Triple-negative breast cancer in West Virginia.
Abstract: In 2007, the American Cancer Society ranked West Virginia 43rd in breast cancer incidence rates for individual states. Despite our improvements in medical care, the advanced pathological characteristics of breast cancer at diagnosis receive little attention. Consequently, we compared the changing pattern of early breast cancer in several cohort studies conducted at regional medical centers in West Virginia. The data used in this analysis was derived from 320 women presenting at West Virginia University Hospital (WVUH) in Morgantown between 1999 and 2004, with a diagnosis of invasive breast cancer. Details of age, tumor size and axillary lymph node status were compared with tumor registry information published from a cohort study of 191 patients from the Charleston Area Medical Center (CAMC) between 1990 and 1991. Only histologically documented adenocarcinomas of the breast were included. Tumor size was characterized using the TNM system and staged according to AJCC criteria. For comparative purposes, details from the two regional centers were compared with tumor characteristics from a large longitudinal cohort of 2,484 breast cancers from the Women's Health Initiative (WHI) study. Baseline median age at diagnosis of women screened at WVUH was younger than patients at CAMC (52 vs. 60). Women diagnosed with triple-negative breast cancer at WVUH and CAMC had similar age distributions. Within the triple-negative patients at WVUH, 44% of patients were less than 50 years of age and 20% were less than 40 years of age. At CAMC, 35% were less than 50 years of age and 7% were less than 40 years of age. For women at WVUH, 61.5% presented with T1 tumors compared to 65.5% at CAMC.

These figures were lower than the WHI average of 80.3%. In contrast, more women presented with larger T2 tumors at our medical centers compared with the national study, 32.6% versus 17.4% respectively. At WVUH, 2.3% of women had T3 tumors (>5 cm) compared with 1% at CAMC. Similar to the WHI study, 3542% of women at WVUH and CAMC were diagnosed at the T1c stage.

Approximately, 30% were diagnosed with positive lymph nodes, compared to 23% in the national study. Combined breast cancer data from our medical centers show an increase in more advanced tumors and positive regional lymph node involvement at the time of diagnosis compared to national reports. Other factors such as obesity, diabetes, poverty and access to mammography screening could be influencing the poorer outcomes for women with breast cancer in West Virginia.
Article Type: Report
Subject: Breast cancer (Risk factors)
Breast cancer (Diagnosis)
Breast cancer (Care and treatment)
Breast cancer (Patient outcomes)
Breast cancer (Demographic aspects)
Cancer (Diagnosis)
Cancer (Usage)
Authors: Abraham, Jame
Flanagan, Melina
Hazard, Hannah
Jubelirer, Steven
Tirona, Maria Tria
Vona-Davis, Linda
Pub Date: 10/01/2009
Publication: Name: West Virginia Medical Journal Publisher: West Virginia State Medical Association Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 West Virginia State Medical Association ISSN: 0043-3284
Issue: Date: Oct, 2009 Source Volume: 105 Source Issue: S1
Geographic: Geographic Scope: West Virginia Geographic Code: 1U5WV West Virginia
Accession Number: 218949142
Full Text: Objectives

After reading this article, the participant will better understand the molecular, genetic and histopathology involved in breast cancer as well as the changing pattern of early breast cancer in West Virginia.

Introduction

Carcinoma of the breast is no longer viewed as a homogenous disease but is now understood as a heterogeneous group of tumor subtypes with distinct clinical presentations, responses to local and systemic treatments, and differences in overall survival. The most common types of breast cancer are invasive ductal and invasive lobular. According to the World Health Organization classification system, tubular and cribriform carcinomas have an overall favorable prognosis, assuming a small tumor size. (1)

Besides histologic type, additional well-established clinical and histopathologic characteristics used for risk assessment, predictive/ prognostic purposes, and treatment decisions, include patient age, tumor size, histologic grade, estrogen and progesterone receptor status, human epidermal growth factor-2 (HER-2/neu) status, and lymph node status. The Nottingham histologic grade, which is the most common grading system, takes into account tubule formation, nuclear grade, and mitotic count. (2)

Most breast cancers are sporadic and due to multiple factors, including hormonal and environmental; however, about 5-10% of breast cancers are familial or hereditary. (3) Mutations of BRCA1 (chromosome 17q21) and BRCA2 (chromosome 13q12-13q13) are responsible for 85% of hereditary breast cancer. The estimated lifetime risk for developing breast cancer in women with a BRCA1 or BRCA2 mutation is 56 to 87%, and the risk for developing bilateral/contralateral breast cancer is about 20% to 40%.

Molecular classification of breast cancer by microarray technique has led to the identification of at least three subtypes--luminal subtype, HER2 subtype and basal-like breast cancer. (4) Luminal subtypes of breast cancer are hormone receptor positive (ER) and are similar to the luminal epithelium of the breast. They are the most common subtypes of breast cancers, estimated to comprise around 60% of all breast cancers. These are divided further into two subtypes--luminal A and luminal B; the former expresses ER more and are associated with decreased expression of genes associated with increased proliferation.

Basal-like breast cancers constitute approximately 15% of all breast cancers, and are associated with poor outcomes (5). Basal-like tumors are usually negative for ER, PR, and HER-2/neu; however, their exact relationship to triple-negative tumors is still being elucidated. While some studies find them to be synonymous, (6) others find significant but not complete overlap (7). These tumors have poor prognosis secondary to the lack of targeted therapeutics due to their triple-negative state and because they are inherently aggressive and tend to be higher grade, high mitotic index, a central necrotic zone, pushing borders, and conspicuous lymphocytic infiltrate (8,9). Features of metaplastic (10) and medullary carcinomas (11) are more common in basal-like than nonbasal carcinomas, and the majority of medullary and metaplastic carcinomas show a basal-like phenotype ( 10,11). Although there is not much literature that specifically addresses the histopathology of triple-negative tumors, it is suggested by inference from experience with basal-like tumors that the majority of triple-negative tumors are high grade invasive ductal carcinomas, as well as metaplastic and medullary carcinomas. (7)

Breast cancer incidence and mortality in West Virginia

The American Cancer Society estimated there were 182,460 new cases of invasive breast cancer and an additional 67,770 newly diagnosed cases of ductal carcinoma in situ across the United States in 2008. (12) Men comprise approximately 1% of all new breast cancer cases (1,990) and an estimated 40,480 patients died of breast cancer in the United States during this same time period. In West Virginia, there were an estimated 1,150 new breast cancers diagnosed and 310 deaths in 2008 as compared to 1,180 new cases with 280 deaths in 2007. (12) While the incidence of breast cancer has decreased nationally by 6.7% since 2002, this is predominantly in the ER receptor positive subtype of breast cancer. (4) In West Virginia, the incidence of breast cancer has remained relatively consistent with national averages, but there are a few counties in the state with higher mortality rates. Table 1 compares the breast cancer incidence rates of three states, including West Virginia, that have a relatively high proportion of the population living below the poverty line, against those of three states with much lower poverty levels. West Virginia is ranked 43rd in the breast cancer incidence rates for individual states published by the American Cancer Society in 2007, whereas more affluent states such as Connecticut, Massachusetts, and Wisconsin were third, fourth, and ninth in rank order. (12) Table 1 also gives the corresponding breast cancer mortality rates. With the exception of Louisiana, mortality rates are similar between the two distinct socioeconomic communities. However, in West Virginia, as in many lower socioeconomic communities, women tend to have more completed pregnancies and tend to be of a younger age at first birth. This might account for the lower incidence of breast cancer indicated in this table but still does not account for the increased mortality seen in some of the more rural Appalachian counties. (13)

There is also a failure to take into account the difference in breast cancer incidence, which itself can influence the risk of dying of the disease. This important point is illustrated in Table 1. When the data are examined as the ratios of incidence to mortality, the low ratio for West Virginia, Louisiana, and Alabama, reflect a reduction in the successful treatment outcome in these states. Furthermore, although the average breast cancer mortality rate for West Virginia over the years 1999-2003 (25.3/100,000) is similar to that for the country as a whole (26.0/100,000), marked variations of breast cancer incidence and mortality rates are observed between individual counties (data not shown). In 2002, more than a quarter of the 55 counties had mortality rates that were higher than the national average, ranging from 26.2 to 45.7/100,000 as reported by the West Virginia Comprehensive Cancer Program.

The purpose of this article was to compare the incidence of breast cancer in West Virginia compiled from two state medical centers to the national data, paying particular attention to tumors that are ER, PR and Her2/neu negative, otherwise known as triple-negative or basal-like tumors.

Epidemiological and clinical features of breast cancer at two state medical centers

With the institution of screening mammography guidelines between 1980 and 1987, there was a doubling in the incidence of small breast cancers ([less than or equal to] 2cm) with a concomitant decrease by 27% in the incidence of larger breast cancer ([greater than or equal to] 3cm).

Additionally, the diagnosis of smaller tumors ([less than or equal to] 2cm) increased 2.1% each year from 1988-99 and stabilized thereafter. (12) We have been able to identify similar trends at two of the large, tertiary referral medical centers in West Virginia. At Charleston Area Medical Center (CAMC), Jubelirer et al. (14) compared tumor registry data from 1990-1991 to the data from the previous 10 years. The author reported an increase in the proportion of breast cancers that were 2 cm or less in diameter and a decrease in the number of patients with positive lymph nodes. The reduction in clinically advanced disease at presentation (Stage III/IV) was associated with an increase in the number of patients whose initial diagnosis was made by mammography alone.

Even with these improvements, breast cancer patients in West Virginia continue to present with relatively more advanced tumors when compared to other subpopulations of women in the United States. Table 2 shows the stage at diagnosis for 191 women at CAMC between 1990 and 1991 as well as previously unpublished data of 369 women treated at West Virginia University Hospital (WVUH) in Morgantown, WV during 1999 and 2002. These findings are compared to the stage at presentation of 2,484 women diagnosed with breast cancer between 1993 and 1998 after their recruitment into the nationwide Women's Health Initiative (WHI) trial. (15)

Baseline median age at diagnosis of women screened at WVUH was younger than patients at CAMC (52 vs. 60). For women at WVUH, 61.5% presented with T1 tumors compared to 65.5% at CAMC. These figures were lower than the WHI average of 80.3%. In contrast, more women presented with larger T2 tumors at our medical centers compared with the national study, 32.6% versus 17.4% respectively. At WVUH, 2.3% of women had T3 tumors ([greater than or equal to] 5cm) compared with 1% at CAMC. Similar to the WHI study, 35-42% of women at WVUH and CAMC were diagnosed at the T1c stage. Approximately, 30% were diagnosed with positive lymph nodes, compared to 23% in the national study.

The women in West Virginia, where a significant number of patients have limited access to health care, were more likely to have tumors greater than 2.0 cm and were more likely to have axillary nodal involvement at presentation as compared to those participating in the nationwide WHI study. This is best explained by the inevitable selection for women who had ready access to health care through the WHI program. (15)

What has emerged from a recent study published in 2008 by VonaDavis, et al., (16) at WVUH is the presence of a high percentage of women with the aggressive triple-negative phenotype. We studied, by retrospective review, 620 patients with invasive breast cancer found in the West Virginia Hospital tumor registry. Of these women, 73.7% had ER positive disease and 67.7% PR positive disease. On the basis of the ER, PR and HER-2/neu assay results, 117 of the 620 (18.9%) breast cancer patients available for study were classified as triple-negative. Four hundred thirty three (69.8%) were ductal carcinomas, 55 (8.9%) were lobular carcinomas, 84 (13.5%) had both lobular and ductal features, and 48 (7.8%) were comprised other tumor types such as mucinous, tubular, and medullary carcinomas. Significantly, more of the ductal carcinomas were ER and PR negative compared to the pure lobular and mixed lobular/ductal tumors (P < 0.001; Table 3). Furthermore, 23.1% of the ductal, but only 5.5% and 7.1%, respectively, of the lobular and mixed lobular/ductal carcinomas were triple-negative tumors. (16)

Women diagnosed with triple-negative breast cancer at WVUH and CAMC had similar age distributions as compared to African American women. Within the triple-negative patients at WVUH, 44% of patients were less than 50 years of age and 20% were less than 40 years of age. At CAMC, 35% were less than 50 years of age and 7% were less than 40 years of age. This age distribution pattern is similar to the triple-negative tumors diagnosed in young, premenopausal, African-American 17-20 and British black (21) women. At WVUH, we found the mean size of the triple-negative tumors at diagnosis was greater than that of the non-triple-negative tumors (P < 0.001; Table 4) and this finding is similar to that observed at CAMC, as well as with a number of other studies. (19,22,23) Dent et al (22) observed a positive correlation between tumor size and the presence of nodal involvement only in the non-triple-negative group thereby indicating metastasis occurred earlier in triple-negative tumors; a similar relationship was observed in the

WVUH study (data not shown). (16) Further validation of these results is warranted as data were compiled using relatively small samples of unequal size taken at different times from two medical centers.

Conclusions

Breast cancers lacking estrogen and progesterone receptors and HER-2/neu expression, otherwise known as triple-negative tumors, have a relatively poor prognosis as compared to tumors with the presence of estrogen and or progesterone receptors. Triple-negative disease is particularly common in young African-American women 17-20 and is more likely to be of high histologic and nuclear grade. (18,22) Additionally, these tumors have shorter relapse-free survival times than patients with other tumor receptor patterns and are more likely to develop distant metastases. (22,23)

Breast cancer patients in West Virginia, a predominantly rural Appalachian community, continue to present with relatively more advanced tumors as compared to other subpopulations of women in the United States. Of particular importance is the high percentage of women with the aggressive triple-negative phenotype occurring in younger women who present with more advanced disease. Since most triple-negative tumors are high grade and have a high incidence of recurrence and distant metastases, there is an urgent need for new therapeutic treatment approaches for triple-negative breast cancer for which, unlike other subtypes of breast cancer, standard adjuvant systemic treatment seems less effective. With improvements to access to care through screening mammography programs such as Bonnie's Bus, the objective is to identify the particularly difficult subtypes of breast cancer at an earlier stage. By doing so, we hope to reduce mortality throughout the state and in particular, those counties with mortality rates higher than the national average.

CME Post-Test

32. Triple-negative beast cancer is defined by negative findings for which of the following:

a. Negative for estrogen, progesterone and HER1 receptors

b. Negative for estrogen, progesterone and HER2 receptors

c. Negative for estrogen, progesterone and BRCA1 receptors

33. Compared to other subtypes of breast cancer, triple-negative breast cancers have a:

a. poorer prognosis

b. better prognosis

c. similar prognosis

References

(1.) Tavassoli FA, Deville P, eds. Pathology and Genetics: Tumours of the Breast and Female Genital Organs. World Health Organization Classification of Tumours. 2003; IARC Press.

(2.) Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991;19:403-410.

(3.) Sharma M, Abraham J. Breast Cancer. Bethesda Handbook of Clinical Oncology.2009; Philadelphia: Lippincott Williams and Wilkins.

(4.) Kanate A, Abraham J. Biology of Breast Cancer. Breast Cancer an Essential Guide.2007; Philadelphia: Lippincott Williams and Wilkins.

(5.) Nielsen TO, Hsu FD, Jensen K et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004;10:5367-5374.

(6.) Kreike B, van KM, Horlings H et al. Gene expression profiling and histopathological characterization of triple-negative/basallike breast carcinomas. Breast Cancer Res. 2007;9:R65.

(7.) Reis-Filho JS, Tutt AN. Triple negative tumours: a critical review. Histopathology. 2008;52:108-118.

(8.) Fulford LG, Easton DF, Reis-Filho JS et al. Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast. Histopathology. 2006;49:22-34.

(9.) Livasy CA, Karaca G, Nanda R et al. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol. 2006;19:264-271.

(10.) Reis-Filho JS, Milanezi F, Steele D et al. Metaplastic breast carcinomas are basal-like tumours. Histopathology. 2006;49:10-21.

(11.) Jacquemier J, Padovani L, Rabayrol L et al. Typical medullary breast carcinomas have a basal/myoepithelial phenotype. J Pathol. 2005;207:260-268.

(12.) American Cancer Society. Cancer Facts and Figures. http://www.acs.org . 2009.

(13.) West Virginia Cancer Registry. West Virginia Bureau of Public Health. http:// www.wvcancerregistry.org . 3-27-2009.

(14.) Jubelirer SJ, Smith JI, Gharib M. The changing pattern of early breast cancer and its primary management at CAMC. W V Med J. 1993;89:442-444.

(15.) Chlebowski RT, Aiello E, McTiernan A. Weight loss in breast cancer patient management. J Clin Oncol. 2002;20:11281143.

(16.) Vona-Davis L, Rose DP, Hazard H et al. Triple-negative breast cancer and obesity in a rural Appalachian population. Cancer Epidemiol Biomarkers Prev. 2008;17:33193324.

(17.) Morris GJ, Naidu S, Topham AK et al. Differences in breast carcinoma characteristics in newly diagnosed African American and Caucasian patients: a single-institution compilation compared with the National Cancer Institute's Surveillance, Epidemiology, and End Results database. Cancer. 2007;110:876 884.

(18.) Carey LA, Perou CM, Livasy CA et al. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA. 2006;295:2492-2502.

(19.) Bauer KR, Brown M, Cress RD et al. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype: a population-based study from the California cancer Registry. Cancer. 2007;109:1721-1728.

(20.) Ihemelandu CU, Leffall LD, Jr., Dewitty RL et al. Molecular breast cancer subtypes in premenopausal and postmenopausal African-American women: age-specific prevalence and survival. J Surg Res. 2007;143:109-118.

(21.) Bowen RL, Duffy SW, Ryan DA et al. Early onset of breast cancer in a group of British black women. Br J Cancer. 2008;98:277-281.

(22.) Dent R, Trudeau M, Pritchard KI et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13:4429-4434.

(23.) Haffty BG, Yang Q, Reiss M et al. Locoregional relapse and distant metastasis in conservatively managed triple negative early-stage breast cancer. J Clin Oncol. 2006;24:5652-5657.

Jame Abraham, MD, FACP [1] Melina Flanagan, MD, MSPH [2] Hannah Hazard, MD [3] Steven Jubelirer, MD, FACP [4] Maria Tria Tirona, MD, FACP [5] Linda Vona-Davis, PhD [6]

[1] Bonnie Wells Wilson Eminent Scholar and Distinguished Professor in Breast Cancer Research, Chief of Hematology /Oncology, Director of Comprehensive Breast Cancer

Program, Medical Director of Mary Babb Randolph Cancer Center, West Virginia University, Morgantown

[2] Assistant Professor of Pathology & Director of Cytopathology, West Virginia University, Morgantown

[3] Assistant Professor of Surgery, Division of Surgical Oncology, West Virginia University, Morgantown

[4] Professor and Chief of Hematology/Oncology, West Virginia University, Charleston Division, Charleston Area Medical Center, Charleston

[5] Professor and Chief of Hematology/Oncology, Director of Medical Oncology, Edwards Comprehensive Cancer Center, Marshall University, Huntington

[6] Associate Professor of Surgery, Breast Cancer Research Program, Mary Babb Randolph Cancer Center, Morgantown
Table 1. Breast cancer incidence and mortality in West Virginia
compared with selected other States ([dagger])

                                              Incidence to
                Incidence *    Mortality *   mortality ratio

West Virginia   116.9 (43) #    25.3 (23)       4.62 (39)

Louisiana        122.8 (36)     30.1 (1)        4.08 (47)
Alabama          115.3 (45)     26.3 (17)       4.38 (45)
Connecticut      140.4 (3)      23.5 (41)       5.55 (6)
Massachusetts    138.8 (4)      26.2 (19)       5.30 (13)
Wisconsin        133.9 (9)      25.1 (24)       5.33 (12)

United States      128.2          26.0            4.93

([dagger]) American Cancer Society. Facts and Figures. American
Cancer Society: Atlanta, 2007

* Average for years 1999-2003 per 100,000 women age-adjusted to
2000 US standard population.

# Numbers in parentheses are the rank orders among the 50 states.

Table 2. Comparison of breast cancer cases at Charleston Area Medical
Center, West Virginia University Hospital in Morgantown with the
Women's Health Initiative Breast Cancer Study.

                                            Women's Health
                      CAMC        WVUH      Initiative (15)
                    1990-1991   1999-2002      1993-1998

Number of Patients     191         369           2,484

Age (median)           60          66             64

Tumor size
  T1 (<2cm)           65.5%       61.5%          80.3%
  T2 (2-5cm)          33.5%       31.6%          17.4%
  T3 (>5cm)            1.0%        6.9%           2.3%

Tumor grade
  I                    7.9%        6.9%           9.6%
  II                  15.7%       18.8%          29.4%
  III                 41.9%       35.8%          41.3%

Lymph node status
Negative               72%         69%            77%
Positive               28%         31%            23%

Table 3. ER, PR, HER2 status of breast cancer patients
with ductal, lobular and mixed ductal/ lobular histologic
types in a recent study by WVUH (1999-2004). (16)

                                            Mixed
                   Ductal     Lobular   lobular/ductal
                   n (%)       n (%)        n (%)

ER+              307 (70.9)  48 (87.3)    68 (81.0)
ER-              126 (29.1)  7 (12.7)     16 (19.0)

PR+              283 (65.5)  46 (86.8)    63 (75.9)
PR-              149 (34.5)  7 (13.2)     20 (24.1)

HER+             67 (22.6)   5 (16.7)     19 (36.5)
HER-             229 (77.4)  25 (83.3)    33 (63.5)

Triple negative  100 (23.1)   3 (5.5)      6 (7.1)

Non-triple
negative         333 (76.9)  52 (94.5)    78 (92.9)

                                             Total
                 Other ([dagger])         population          P value
                      n (%)                    n

ER+                 34 (70.8)               457/620           0.0153
ER-                 14 (29.2)               163/620

PR+                 28 (59.6)      420/620 ([double dagger])  0.0015
PR-                 19 (40.4)               195/620

HER+                12 (35.3)      103/620 ([double dagger])  0.0511
HER-                22 (64.7)               309/620

Triple negative      8 (16.7)               117/620           <0.0001

Non-triple
negative            40 (83.3)               503/620

ER estrogen receptor; PR progesterone receptor

([dagger]) Mucinous, tubular and medullary carcinomas

([double dagger]) Some tissues were not tested for all characteristics.

Table 4. Clinical characteristics of triple-negative status

                        WVUH
                      1999-2004

                                      Non-triple
                   Triple-negative     negative

Age at diagnosis        n (%)           n (%)
  <40                 22 (20.0)       41 (9.4) *
  40-49               27 (24.5)       76 (17.3)
  50-59               33 (30.0)       128 (29.2)
  60-69               18 (16.4)       95 (21.7)
  >70                 10 (9.1)        98 (22.4)

Tumor size
  T1 (<2cm)           34 (30.9)      245 (52.2) *
  T2 (2-5cm)          58 (52.7)       179 (38.2)
  T3 (>5cm)           18 (16.4)        45 (9.6)

Tumor grade
  I                    2 (1.9)       63 (14.5) *
  II                  19 (17.6)       161 (36.9)
  III                 87 (80.5)       212 (48.6)

Nodal status
  Negative            54 (67.5)       277 (69.8)
  Positive            26 (32.5)       120 (30.2)

                         CAMC
                      2002-2004

                                      Non-triple
                   Triple- negative    negative

Age at diagnosis        n (%)           n (%)
  <40                  4 (7.1)         5 (2.7)
  40-49               13 (28.2)       42 (23.3)
  50-59               11 (19.6)       40 (22.2)
  60-69                9 (16.1)       40 (22.2)
  >70                  9 (16.1)       53 (29.4)

Tumor size
  T1 (<2cm)           15 (32.6)       75 (41.6)
  T2 (2-5cm)          25 (54.3)       34 (18.8)
  T3 (>5cm)            2 (4.3)         7 (3.8)

Tumor grade
  I                    1 (2.1)        33 (18.3)
  II                   8 (17.3)       70 (38.8)
  III                 26 (56.5)       38 (21.1)

Nodal status
  Negative            24 (52.2)       131 (72.8)
  Positive            22 (47.8)       49 (27.2)

* Represents a significant difference between groups where P < 0.001.
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