Giordano, Karin F.

Abstract CLL/SLL and other CLPD are associated with an increased risk of autoimmune hemolytic anemia (AIHA), immune thrombocytopenic purpura (ITP), pure red blood cell aplasia (PRBCA), and autoimmune agranulocytosis (AIAG). CLL patients with AIHA and ITP have advanced stage disease according to the Rai and Binet clinical classifications, implying a poor prognosis. However, previous community based studies suggest that patients with CLL and immune cytopenias could have a similar prognosis to other patients with CLL. To further define the prognostic significance of autoimmune cytopenia, we studied a cohort of patients seen at the Mayo Clinic Rochester (MCR). Methods The MCR Division of Hematology Chronic Lymphoproliferative Disease Database was used to identify patients with CLPD and autoimmune complications (AID) over a 10 year period (1 January 1995 to 30 December 2004). The clinical records of all patients with AID were thoroughly reviewed and collated with additional biological data. Results Forty-one (6%) of 665 patients with CLPD had AID. Of the patients with CLPD and AID, 21 (51%) had AIHA, 19 (46%) had ITP, 8 (20%) had PRBCA and 2 (5%) had AIAG. 17% of patients had 2 or more AID diagnoses. In 4 patients (10%) the diagnosis of AID preceded the diagnosis of CLPD, and in 7 patients (17%) the diagnoses were made concurrently. In the 32 patients with CLL, the median lymphocyte count at diagnosis was 24.8 x 109/L (5.18 – 121.8). Of 27 CLL patients who had interphase FISH analysis performed, 12 (44%) had abnormalities associated with poor prognosis CLL (17p13–11% 11q22–22%, 12+ 11%), and 15 (56%) had results associated with a more favorable prognosis (no detectable abnormalities or 13q14 deletion as the sole abnormality). CD38 was positive (> 30% of cells) in 38% of 21 tested patients. 39 patients required treatment for their AID. Treatment included corticosteroids (90%), splenectomy (31%), chemotherapy (31%), and rituximab (46%). Response rates following the first treatment for AID were: AIHA 53% complete response (CR) and 26% partial response (PR); ITP 56% CR and 22% PR; PRBCA 38% CR and 38% PR; AIAG no responses. Of the 41 patients seen over the 10 year period, there were 4 deaths (2 from CLPD). Conclusions This study of a large cohort of community and referred patients showed a prevalence of AID similar to that reported in community based studies. We found an equivalent prevalence of AIHA and ITP as well as a substantial number of patients with 2 or more AID complications. This could be due in part to detailed workup of all cytopenias. In addition, 27% of patients did not have a prior diagnosis of a B cell malignancy when presenting with AID. The measured biological risk factors (FISH anomalies and CD38) suggest that CLL patients with AID do not have a higher percentage of high risk CLL. The majority of patients responded well to AID treatment. Mortality appears to be lower than CLL patients with non-autoimmune cytopenia. These preliminary data suggest that AID is a common and often presenting complication of CLL/SLL and other CLPD. Early diagnosis and management of AID could lead to improved outcomes.

Abstract Background: In B-CLL, the observation of interphase cells with hemizygous D13S319 deletion at 13q14 (13q-x1) as a sole anomaly in blood is widely considered a favorable prognosis. The observation of cells with 11q-, +12 or 17p- has been associated with a relatively poor prognosis. Over the past 1.5 yrs, 16.2% (174/1,076) of patients (pts) referred for fluorescence in situ hybridization (FISH) testing for B-CLL in our clinical practice had a clone with homozygous D13S319 deletion (13q-x2), but the prognostic significance of this observation is poorly understood. Moreover, 39.3% (142/361) of pts with unfavorable FISH anomalies have 13q-x1 and/or 13q-x2 and the clinical significance of this observation is also unknown. Thus, we investigated pts with 13q- (with or without other chromosome anomalies) to establish the relative clinical significance of 13q- in B-CLL. Methods: We studied 333 pts with B-CLL sampled between 9/1999 and 6/2004 who had FISH performed on interphase nuclei from blood. The FISH probe set was designed to detect 6q-, 11q-, +12, 13q-, 17p-, and translocations involving IgH at 14q32. We classified pts into four groups: 13q-x1 only (group 1), 13q-x1 and 13q-x2 only (group 2), 13q-x2 only (group 3) and 13q-x1 and/or 13q-x2 plus other FISH anomalies (group 4). FISH groups were compared with gender, age, Rai stage, treatment status, time to treatment, CD38 and IgVH mutation. Results: Of the 333 pts, 171 (51.3%) had a 13q-: 71 were in group 1, 25 in group 2, 26 in group 3 and 49 in group 4. %CD38+ differed significantly across FISH groups; in pairwise analyses, the proportion of pts with >30% CD38+ was significantly greater for pts in group 4 vs. group 3 (p=0.0015) although no significant differences were observed for group 3 vs. group 1 or vs. group 2. Pts in group 3 were not significantly different from other FISH groups for Rai stage, IgVH mutation or gender. The median percentage of abnormal nuclei for pts with group 1 was 54.5% vs. 79.5% for pts in group 4 (p<0.0001). The median percent abnormal nuclei for pts in groups 3 and 2 was 72.5% and 68.5%, respectively. Median % abnormal nuclei for group 3 was not significantly different than the other FISH groups. Treatment status was available on 147 pts, where the proportion of pts who had treatment in each group was as follows: group 1, 15/66; group 2, 2/22; group 3, 4/21; and group 4, 9/38. Due to limited sample size and heavy censoring, any analysis on time to treatment is preliminary; however, these early analyses suggest group 4 pts have a lower median time to treatment (9 yrs) compared to groups 3 and 1 (12.3 and 13 yrs, respectively). Conclusions: This study has generated new information about the 13q- anomaly in B-CLL. First, known prognostic markers for B-CLL pts with 13q-x2 are not significantly different than for pts with 13q-x1 or 13q-x1/13q-x2. Second, 13q-x1 and/or 13q-x2 occurring with other unfavorable FISH anomalies have an unfavorable prognosis; i.e. potential benefits of 13q- are trumped when it is observed with unfavorable FISH anomalies. Thus, patients with any form of 13q- alone may have indolent disease while patients with 13q- and unfavorable FISH anomalies should be considered to be in a more aggressive phase.

Abstract Patients with CLL complicated by autoimmune diseases causing cytopenias (AID) are often considered to have clinically advanced disease (Rai stage III - IV, Binet C). With the use of automatic differential counts and flow cytometric immunophenotyping of peripheral blood, CLL is now most often diagnosed in asymptomatic patients with early stage disease. These changes in clinical practice could have an impact on clinical presentation and prognosis of CLL patients with AID. To more fully define AID complicating CLL, we conducted an observational study of a large cohort of CLL patients. Methods All 1737 patients with chronic B cell lymphoproliferative diseases seen in the Division of Hematology at Mayo Clinic Rochester from January 1, 1995 to December 30, 2004 were included. Lymphoid malignancies were classified using WHO and modified NC-WG 1996 criteria (CLL = 1649, “atypical” CLL = 4, leukemic phase of lymphoma = 9, chronic B cell lymphoproliferative disorders (not otherwise classified) = 75). Autoimmune hemolytic anemia (AIHA) was defined as a Hgb ≤ 10 g/dL with at least one appropriate marker of hemolysis (elevated indirect bilirubin, LDH, or reticulocyte count; or increased bone marrow erythropoiesis without bleeding) and evidence of an autoimmune mechanism (positive DAT, cold agglutinins), or at least 2 markers of hemolysis in absence of evidence of bleeding or hypersplenism. Immune thrombocytopenia (ITP) was defined as a platelet count of ≤100 x 109/L with normal/ increased bone marrow (BM) megakaryocytes or normal/increased absolute reticulocyte count. Pure red blood cell aplasia (PRBCA) was defined as a Hgb ≤ 10 g/dL, reticulocytopenia, and an isolated absence of BM erythrocyte precursors. Autoimmune granulocytopenia (AIG) was defined as sustained neutropenia (< 0.5 x 109/l) in the absence of chemotherapy for at least 8 weeks and with decreased/absent BM granulocyte precursors. Results Seventy four (4.5%) CLL patients had AID (78% male, median age at diagnosis 67 yr). The diagnosis of AID preceded CLL in 12% and was concomitant with the diagnosis of CLL in 15% of patients. Of the CLL patients with AID, 55% had AIHA, 47% ITP, 10% PRBCA and 4% AIG; 10% had coincident AIHA and ITP (Evans syndrome). Most AIHA (74%) patients presented with symptomatic anemia but 68% of the ITP patients were asymptomatic at diagnosis. Forty one patients (55%) developed AID prior to receiving any treatment for CLL. Only 9 of the 33 patients who developed AID after treatment for CLL had received purine analogues. Eighteen (24%) patients with CLL and AID died; the estimated median survival was 12.4 yr. Discussion In this CLL population with predominantly early stage disease at diagnosis (> 60% Rai stage 0), AID is clinically distinct from that previously reported in patients with more advanced disease. A higher proportion of patients had ITP which was usually asymptomatic. Most patients with AID had not previously required therapy for CLL. Purine analogue therapy did not appear to be a major cause of AID in this population. Overall survival was considerably more favorable than that previously reported with CLL induced bone marrow failure (Rai stage III-IV, Binet C). We conclude that the earlier diagnosis and subsequently longer follow up of patients with CLL has altered the clinical presentation and prognostic consequences of AID. A more detailed analysis of the correlation between novel prognostic risk factors and the risk of AID in CLL patients is planned.

Abstract Background: Historically, CLL patients with cytopenia were thought to have a poor prognosis and cytopenia is a key feature of CLL clinical staging systems. The implications of cytopenia in CLL could have changed because of improved diagnostic accuracy, increased recognition of early stage disease, and better treatments. Newer data suggest that the etiology of cytopenia determines its prognostic importance in CLL. Patients with cytopenia caused by autoimmune disease (AID) are less likely to have the poor prognosis associated with patients with bone marrow (BM) failure. To determine the prognostic significance of cytopenia in patients with CLL, we performed an observational study at Mayo Clinic Rochester (MCR). Methods: We studied all patients with CLL seen in the Division of Hematology at MCR from 1 January 1995 to 31 December 2004 (n = 1,750). Cytopenia (hemoglobin < 10 g/dL or platelet count < 100 × 09/L) was considered due to BM failure in patients with extensive BM involvement by CLL (< 20% residual myeloid tissue on BM biopsy) or reticulocytopenia without other causes persisting > 3 months after CLL therapy. Cytopenia was attributed to AID in patients with autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), pure red blood cell aplasia (PRCA), or autoimmune granulocytopenia (AIG). Results: Median age at diagnosis of CLL was 63.7 years (69% male). Cytopenia occured in 423 (24.2%) patients. In 303 patients, cytopenia was due to CLL (228 BM failure and 75 AID). In 120 patients cytopenia was due to CLL related factors (splenomegaly, n = 11; possible AID, n = 18; treatment of CLL, n = 16) or non CLL causes (other malignancy, n = 12; iron deficiency, n = 11; anemia of chronic disease, n = 10; renal failure, n = 6; drugs effect, n = 4; surgical blood loss n = 4). In 28 (6.6%) patients the cause of cytopenia was not definitively determined. Of the patients with BM failure, 97 (43%) had cytopenia at CLL diagnosis (70% male, median age 63.3 years [range 32–95]), The AID patients the median age of CLL diagnosis was 66.7 years (range 30–85) with 79% male. AID diagnoses were AIHA (n = 41), ITP (n= 35), PRCA (n=8), and AIG (n=3); 9 patients had > 1 AID. Survival from diagnosis of cytopenia was significantly better for patients with AID (median 9.1 years) than BM failure (median 4.4 years, p < 0.001). AID patients had a longer survival from diagnosis of CLL (median 12.4 years) versus the rest of the CLL population (median 9.5 years, p = 0.020) but patients with BM failure at diagnosis had a worse prognosis (median 6.2 years, p < 0.001). Conclusion: We show that the etiology of cytopenia in CLL patients determines its prognostic importance. Anemia and thrombocytopenia caused by BM failure, but not AID, are associated with a significantly worse prognosis. These data show the importance of accurate determination of the etiology of cytopenia in all patients with CLL. Cytopenia in patients with AID is not a marker of advanced stage disease; these patients often need different management compared to those with BM failure. Cytopenia due to AID versus BM failure can be difficult to distinguish and a BM study should be done in all patients considered for treatment. Patients with cytopenia due to AID cannot be meaningfully classified by current clinical staging systems. We believe that efforts to review the NCI-WG 96 criteria are timely and should consider the etiology of cytopenia in CLL patients.

Abstract Metaplastic breast carcinoma, a rare tumor composed of adenocarcinomatous and nonglandular growth patterns, is characterized by a propensity for distant metastases and resistance to standard anticancer therapies. We sought confirmation that this tumor is a basal-like breast cancer, expressing epidermal growth factor receptor (EGFR) and stem cell factor receptor (KIT). EGFR activating mutations and high copy number (associated with response to tyrosine kinase inhibitor gefitinib) and KIT activating mutations (associated with imatinib sensitivity) were then investigated. Seventy-seven metaplastic cases were identified (1976-2006); 38 with tumor blocks available underwent pathologic confirmation before EGFR and KIT immunohistochemical analyses. A tissue microarray of malignant glandular and metaplastic elements was constructed and analyzed immunohistochemically for cytokeratin 5/6, estrogen receptor, progesterone receptor, and p63, and by fluorescence in situ hybridization for EGFR and HER-2/neu. DNA isolated from individual elements was assessed for EGFR and KIT activating mutations. All assessable cases were negative for estrogen receptor, progesterone receptor, and (except one) HER2. The majority were positive for cytokeratin 5/6 (58%), p63 (59%), and EGFR overexpression (66%); 24% were KIT positive. No EGFR or KIT activating mutations were present; 26% of the primary metaplastic breast carcinomas were fluorescence in situ hybridization-positive, displaying high EGFR copy number secondary to aneusomy (22%) and amplification (4%). We report here that metaplastic breast carcinoma is a basal-like breast cancer lacking EGFR and KIT activating mutations but exhibiting high EGFR copy number (primarily via aneusomy), suggesting that EGFR tyrosine kinase inhibitors should be evaluated in this molecular subset of breast carcinomas. [Mol Cancer Ther 2008;7(4):944–51]