hiv aids related lymphoma immunophenotypes in biopsy samples

HIV/AIDS-related Lymphoma immunophenotypes in biopsy samples from patients during pre HAART and HAART treatment periods Ayers LW1, Hackman BA1, McGrath MS2, Silver S3, Orenstein J3, AIDS and Cancer Specimen Resource (ACSR) and Bhatia K4 Office of AIDS Malignancy Program 1Ohio State University, Columbus, OH; 2University of California San Francisco (UCSF), San Francisco, CA; 3George Washington University, Washington, DC; 4National Cancer Institute, Bethesda, MD. USA. ayers.1@osu.edu Abstract AIDS-associated malignant lymphoma tissue micro-arrays (TMAs) allow parallel immunohistochemical (IHC) testing for detection of genetic/protein targets and in-situ hybridization (ISH) for other targets. Archived formalin-fixed paraffin-embedded tissues (FFPET) are a resource for rapid translation from molecular target discovery to clinical disease application. The NCI ACSR provides HIV/AIDS-related biological samples and negative controls to approved investigators. The inclusion in these TMAs of lymphoma tissues from the beginning of the AIDS epidemic in the USA allows a retrospective comparison of early lymphoma phenotypes (preHAART) with current lymphoma phenotypes. TMA blocks prepared by the ACSR from FFPET of AIDS-related lymphoma (108 cases), control lymphoma (1 case), control lymph node (6 cases) from 1980-2005 were sectioned and stained with H&E, IHC (Dako): CD 45, CD3, CD20, CD10, CD138, MIB1, MUM-1, Bcl-6 and ISH for EBV (EBER) as part of an ACSR TMA quality control protocol. The results of IHC and ISH staining from pre (1980-1995) and post (1996-2005) HAART intervals were compared for patient demographics, immunophenotypes and Epstein Barr virus (EBER) presence within the lymphoma cells. Pre-HAART cases n=65, (91% male) vs post-HAART cases n=43, (77% male). Age range of both groups was 21-62 years with a median age of 38 and 39 years respectively. Lymphomas in females increased (9 to 23%). Overall, 53% of lymphomas were found in cases 30-39 years of age. EBER rich tumors decreased (31 to 21%). Plasmablastic or plasma cell rich lymphomas with CD138 marker decreased (31 to 16%). CD10 ,decreased (23 to 9%). Frequency of other analytes increased: CD45 (52 to 77%), MUM-1 (22 to 42%), MIB-1 (57 to 95%), Bcl-6 (26 to 40%), There was little change in frequency of CD3 (26 to 33%) and CD20 (68 to 63%). A few samples showed loss of target integrity within each time interval, but not necessarily over time. The number of women with HIV related lymphoma has increased over the study period. The age at which lymphoma appears has not yet been extended with HAART although the total number of lymphomas available has decreased in the post HAART period. The change in the overall immuno-staining analytes may represent an actual change in phenotypes, such as might be associated with a change in the prevalence of Epstein Barr virus and/or Kaposi’s Sarcoma associated virus (KSHV). Or changes in phenotypes may represent more highly differentiated tumors or better performance of stains in more recent tissue. Diminished molecular target integrity appears to be a function of individual sample potential/quality rather than sample age. The NCI ACSR provides HIV/AIDS-related biological samples and negative controls to approved investigators. http://acsr.ucsf.edu Table 2: Comparison of 9 IHC stains in pre HAART period (1980-1995) Study Number 1000720 5245 5253 1003150 5233 5239 5241 5260 420042 420108 420110 4000770 5254 4000900 4305050 1000690 5247 1000530 5252 5256 5263 5268 420051 5244 420106 5255 4000770 4000500 5269 10002860 4000860 420039 4000500 420031 420149 4004234 1001120 5242 5249 5251 5266 420040 420043 420028 5258 5261 5265 5267 420151 5271 1000520 1001490 1009730 420115 10001130 4309313 4305040 5246 420087 5270 4000460 4000770 5234 420101 4100160 Tissue Type axillary mass LN neck mass LN LN abdom tumor submandib mass LN neck mass skin-abdominal wall spinal mass lung nodule neck mass LN neck mass inguinal mass LN sm bowel back mass neck mass LN colon lung nodule thoracic tumor liver abdom nodule brain LN submandib mass mouth brain back LN nasopharynx lung nodule spleen testis LN LN neck mass LN hard palate lung lung nodule tonsil LN neck mass LN LN colon testis LN axillary mass parotid gland liver vulva bone marrow medias mass skin & sub Q LN L ventricle tracheal nodule LN lung nodule neck mass HIV pos pos pos Age 44 36 32 32 22 52 40 33 46 37 53 39 31 62 45 43 39 33 36 39 36 39 29 26 25 31 34 39 38 37 34 38 39 40 53 38 57 42 56 42 46 35 43 36 39 45 38 35 54 43 40 38 37 61 43 50 39 28 21 36 33 38 46 31 36 Sex M M M M M M M M M M M M M F F M M M M M M M M M M M M M M M M M M M M M M M M M M M F M M M M M M M M M M F M F M F M M M M M M M Surgery Date 1995 1985 1987 1995 1981 1983 1984 1988 1990 1990 1990 1990 1987 1993 1994 1995 1985 1995 1987 1987 1990 1992 1987 1985 1989 1987 1990 1988 1992 1995 1992 1990 1988 1992 1992 1992 1995 1984 1986 1986 1990 1990 1990 1993 1988 1989 1990 1991 1993 1994 1995 1995 1995 1993 1995 1991 1993 1985 1990 1992 1993 1990 1981 1987 1994 MIB1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 1 1 1 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 CD45 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 0 0 0 0 4 4 4 4 4 4 4 2 2 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 2 1 0 0 CD20 4 4 4 4 4 4 4 4 4 4 4 4 2 0 0 0 4 4 4 4 4 4 4 4 4 1 1 0 0 0 0 1 0 4 4 4 0 4 4 4 4 4 4 1 4 4 4 4 4 4 4 4 4 1 1 0 0 4 4 4 4 1 4 0 0 MUM1 4 4 2 1 0 0 0 0 0 0 0 0 0 0 4 4 4 4 4 2 2 2 0 0 2 4 2 0 4 4 0 0 0 0 0 0 2 2 4 0 0 0 0 4 4 2 4 2 0 0 0 0 0 3 1 0 4 2 0 0 0 0 0 0 0 Bcl6 4 1 2 4 4 0 4 0 4 4 4 0 1 0 0 0 1 0 2 4 4 2 0 0 0 0 0 2 0 0 0 0 0 0 0 1 1 1 1 0 0 0 1 4 2 2 0 2 4 4 4 4 4 0 4 1 0 4 0 0 0 0 CD138 2 0 2 4 4 4 0 0 4 0 0 0 0 0 0 0 0 0 0 4 4 0 0 0 0 0 4 4 4 0 0 4 4 0 0 4 4 4 0 0 4 4 0 4 0 2 4 4 0 0 0 0 1 1 1 0 0 4 0 0 0 0 0 CD10 0 0 4 0 4 3 4 0 0 0 0 0 4 0 0 0 0 0 0 4 0 0 0 4 0 0 0 0 4 4 0 0 0 0 0 0 0 0 0 0 0 4 0 4 0 4 0 0 0 4 4 0 0 0 0 0 0 0 0 4 0 0 2 0 0 CD3 EBER 2 0 0 4 0 4 0 0 0 4 0 2 0 4 4 4 4 2 2 1 0 2 1 2 4 4 2 4 4 4 2 0 4 1 1 2 0 0 0 1 4 2 4 2 0 0 2 2 2 4 4 4 2 2 2 4 0 0 4 1 0 0 4 2 Results Figure 2: Comparison of percentage of IHC grades of each IHC stain in pre-HAART (1980-1995) vs HAART (1996-2005) periods. Diagnosis immunoblastic, large cell 100% 90% 2 4 2 0 2 1 diffuse large B cell pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos 80% large cell diffuse large cell diffuse large B cell immunoblastic, large B cell B cell, intermediate grade diffuse large T cell, Ki-1 pos immunoblastic, large cell immunoblastic, diffuse large cell 0 IHC grade 3-4 IHC grade 2 IHC grade 1 IHC grade 0 70% 60% 50% 40% 2 1 4 2 1 0 2 large cell large cell and small cleaved immunoblastic, large B cell immunoblastic, large B cell immunoblastic, large cell B cell angioinvasive large cell immunoblastic, large B cell diffuse large cell diffuse large cell immunoblastic, diffuse large B cell immunoblastic, diffuse large cell 30% 20% 10% 0% 3 0 2 3 4 pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos 2 1 4 4 2 immunoblastic, diffuse large cell immunoblastic, diffuse large cell immunoblastic, diffuse, high grade diffuse large B cell large cell immunoblastic, diffuse large cell B cell, low grade immunoblastic with plasmacytoid differentiation diffuse large cell high grade B cell immunoblastic large B cell large cell lymphoma Figure 3: Common IHC/ISH stain phenotypes are demonstrated* by patient case in rows below. Reactions are usually strong when present. pr e immunoblastic large cell diffuse large cell large cell Introduction • • Archived formalin-fixed paraffin-embedded tissues (FFPET) are a resource for rapid translation from molecular target discovery to clinical disease application. The NCI ACSR provides HIV/AIDS-related biological samples and negative controls to approved investigators.1 Tissue Microarray characteristics • Many samples (up to 700) processed per slide • Saves reagents, effort, space • Precious tissue conserved by using small cores Figure 1: Constructing TMA Methods/Tools • TMA blocks prepared by the ACSR from FFPET of AIDS-related lymphoma (108 cases), control lymphoma (1 case), control lymph node (6 cases) from 19802005 were sectioned and stained with H&E, IHC (Dako): CD 45, CD3, CD20, CD10, CD138, MIB1, MUM-1, Bcl-6 and ISH for EBV (EBER) as part of an ACSR TMA quality control protocol. • Results of IHC and ISH staining from pre (1980-1995) and HAART(1996-2005) intervals were compared for patient demographics, immunophenotypes and tumor associated Epstein Barr virus (EBER). Table 1: Demographic summary of HIV+ cases with lymphoma total male female age range median age age by decade 21-29 30-39 40-49 50-59 60-62 pre HAART 1981-1995 65 58 (91%) 6 (9%) 21-62 38 6 34 15 8 2 HAART 1996-2005 43 33 (77%) 10 (23%) 21-60 39 3 19 14 5 1 2 2 Case 1 2 Case 2 Table 3: Comparison of 9 IHC stains in HAART period (1996-2005) Study Number 420246 4301250 5277 4006967 4007262 1007150 420251 4002750 4100400 1053050 1053628 5301 420247 1016590 5281 5283 5287 5291 5293 1053010 5284 1007690 1009920 4309286 4004324 5303 420252 4307880 4309053 4305000 4305000 5296 4202825 5304 4004303 1003370 4305080 5278 4308100 1020550 4000120 4100270 4301580 Tissue Type HIV Age Sex Surgery Date 1997 1998 1996 1996 1996 1997 1998 1997 1999 2001 2002 2003 1997 1997 1998 1999 1999 2000 2000 2001 1999 1998 1997 2001 2003 2004 1998 2000 2001 1997 1997 2002 1999 2004 2003 1996 1998 1998 1996 1997 1997 1997 1999 MIB1 CD45 CD20 MUM1 Bcl6 CD138 CD10 CD3 EBER Case 3 Diagnosis diffuse large B cell large B cell, plasmacytoid features diffuse large B cell diffuse large B cell immunoblastic, diffuse large cell diffuse large B cell diffuse large B cell lymphoma diffuse large B cell diffuse large B cell diffuse large B cell diffuse large B cell Case 4 TMA Construction2 • Extract tiny (0.6 mm - 2.0 mm diameter) core from donor (source) tissue block • Insert tissue core into recipient paraffin block • Cut paraffin sections from the recipient block • Place on slide, process and stain bone marrow brain gastric tumor LN inguinal mass sm bowel sm bowel LN neck mass thyroid LN LN testis axillary mass groin mass medias mass medias mass LN LN parotid mass LN LN LN scrotum LN LN LN sm bowel LN mouth floor neck mass LN endometrium LN tracheal fistula nasal tissue heart LN lung tumor LN LN LN GE junction pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos 32 37 58 31 37 33 60 44 49 37 55 44 41 57 33 24 21 44 43 43 38 52 37 36 29 46 32 38 53 40 45 40 36 46 30 38 48 34 39 39 35 42 F M F M F M M M M M M F M M M F F M M M M M M M M M M M F F F M F M M M M M M M M M M 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 2 2 1 1 0 0 0 0 4 4 4 0 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 0 0 0 0 4 2 2 1 0 0 4 0 0 0 1 0 0 0 2 4 4 4 4 4 4 4 4 4 2 1 2 2 2 2 2 0 0 0 0 0 0 2 4 4 4 0 0 2 0 4 4 4 4 0 4 4 0 1 0 0 1 4 4 0 0 0 4 4 4 2 4 4 4 4 1 4 1 4 3 4 4 4 4 2 0 0 0 0 2 0 2 0 0 0 4 4 0 0 2 0 1 0 0 1 1 0 0 0 1 0 0 0 0 4 4 0 0 0 0 4 0 0 0 0 0 2 1 0 4 0 0 0 1 0 4 4 0 0 4 0 1 0 4 0 0 0 0 0 0 0 1 0 0 0 0 2 0 0 2 2 1 0 0 0 0 0 0 0 0 1 4 4 0 0 0 0 1 0 4 4 0 0 0 0 3 2 4 2 2 4 4 3 2 2 2 0 0 0 1 0 4 4 3 0 0 0 0 0 B 0 0 0 0 0 0 0 0 0 0 0 0 4 4 4 B 1 4 0 2 0 4 0 0 0 4 0 2 0 Case 5 Case 6 Case 7 *Scanned with ScanScope (Aperio, Vista, CA), displayed with AnalyzeCase3. large B cell large B cell large B cell diffuse large B cell diffuse large B cell immunoblastic, diffuse large B cell immunoblastic, diffuse large B cell, high grade large B cell NK type NK type 1. AIDS and Cancer Specimen Resource web site [http://acsr.ucsf.edu] 2. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998 Jul;4(7):844-7. 3. Mid-Region AIDS and Cancer Specimen Resource web site [http://www.acsr.mid-region.org] 4. Chu PG, Loera S, Huang Q, Weiss LM. Lineage determination of CD20- B-Cell neoplasms: an immunohistochemical study. Am J Clin Pathol. 2006 Oct;126(4):534-44. 5. Hans CP, Weisenburger DD, Greiner TC, et. al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004 Jan 1;103(1):275-82. Epub 2003 Sep 22. diffuse large B cell, plasmacytoid features immunoblastic high grade B cell diffuse large B cell immunoblastic, diffuse large cell anaplastic large cell, Ki-1 pos, null type Hodgkin's large cell and small cleaved -H AA H R AA T R EB T E pr EB R eH ER AA H R AA T pr R CD eT 3 H AA CD 3 H R AA T pr R CD eT 1 H AA CD 0 10 H R AA T R CD T 1 pr C 38 eD 13 H AA 8 H R AA T pr R Bc eT l6 H Bc AA l6 H R AA T R MU pr T M eM 1 H AA UM 1 H R AA T R CD pr T 2 eC 0 H D AA 20 H R AA T R CD pr T 4 eC 5 H AA D45 H R AA T R MI T B M 1 IB 1 Conclusions • The change in the overall immunotyping analytes may represent an actual change in phenotypes, such as might be associated with a change in the prevalence of Epstein Barr virus and/or Kaposi’s sarcoma associated virus (KSHV). Changes in phenotypes may represent more highly differentiated tumors or better performance of stains in more recent tissue. Diminished molecular target integrity appears to be a function of individual sample potential/quality rather than sample age. The NCI ACSR provides HIV/AIDS-related biological samples and negative controls to approved investigators. The ACSR (U01-CA066531) is a NCI supported resource. • • • • References