Kathleen Donohue

A Quantitative Exploration of Surface Antigen Expression in Common B-Cell Malignancies Using Flow Cytometry

The use of flow cytometry to diagnose hematological malignancies has become routine due to its ability to often differentiate between morphologically similar diseases based on antigens expressed on the surface of malignant cells. In an attempt to expand on the utility of flow cytometry in the study of B-cell malignancies we have used the most reliable quantitative methodology, QIFI (quantitative indirect immunoflourescence assay), to study the expression of CD5, CD10, CD11c, CD19, CD20, CD22, CD23, and CD79b in 384 cases of several common B-lineage malignancies, including: B-ALL, CLL, SLL, hairy cell leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. The impetus behind this extensive, single institution study of surface antigens was two-fold: evaluating similarities and differences of antigen expression between B-cell neoplasms and finding additional clinical utility for the quantitative flow cytometric data generated. Our results show that each distinct malignant histology has its own quantitative pattern of surface antigen expression. In most cases, these quantitative patterns do not increase the ability of flow cytometry to distinguish between them. However, a high expression of specific antigens on a given B-cell malignancy may potentially identify optimal therapeutic targets for current and/or future monoclonal antibody-based therapies.

HLA class I antigen cell surface expression is preserved on acute myeloid leukemia blasts at diagnosis and at relapse

Human leukocyte antigens (HLA) class I molecules restrict the interaction between cytotoxic T cells and target cells. Abnormalities in HLA class I antigen expression and/or function may provide tumor cells with a mechanism for escaping immune surveillance and resisting T cell-based immunotherapies. The potential for applying T cell-based immunotherapy in the treatment of acute myeloid leukemia (AML) has stimulated interest in analyzing HLA class I antigen expression on leukemic blasts in this disease. Little information is available in the literature. We have analyzed HLA class I antigen expression on bone marrow samples from 25 newly diagnosed AML patients by indirect immunofluorescence staining with monoclonal antibodies. Five of these patients were also studied at relapse. Leukemic blasts were resolved from normal lymphocytes by staining with anti-CD45 antibody; CD45 expression is dim on leukemia cells, but bright on lymphocytes. HLA class I antigen expression was higher on leukemic blasts than on autologous lymphocytes in all but one case. Moreover, there was no significant change in HLA class I antigen expression at relapse. These results suggest that abnormalities in HLA class I antigens are infrequent in AML and should not represent a major obstacle to the application of T cell-based immunotherapies in this disease.

Truncated STAT proteins are prevalent at relapse of acute myeloid leukemia

Signal transducer and activator of transcription (STAT) proteins are implicated in the control of cell survival, proliferation and differentiation in response to hematopoietic cytokines. C-terminally truncated STAT isoforms (STATbeta), as opposed to the full length form (STATalpha), have a competitive or even transdominant negative effect on gene induction mediated by the STAT pathway. We have previously demonstrated that while constitutively active STAT proteins were detected in ten of 36 (28%) for STAT3 and eight of 36 (22%) for STAT5 in pretreatment samples from newly diagnosed acute myeloid leukemia (AML) patients, a significantly larger fraction of samples [21 of 27 (78%)] expressed STATbeta proteins. To determine whether STATbeta expression was maintained or increased after relapse in AML, we compared STAT activity and isoform expression at diagnosis and at relapse in 17 patients. In this selected group, constitutively active STAT3 was detected in 13 of 17 (76%) AML samples at diagnosis but was detected in only four of these patients at relapse. Constitutively active STAT5 was detected in three of 17 (18%) AML samples at diagnosis; but only two at relapse. In contrast, STATbeta protein expression was observed in 12 of the 17 pretreatment samples (71%) and in 16 of 17 samples at relapse. Only one patient did not express STATbeta at relapse. Our results suggest that STATbeta isoform expression, rather than level of constitutive activity, may be involved in disease progression in AML.

Arsenic trioxide affects signal transducer and activator of transcription proteins through alteration of protein tyrosine kinase phosphorylation

Purpose: Arsenic trioxide decreases proliferation of acute myeloid leukemia (AML) cells, but its precise mechanism of action is unknown. Experimental Design: We studied the effect of arsenic trioxide on patient samples and the AML cell line HEL, which, like leukemic blasts from 50% of AML cases, has constitutively activated signal transducer and activator of transcription (STAT) proteins. Results: Arsenic trioxide induced mitotic arrest starting at 24 hours and significant cell death at 48 hours. These events were preceded by an arsenic trioxide dose-dependent down-regulation of activated STAT proteins starting at 6 hours. We hypothesized that arsenic trioxide inhibits protein tyrosine kinases (PTK), which, among others, phosphorylate and activate STATs. We therefore studied arsenic trioxide effects on Janus kinases and on three oncogenic PTKs that are known to activate STATs [FLT3, ZNF198/fibroblast growth factor receptor 1 (FGFR1), and BCR/ABL]. Arsenic trioxide reduced STAT3 activation by Janus kinases, altered phosphorylation and electrophoretic mobility of ZNF198/fibroblast growth factor receptor 1, reduced kinase protein level, and decreased STAT3 protein phosphorylation. Arsenic trioxide also reduced the phosphorylation of BCR/ABL and FLT3 with corresponding decreased STAT5 phosphorylation. Conclusions: These results suggest a selective activity of arsenic trioxide on PTKs and will assist in developing clinical trials in AML.

Expression of intestinal trefoil factor (TFF-3) in hepatocellular carcinoma

AbstractBackground: Trefoil peptides (TFF-1, 2, 3) are a family of protease-resistant regulatory factors that play a role in mucosal restitution, angiogenesis, apoptosis, and tumor progression. Intestinal trefoil peptide (TFF-3) expression has been demonstrated in benign hepatobiliary diseases, but there are limited data regarding its expression in HCC.n Methods: Thirty consecutive cases of HCC from 1998 to 2003 were studied. Immunohistochemistry was performed on formalin-fixed paraffin-embedded blocks of HCC using polyclonal antibody to TFF-3. TFF-3 expression was classified as strong, moderate, weak, focal, and negative. Clinical data were obtained per an IRB-approved protocol.n Results: Median age was 69 yr (range: 39–83 yr). Twenty- three patients were males and 7 were females. Treatments included hepatic resection (n=16), chemo-embolization (n=4), combined modality therapy (n=5) and no treatment (n=4). HCC was well differentiated in 12 (40%), moderately differentiated in 13 (43%), and poorly differentiated in 5 (17%) patients. TFF-3 expression was detected in 28/30 (93.3%) patient samples. Sixteen patients (53%) had moderate and 1 (3%) patient had strong TFF-3 expression. Tumor/normal tissue interface was assessable in 21 cases; 11 cases expressed TFF-3 at the interface. There was a strong correlation between tumor grade and TFF-3 expression, wherein poorly differentiated tumors had moderate/strong TFF-3 expression (p=0.008). There was no correlation between TFF-3 expression and survival (p=0.77). Furthermore, there was no correlation among age, disease stage, and survival.n Conclusion: TFF-3 is commonly expressed in HCC and its expression correlates with tumor grade.

Long-term outcome of stage I seminoma.

Purpose:To report on long-term outcomes among patients with stage I seminoma treated by orchiectomy with or without adjuvant radiation. Materials and Methods:A retrospective review of medical records of patients treated between 1974 and 2002 was undertaken to identify factors associated with patient outcomes. Results:With a median follow-up of 7.7 years, 80% (4 of 5) of the surveillance group experienced a disease relapse, while only 3% (2 of 70) in the radiation therapy group had disease relapse. This difference in relapse rates was statistically significant, but there was no significant difference in overall survival between the 2 groups. There was a significant relationship between patient age and disease relapse, whereby all of the relapses were seen in patients younger than 36 years at diagnosis (P = 0.03). Of the total 75 patients, 7 (9%) developed second primary tumors. Six of them (6 of 7) were treated with adjuvant radiation, and 1 patient (1 of 7) was on surveillance. Conclusion:In this study, risk of relapse was significantly associated with surveillance and in patients younger than 36 years at diagnosis. These results suggest that surveillance can only be safely adopted for patients who can be followed up closely. We consider adjuvant radiation a very effective choice despite the low risk of associated secondary malignancies.

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy.

Pure Tubular Breast Carcinoma: A 34 year Study of Outcomes

To the Editor: Tubular carcinoma of the breast is a rare, well-differentiated variant of invasive ductal carcinoma which is thought to carry an excellent prognosis (1–3). The purpose of this study is to review the Roswell Park Cancer Institute experience with tubular breast cancers. Between September of 1971 and January 2004, 8,832 patients were treated for both invasive and noninvasive breast cancer, and 44 (0.5%) were coded as tubular carcinoma or invasive ductal carcinoma with tubular features. Tumors were considered tubular only if the pathology report specifically identified the histology as tubular carcinoma, and did not include a description of other histologic types. This process identified 27 of the 44 cases as tubular cancers, while 17 were considered of mixed tubular or other histology. Staging was according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, 6th edition (4). Patient follow-up time was calculated using the potential follow-up method (5). The Kaplan–Meier method was used to derive survival probabilities (6). The mean patient age was 59.3 years and all patients were Caucasian. Seventy-eight percent (21 ⁄ 27) of patients had stage I disease, while 19% (5 ⁄ 27) had stage IIA disease, and 4% (1 ⁄ 27) was stage IIIA. Forty-one percent (11 ⁄ 27) of patients had a first-degree relative with breast or ovarian cancer. The initial detection of a breast tumor was made by mammogram in 52% (14 ⁄ 27) of cases, while a mass was palpated by the patient or a physician in 37% (10 ⁄ 27) of cases. Tumor characteristics are summarized in Table 1. The mean tumor diameter was 1.09 cm (range, 0.2–4.2 cm). The majority of patients, who had lymph nodes assessed by either axillary node dissection or a sentinel node biopsy, had no positive lymph nodes on presentation (84%), while 11% (2 ⁄ 19) had 1–3 positive nodes and 5% (1 ⁄ 19) had four positive axillary node metastases. Ductal carcinoma in situ was reported in 59% (16 ⁄ 27) of patients. All patients who had hormone receptor status assessed (16 ⁄ 16) were estrogen receptor (ER) positive, while the majority of patients (10 ⁄ 14) were also progesterone receptor positive. The upper outer quadrant was the site of occurrence in 67% (18 ⁄ 27) of patients and with 89% (24 ⁄ 27) of patients having unifocal disease and 11% (3 ⁄ 27) of patients having multifocal distribution. Seventy-four percent (20 ⁄ 27) of patients were treated with breast-conserving surgery, while 22% (6 ⁄ 27) underwent modified radical mastectomy (MRM), and one patient received no additional surgery after biopsy. In the post-operative setting, one third of patients (9 ⁄ 27) received no adjuvant treatment. Over half (15 ⁄ 27) of patients received post-operative radiation (XRT), 14 of which had undergone a previous breast conserving surgery, and one of which had a previous MRM. Thirty percent of patients (8 ⁄ 27) had XRT alone in the adjuvant setting, while 19% (5 ⁄ 27) receive a combination of XRT and hormonal therapy (HT), and 7% (2 ⁄ 27) received XRT and HT in combination with systemic chemotherapy. Eleven percent (3 ⁄ 27) of patients received HT alone in the adjuvant setting. With a median follow-up period was 5.3 years (range; <1–24 years), none of the patients had evidence of either local or systemic recurrence, and no patients had died of breast cancer. Seventy-eight percent (21 ⁄ 27) of patients were alive at the time of last follow-up and 22% (6 ⁄ 27) had died of other causes not related to breast cancer. Of patients with at least five-years of follow-up, overall survival at 5-years was 89% (16 ⁄ 18) while for those with at least 10-year follow-up, overall survival is 70% (7 ⁄ 10). Twenty-two percent of patients (6 ⁄ 27) were diagnosed with a second cancer, two of which developed infiltrating ductal carcinoma. Of those who developed a second malignancy, the only patient who received XRT, was a patient who developed infiltrating ductal carcinoma in the contralateral breast 7 years after completing XRT. Address correspondence and reprint requests to: Gary Y. Yang, MD, Department of Radiation Medicine, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263, or e-mail: Gary.yang@roswellpark.org.