases of MDS/AML and t-mds/t-aml, deletion of EGR1 as part of a complex karyotype is [4, 5] associated with a worse prognosis and unfavorable outcome. 5 EGR1/5p15 5p15 ~554kb EGR1 5 3 ~222kb 1. NCBI/Gene database Herry, A., et al. Eur J Haematol, (6): p Schoch, C., et al. Genes Chromosomes Cancer, (1): p List, A., et al. N Engl J Med, (14): p Haase, D. Ann Hematol, (7): p ERBB2/Cen17 Two Color, Enumeration Probe Ref: The ERBB2/Cen17 DNA-FISH Probe is designed to detect the amplification of the ERBB2 gene (also named HER2/neu) on chromosome 17q12 relative to the control Cen17 using fluorescence in situ hybridization (FISH) in formalin-fixed, paraffinembedded (FFPE) breast cancer tissues. Overexpression of the ERBB2 gene occurs in 25-30% of human breast carcinomas, and ~90-95% of these cases result directly from gene amplification. [1,2] Patients showing such a rearrangement are at high-risk for relapse and lower overall survival. [2-4] Amplification of the ERBB2 gene predicts a favorable response to certain chemotherapy regimens and selective monoclonal antibody therapy with trastuzumab. [1-6] ERBB2 amplification is also seen in other solid tumors such as gastric, esophageal, gynecologic, bladder, and non-small cell lung cancer and correlates with a poor prognosis. [1,7] 17q12 17 Cen17 ERBB2 5 3 ~ 160 kb Breast 1. Casalini, P., et al Pauletti, G., et al. J Clin Oncol, (21): Harries, M., et al. Endocr Relat Cancer, (2): Kallioniemi, O. P., et al. Proc Natl Acad Sci USA, (12): Slamon, D. J., et al. Science, (4785): Wolff, et al. J Clin Oncol, (1): Mano, M. S., et al. Cancer Treat Rev, (1): ERBB2 & Cen17 Page 21

22 FGFR3/IGH FGFR3/IGH Two Color, Two Fusion Translocation Probe Ref: The FGFR3/IGH translocation probe is designed to detect the translocation between the FGFR3 gene located on 4p16 and the IGH gene located on 14q32 using fluorescence in situ hybridization (FISH). [1] The t(4;14) rearrangement has been observed in ~15% of multiple myeloma (MM) patients. Detection of the t(4;14) translocation is clinically relevant because it confers an aggressive phenotype with a poor prognosis and a rapid relapse after high-dose chemotherapy. [2,3] 4p16 FGFR ~593 kb 4 ~644 kb MM 14q32 3 C segments ~561kb J segments D segments IGH V segments ~420kb 5 1. Huret, J. L., et al Chen, L., et al. Exp Oncol, (2): Moreau, P., et al. Blood, (5): FHACT TM Four Color, Enumeration Probe Ref: The FISH-based HPV-Associated Cancer Test (FHACT TM ) is a probe designed to determine copy number changes of the 3q26 (TERC), 5p15 (D5S2095), 20q13 (D20S911), and Cen7 regions by fluorescence in situ hybridization (FISH). [1] Cervical 3 5 FHACT TM 3q26 7 ~904 kb TERC Cen7 5p15 20q13 20 D5S2095 ~601 kb D20S Huret, J. L., et al. ~493 kb Page 22

23 Cancer Genetics Italia DNA-FISH Probe Catalog IGH/BCL2 Two Color, Two Fusion Translocation Probe Ref: The IGH/BCL2 DNA-FISH Probe is designed to detect the translocation between the IGH gene located on 14q32 and the BCL2 gene located on 18q21, using fluorescence in situ hybridization (FISH). [1] The translocation between the IGH and BCL2 gene is designated as t(14;18)(q32;q21) and is the hallmark of follicular lymphoma (FL). The t(14;18) translocation is more frequent in lower FL grades, such as FL grades 1 and 2 (88%), than in higher grades, such as FL 3b (4-13%). [2] The translocation is also detected in 30% of diffuse large B-cell lymphoma (DLBCL) cases, but is less frequently detected in other non-hodgkin lymphomas. [2,3] By conventional cytogenetics, the t(14;18)(q32;q21) translocation that involves the IGH and BCL2 genes is indistinguishable from the t(14;18)(q32;q21) translocation that involves the IGH and MALT1 genes. [2] Such translocations can be distinguished by using the respective DNA-FISH probes. [2] 14 ALL NHL IGH/BCL2 14q32 3 C segments J segments D segments IGH V segments 5 1. Huret, J. L., et al Heim, S., et al. Cancer Cytogenetics, 2009 (3 rd Edition). 3. Jaffe, E. S., et al. Hematopathology, 2011 (1 st Edition). ~561kb ~420kb 18 18q21 BCL2 3 5 ~441kbp ~575kbp IGH Break Apart Two Color, Two Fusion Translocation Probe Ref: The IGH Break Apart DNA-FISH Probe is designed to detect the translocation involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32 using fluorescence in situ hybridization (FISH). At least 40 translocation gene partners to the IGH locus have been identified. [1] Rearrangements involving the IGH locus and specific partners are mainly found in multiple myeloma (MM) [2,3] and non-hodgkin s lymphoma (NHL) subtypes [4,5] The prognosis is dependent upon the translocation partner and the type of malignancy. The design of the IGH Break Apart DNA-FISH Probe allows the visualization of a break between the C domain (red) and the V domain (green) of the IGH locus and the resulting translocation. 14q C segments ~561kb J segments D segments IGH V segments ~420kb 5 ALL MM NHL 1. Huret, J. L., et al Streubel, B., et al. Blood, (6):p Streubel, B., et al. Leukemia, (10):p Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). IGH Break Apart Page 23

24 IGH/MAF IGH/MAF Two Color, Two Fusion Translocation Probe Ref: The IGH/MAF DNA-FISH Probe is designed to detect the translocation between the IGH gene located on 14q32 and the MAF gene located on 16q23 by fluorescence in situ hybridization (FISH). [1] The translocation between the IGH and MAF gene, designated as t(14;16)(q32;q23), is found in 2-10% of multiple myeloma (MM) cases and is associated with a more aggressive disease along with an unfavorable prognosis and outcome. [1-3] 14 14q32 3 C segments ~561kb J segments D segments IGH V segments ~420kb 5 MM 16q23 16 MAF Liebisch, P., et al. Eur J Cancer, (11): p Kapoor, P., et al. Mayo Clin Proc, (6): p Terpos, E., et al. Leuk Lymphoma, (5): p ~655kbp IGH/MALT1 IGH/MALT1 Two Color, Two Fusion Translocation Probe Ref: The IGH/MALT1 DNA-FISH probe is designed to detect the translocation between the IGH gene located at 14q32 and the MALT1 gene located at 18q21 by fluorescence in situ hybridization (FISH), designated as t(14;18)(q32;q21). [1] The rearrangement of IGH/MALT1 has been observed in 10-20% of mucosa-associated lymphoid tissue (MALT) lymphoma, predominantly occurring in liver, skin, and ocular adnexa. [2,3] By conventional cytogenetics, t(14;18)(q32;q21) involving the IGH and MALT1 genes is indistinguishable from the t(14;18)(q32;q21) involving the IGH and BCL2 genes (the hallmark of follicular lymphoma). [4] These translocations can be distinguished by FISH, using the respective DNA-FISH probes. 14q ~561kb J segments D segments IGH C segmentsv segments ~420kb 5 NHL 1. Huret, J. L., et al Streubel, B., et al. Blood, (6):p Streubel, B., et al. Leukemia, (10):p Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). 18q21 MALT1 5 3 ~641kb ~738kb Page 24

25 Cancer Genetics Italia DNA-FISH Probe Catalog MDM2/D12S1837 Two Color, Enumeration Probe Ref: The MDM2/D12S1837 DNA-FISH Probe is designed to detect both the polyploidy of chromosome 12 and the amplification of the MDM2 gene located on 12q14.3-q15 relative to the control locus D12S1837 located on 12p11 by fluorescence in situ hybridization (FISH). [1,2] Well-differentiated liposarcomas has been found by molecular and cytogenetic studies to contain amplification of the 12q13-15 region, including the MDM2 gene. [3,4] Precise recognition of benign lipoma and well-differentiated liposarcoma by core needle biopsy can facilitate appropriate clinical management. Trisomy of chromosome 12 (+12) is a commonly observed numerical aberration in non-hodgkin lymphomas (NHL) cases, mostly in mantle cell lymphoma (MCL), and in chronic lymphocytic leukemia (CLL) cases. Trisomy 12 has been observed in ~20-30% of MCL patients and in ~15-50% of CLL patients. [2,5,6] In CLL, trisomy 12, when observed as a sole abnormality, appears to have limited prognostic implications and is often associated with an atypical morphology, although it is only observed in a subset of tumor cells. [6,7] 12 D12S1837 MDM2 5 3 CLL NHL 1. NCBI/Gene database Huret, J. L., et al Weaver, J., et al. Modern Pathology: An Offic J of the U.S. and Canadian Acad of Pathol, Inc., (10): p Dal Cin, P., et al. Cancer Genetics and Cytogenetics, (2): p Heim, S., et al. Cancer Cytogenetics, 2009 (3rd Edition). 6. Jaffe, E. S., et al Hematopathology, 2011 (1st Edition). 7. Dohner, H., et al. J Mol Med, (2):p MDM2/D12S1837 ~304kb ~289kb MLL Break Apart Two Color, Two Fusion Translocation Probe Ref: The MLL Break Apart probe is designed to detect the translocation involving the MLL gene on chromosome 11q23 using fluorescence in situ hybridization (FISH). At least 54 translocation partner genes have been identified. [1] Translocation of MLL is found in ~3-10% of acute lymphoblastic leukemia (ALL) cases and in ~8-10% of acute myeloid leukemia (AML) cases and is clinically relevant in both cases. [2,3] However, the prognostic implication is dependent on the age and phenotype of the leukemia. MLL rearrangement has been observed in ~80% of infant ALL cases and is associated with a high risk in such cases and requires aggressive treatment. In AML, the prognosis is intermediate regardless of age. MLL translocations are also found in ~25% of patients with therapy-related leukemias, particularly following treatment with DNA topoisomerase II inhibitors and the prognosis in such patients is poor. [2,3] In addition to translocations, deletions of 3 MLL and amplification of MLL also occurs in a subset of ALL and AML cases. [4,5] 11q23 11 ~770kb MLL 5 3 ~820kb ALL AML 1. Marschalek, R Raimondi, S. C Chowdhury, T., et al. Blood Cells Mol Dis, : Barber, K. E, et al. Genes Chromosomes Cancer, : Andersen, M. K, et al. Genes Chromosomes Cancer, : MLL Break Apart Page 25

26 MYB/SHGC MYB/SHGC Two Color, Enumeration Probe Ref: The MYB/SHGC DNA-FISH Probe is designed to detect copy number changes of the MYB locus located on 6q23 relative to a 6p12 control locus, using fluorescence in situ hybridization (FISH). [1] Loss of MYB has been observed in ~5% of chronic lymphocytic leukemia (CLL) cases [2] and in 6-16% of T-cell acute lymphoblastic leukemia (T-ALL) cases. [3] Deletion of 6q, encompassing the MYB gene, has been associated with a poor prognosis in CLL and T-ALL cases, [2,3] while gain of the MYB gene has been observed in ~ 30% of hereditary BRCA1 positive breast tumors. [4] 6 SHGC ~494kb ~777kb MYB 5 3 ALL CLL 1. Huret, J. L., et al Reddy, K. S. Br J Haematol, (6):p Sinclair, P., et al. Haematologica, (5):p Kauraniemi, P., et al. Cancer Res, (19):p MYC Break Apart MYC Break Apart Two Color, Break Apart Probe Ref: The MYC Break Apart DNA-FISH Probe is designed to detect the translocation between the MYC gene located at 8q24 and one of 11 known translocation partner loci using fluorescence in situ hybridization (FISH). [1] The most common translocation, t(8;14)(q24;q32), is found in 60-80% of Burkitt lymphoma (BL) cases and is the cytogenetic hallmark of BL. [2,3] The t(8;14)(q24;q32) in BL is associated with an aggressive clinical course that responds well to high-intensity, brief-duration drug regimens with an overall favorable outcome. [3,4] Translocation of MYC is often detected as a secondary genomic abnormality at low frequencies in high-grade B-cell lymphomas, such as diffuse large B-cell lymphoma (DLBCL) (5-16%) and chronic lymphocytic leukemia (CLL) (0.1-2%). [2,4-6] In DLBCL, the presence of MYC translocation is associated with an aggressive disease with a poor prognosis and an unfavorable outcome. [5] MYC translocation has also been observed in 4-6% of acute lymphoblastic leukemia (ALL). [7] 8 8q24 ~647kb MYC 5 3 ~594kb ALL CLL NHL 1. Huret, J. L., et al Heim, S., et al. Cancer Cytogenetics, 2009 (3 rd Edition). 3. Blum, K. A., et al. Blood, (10): p Lones, M. A., et al. J Pediatr Hematol Oncol, (3): p Snuderl, M., et al. Am J Surg Pathol, (3): p Tibiletti, M. G., et al. Hum Pathol, (5): p Moorman, A. V., et al. Blood, (2): p Page 26

27 Cancer Genetics Italia DNA-FISH Probe Catalog MYC/IGH Two Color, Two Fusion Translocation Probe Ref: The MYC/IGH DNA-FISH Probe is designed to detect the translocation between the MYC gene located at 8q24 and the IGH gene located at 14q32, using fluorescence in situ hybridization (FISH). [1] The translocation between the MYC and IGH gene is designated as t(8;14)(q24;q32) and is the cytogenetic hallmark of Burkitt lymphoma (BL), which is found in 60-80% of patients. [2] In BL, t(8;14)(q24;q32) is associated with an aggressive clinical course that responds well to high-intensity, brief-duration drug regimens with an overall favorable outcome. [2,3] This rearrangement has been observed at lower frequencies in other non-hodgkin lymphomas (NHLs), such as diffuse large B-cell lymphoma (DLBCL) (<10%). [4,5] It also occurs less frequently in acute lymphoblastic leukemia (ALL) where it has been associated with an unfavorable outcome. [4,6] 8 ALL NHL MYC/IGH 14q q24 3 C segments J segments D segments ~647kbp MYC 5 3 IGH V segments ~594kbp 5 1. Huret, J. L., et al Blum, K. A., et al. Blood, (10): p Lones, M. A., et al. J Pediatr Hematol Oncol, (3): p Heim, S., et al. Cancer Cytogenetics, 2009 (3 rd Edition). 5. Akasaka, T., et al. J Clin Oncol, (3): p Moorman, A. V., et al. Blood, (2): p ~561kb ~420kb MYH11/CBFB Two Color, Two Fusion Translocation Probe Ref: The MYH11/CBFB DNA-FISH Probe is designed to detect the pericentric inversion of chromosome 16 (inv(16)) involving the MYH11 gene on 16p13 and the CBFB gene on 16q22 using fluorescence in situ hybridization (FISH). [1] The rearrangement of the MYH11 and CBFB gene results in a fusion of both genes. The inv(16) abnormality is found in ~5-8% of all of de novo acute myeloid leukemia (AML) cases and is associated with AML-M4eo subtype (based on FAB classification). [2,3] Inv(16) has also been observed in therapy related myelodysplastic syndrome (t-mds) cases and in eosinophilic blast crisis of chronic myelogenous leukemia (CML) cases. [1] Whether alone or as part of a complex patient karyotype, inv(16) is indicative of a good prognosis in AML cases. [1,4] MYH11 CBFB q22 16 ~444 kb ~472 kb p13 ~472 kb ~233 kb AML CML MDS 1. Huret, J. L., et al van der Reijden BA, et al. Oncogene 18: , Huret JL. inv(16)(p13q22),t(16;16)(p13;q22), del(16) (q22). Atlas Genet Cytogenet Oncol 4. Haematol. URL : Anomalies/inv16.html Andersen MK, et al. Genes Chromosomes Cancer 33: , Mrozek K, et al. Blood Rev 18: , 2004 MYH11/CBFB Page 27

28 PBX1/E2A PBX1/E2A Two Color, Two Fusion Translocation Probe Ref: The PBX1/E2A DNA-FISH Probe is designed to detect the translocation between the PBX1 gene located on 1q23 and the E2A gene located on 19p13, using fluorescence in situ hybridization (FISH). [1] The translocation between the PBX1 and E2A gene is designated as t(1;19)(q23;p13) and occurs in ~ 6% of pediatric and adult acute lymphoblastic leukemia (ALL) cases; as determined by conventional cytogenetics and reverse transcription-polymerase chain reaction. [2,3] In both pediatric and adult ALL, the translocation is correlated with a negative prognosis. [4,5] It may occur as a balanced translocation, der(19)t(1;19)(q23;p13), or as an unbalanced translocation, der(19), where only the derivative chromosome 19 is observed. [2] The unbalanced translocation, der(19), is the most common form and accounts for 75% of all PBX1/E2A rearrangements. [2] Both balanced and unbalanced translocations are sometimes observed in the same patient as separate clones. [2] 1 ALL 1q23 ~255kb 19 PBX1 5 3 ~326kb 1. Huret, J. L., et al Heim, S., et al. Cancer Cytogenetics, 2009 (3 rd Edition). 3. Shearer, B. M., et al. Br J Haematol, (1): p Soszynska, K., et al. Ann Hematol, (12): p Chiaretti, S., et al. Blood, (1): p p13 E2A 3 5 ~275kb ~307kb PML/RARA PML/RARA Two Color, Two Fusion Translocation Probe Ref: The PML/RARA DNA-FISH Probe is designed to detect the translocation between the PML gene on chromosome 15q24 (previously assigned to band 15q22) and the RARA gene on chromosome 17q21, using fluorescence in situ hybridization (FISH). The t(15;17) translocation is the diagnostic hallmark of acute promyelocytic leukemia (APL) and results in the fusion of the PML and RARA genes. [1] The presence of a PML-RARA fusion predicts a favorable response to differentiation therapy with all-trans retinoic acid (ATRA) and is currently the most curable subtype of acute myeloid leukemia (AML). [1-5] The t(15;17) translocation has also been identified in cases of chronic myeloid leukemia (CML) cases with promyelocytic blast crisis. 15q24 15 PML 5 3 AML CML 17q21 ~270kb 17 RARA 5 3 ~350kb 1. Melnick, A., et al. Blood, : Wan, T. S, et al. Oncol Rep, : Grimwade, D., et al. Blood, : Brockman, S. R, et al. Cancer Genet Cytogenet, : Huret, J. L., et al. ~570kb ~510kb Page 28

29 Cancer Genetics Italia DNA-FISH Probe Catalog RB1/D13S1009 Two Color, Enumeration Probe Ref: The RB1/D13S1009 DNA-FISH Probe is designed to detect loss of the RB1 gene on chromosome 13q14 relative to the control marker, D13S1009 on chromosome 13q34, using fluorescence in situ hybridization (FISH). The RB1 gene is a well characterized tumor-suppressor gene and bi-allelic inactivation of the gene due to mutations and/or deletions is causal for the development of Retinoblastoma (RB). Deletion of the RB1 gene is also common in a wide variety of solid tumors and hematologic malignancies such as chronic lymphocytic leukemia (CML), multiple myeloma (MM), acute myelocytic leukemia (AML), myelodysplastic syndrome (MDS), and chronic myeloproliferative disorders. [1-4] The RB1 gene is proximal to the D13S25 locus at 13q14, which is often co-deleted with the RB1 gene in some B-cell hematologic malignancies. t 13 RB1 5 3 ~305kb D13S1009 ~368kb elomere AML MDS CML MM 1. Khidr, L., et al. Oncogene, : Goodrich, D. W. Oncogene, : Genovese, C., et al. Oncogene, : Lohmann, D. R. RB1/D13S1009 Breast TP53/RARA Two Color, Enumeration Probe Ref: The TP53/RARA DNA-FISH Probe is designed to detect the deletion of the TP53 gene located on 17p13, relative to the control marker gene RARA located on 17q21, using fluorescence in situ hybridization (FISH). The TP53 gene is a known tumor suppressor gene. [1] Deletion of the TP53 gene is common in a wide variety of solid tumors [1, 2] and hematologic malignancies such as mature B-cell neoplasms, [3] myeloid disorders such as acute myeloid leukemia (AML), [4] and myelodysplastic syndrome (MDS). [5] Deletion of the TP53 gene has been associated with advanced stage, shortened survival, and resistance to treatment in several malignancies and solid tumors. [3,6] The loss of TP53 is also found in chronic lymphocytic leukemia (CLL) cases and is associated with a very poor outcome in CLL cases. [7] TP53 RARA ~226 kb 17 ~233 kb ~472 kb Colon/Rectal Lung AML CLL MDS 1. Bertheau, et al. Pathobiology, 2008 (Review). 75(2): Doak, S. H, et al. Br J Cancer, (9): Döhner, H., et al. J Mol Med, 1999 (Review). 77(2): Schoch, C., et al. Genes Chromosomes Cancer, (1): Silveira, C. G., et al. Leuk Res, (1): Carlebach, M., et al., Cancer Genet Cytogenet, (1): Laurenti, L., et al. Ann Hematol (1): TP53/RARA Page 29

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31 APPENDIX

32 Scoring Guidance & Signal Interpretation Signal interpretation depends on the DNA-FISH Probe type and should be made with the full knowledge of the product design. The following table divides the DNA-FISH Probe into two types: enumeration and translocation probes. Count as R=red, G=green, F=fusion Scoring Guidance Enumeration Probes Translocation Probes The boundary of each nucleus is clear; for each nucleus count as 2R2G signals. Count as 2R2G signals. Depending on the overlap, the red and green signal overlap can appear as yellow. Count as 2R2G signals; the green signal is a split. Count as 2R2G signals; the distance between the red and green signals is less than one signal width apart. Count as 2R2G signals; the green signal is stringy or dispersed. ** Count as >6R2G signals; one red signal is focally amplified. ** As is characteristic of certain probes that target a highly transcribed region (i.e. IGH). Count as 2F signals. Depending on the overlap, the red and green signal overlap can appear as yellow. Count as 2F signals; the distance between the red and green signals is less than one signal width apart. Count as 1R2G1F signals. Count as 2R2G signals; the distance between the signals is more than one signal width apart. Count as 1R2G signals. Do Not Count Nucleus is physically damaged and nuclei overlap prevents distinction of which signals belong to which nucleus. The true green signal and the green artifact are indistinguishable. The green artifact is so bright, it interferes with a proper evaluation. The signals are indistinguishable from the background. The irregular red signal and the green signal on the periphery could be artifacts. Page 32

33 Cancer Genetics Italia DNA-FISH Probe Catalog Filter Specifications The following table shows the filter requirements for Cancer Genetics Italia DNA-FISH Probes. Fluorophore Excitation max Emission max Green 496 nm 520 nm Red nm nm DAPI 360 nm 460 nm Gold 525 nm 551 nm Aqua 431 nm 480 nm Filter Maintenance Recommendation Filters age with use and should be regularly examined for dirt and/or imperfections which may occur over time. Heat from the high intensity transmitted light can etch and damage frequently used filters (such as the DAPI filter), resulting in dimmer images and unevenly illuminated areas. Such filters should be replaced. Handle filters according to manufacturers recommendations. Filter Specifications Troubleshooting 1. Why do I observe weak signals and how can I correct this? Weak signals may be observed due to use of an old mercury lamp ( run time >200 hours), unsuitable immersion oil, or unsuitable filters. Other causes may include an inadequate denaturation of the DNA-FISH Probe or insufficient pretreatment of the specimen (visible cytoplasm on the specimen appearing as a green haze). Attention should be paid to replace the mercury lamp after 200 hours of run time, to use immersion oil with no auto-fluorescence in the UV range, and to use the appropriate filters. The filter specifications table (see above) identifies the filter requirements for Cancer Genetics Italia DNA-FISH Probes. Also, check that the excitation and emission of the dyes fall within the range claimed by your individual filter manufacturer. In order to allow a better denaturation of the probe, an increased denaturing temperature and/or time may be used. Optionally, the specimen may be treated with pepsin prior to denaturation. 2. Why are there areas without signals? Some possible reasons for areas without signals include air bubbles during hybridization or application of an insufficient volume of probe. Another reason includes low permeability of the specimen to the probe, which results in resistance to hybridization. In this case, specimen may be treated with pepsin prior to denaturation. Before adding the coverslip, gently remove all air bubbles with a pipette tip or a needle. After adding the coverslip, gently remove any air bubbles by rolling a pencil eraser evenly across the coverslip like a rolling pin. Also, ensure there is sufficient probe volume (10 μl Probe/22 22mm target area). If the target area is larger or smaller than 22 22mm, then the volume of the probe should be proportionally increased or decreased. Troubleshooting 3. Why are the signals fading? The signals could be fading due to immersion oil, expired or oxidized Antifade/DAPI (deep purple/brown in color), or image acquisition (extended exposure to UV source). Page 33

34 Solutions to this problem could include using immersion oil with no auto-fluorescence in the UV range. The expiration date of the Antifade should also be verified. A slide can be saved by briefly washing off old Antifade with 2xSSC, drying, then applying fresh Antifade. If the Antifade does not contain DAPI, be sure to re-incubate the slides in DAPI prior to drying and mounting with Antifade. Another solution would be to close the shutter when not visualizing or analyzing the specimen, when acquiring images. It is important that the hybridized specimens are stored at 4 C, protected from light. In general, fluorescently labeled probes, both before and after hybridization, are readily photobleached by exposure to light. To limit this, handle probes and hybridized specimens in reduced light when possible. Incubation periods and slide drying should be done in the dark, such as in a closed drawer or box. 4. Why are there no signals? There may be no signals due to insufficient probe volume and/or lack of target/probe denaturation. Other reasons for absence of signals could be an inappropriate duration and/or temperature of incubation. Solutions of the issue include ensuring denaturation of the Probe and target DNA. When denaturing the target and the DNA-FISH Probe, make sure to respect the time and/or temperature recommended in the instructions for use, which is provided with the product. The temperature of the controlled hot plate must be checked prior to use and ensure that the hot plate is calibrated regularly. If a green or yellow haze is observed, there may be too much cytoplasm present, which impedes the probe from hybridizing to the target DNA. Optionally, the specimen may be treated with pepsin prior to denaturation. Troubleshooting 5. Why is the nuclear morphology compromised? The nuclear morphology can be compromised due to specimen over-denaturation or excessive pretreatment. To solve the over-denaturation problem, reduce the time and/or temperature of denaturation. Depending on the extent of the chromatin loss, it is possible to reduce or eliminate the pretreatment with pepsin (the pretreatment with pepsin is an optional step). The slides should be prepared according to the procedures established per the laboratory performing the FISH test. 6. Why is there a high background? A high background could be due to a number of items such as an unclean slide, the presence of cellular debris in the specimen, or allowing the slide to dry during either the hybridization or washing process. Another reason may be due to the wash buffers not being warm enough. Prior to dropping the specimen on the slides, the slides may be soaked in 70% ethanol and wiped with a Kimwipe. The pellet of specimen could be washed in fresh fixative. The specimen may be allowed to stand at room temperature (RT) for 2-3 minutes and allow the residual debris to settle down. When placing coverslips over slides, ensure a proper seal is made with the rubber cement so that it does not dry out. Also, when transferring slides in between washes, do not allow them to air dry prior to completion of protocol. 7. Why is there a high nuclear background? A high nuclear background could be the result of inadequate post-hybridization washing. Although rarely observed, it is advisable to increase the wash stringency and/or duration. Page 34

35 Cancer Genetics Italia DNA-FISH Probe Catalog Ordering Products CGI Italia s DNA-FISH Probes are available in a ready to use, 10 tests per vial format. Because we are dedicated to provide professionals the most clinically relevant products, we are constantly developing new reagents. If you would like more information about our pipeline or if you have specific inquiries, please contact us at support@cancergeneticsitalis.com. To place an order, please contact your local authorized distributor. To locate your local authorized distributor please visit our website at if a distributor is not located within your region, please contact us at purchase@cancergeneticsitalia.com. Ordering Productts If you are interested in distributing our DNA-FISH Probes or would like more information, please contact us at support@cancergeneticsitalia.com. Page 35

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37 DNA-FISH PROBE INDEX

38 Index By Probe Name Index By Probe Name vprobe Name Reference # Lesion Page 5p15, 9q34, 15q Hyperdiploidy 5, 9, 15 A20/PRDM1/SHGC del(6q23),del(6q21)/6p12 14 ABL1/BCR t(9;22) ALK Break Apart t(2p23) 15 ALK/NPM t(2;5) AML1/ETO t(8;21) 16 API2/MALT t(11;18) ATM/D11S del(11q22)/11p15 17 BCL6 Break Apart t(3q27) CCND1/IGH t(11;14) 18 D7S486/Cen del(7q31)/cen7 D13S25/D13S del(13q14)/13q34 19 D20S108/8q del(20q12) & trisomy 8 EGFR/Cen EGFR Amplification 20 EGR1/5p del(5q31)/5p15 ERBB2/Cen ERBB2 Amplification 21 FGFR3/IGH t(4;14) FHACT TM q26/5p15/20q13/Cen7 22 IGH/BCL t(14;18) IGH Break Apart t(14q32) 23 IGH/MAF t(14;16) IGH/MALT t(14;18) 24 MDM2/D12S q15/12p11 MLL Break Apart t(11q23) 25 MYB/SHGC del(6q23)/6p12 MYC Break Apart t(8q24) 26 MYC/IGH t(8;14) MYH11/CBFB inv(16) 27 PBX1/E2A t(1;19) PML/RARA t(15;17) 28 RB1/D13S del(13q14)/13q34 TP53/RARA del(17p13)/17q21 29 Page 38

39 Cancer Genetics Italia DNA-FISH Probe Catalog Index By Chromosome Number Chromosome Probe Name Reference Number Page 1;19 PBX1/E2A ALK Break Apart ;5 ALK/NPM BCL6 Break Apart , 5, 7, 20 FHACT TM ;14 FGFR3/IGH EGR1/5p , 9, 15 5p15, 9q34, 15q A20/PRDM1/SHGC MYB/SHGC D7S486/Cen EGFR/Cen MYC Break Apart ;14 MYC/IGH , 20 D20S108/8q ;21 AML1/ETO ;22 ABL1/BCR ATM/D11S MLL Break Apart ;14 CCND1/IGH ;18 API2/MALT MDM2/D12S D13S25/D13S RB1/D13S IGH Break Apart ;16 IGH/MAF ;18 IGH/BCL ;18 IGH/MALT ;17 PML/RARA MYH11/CBFB ERBB2/Cen TP53/RARA Index By Chrom. Number Page 39

40 Notes

41 Notes

42 Notes

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