Cancer-testis antigen expression is shared between epithelial ovarian cancer tumors.

OBJECTIVES: Cancer-testis (CT) antigens have been proposed as potential targets for cancer immunotherapy. Our objective was to evaluate the expression of a panel of CT antigens in epithelial ovarian cancer (EOC) tumor specimens, and to determine if antigen sharing occurs between tumors. METHODS: RNA was isolated from EOC tumor specimens, EOC cell lines and benign ovarian tissue specimens. Real time-PCR analysis was performed to determine the expression level of 20 CT antigens. RESULTS: A total of 62 EOC specimens, 8 ovarian cancer cell lines and 3 benign ovarian tissues were evaluated for CT antigen expression. The majority of the specimens were: high grade (62%), serous (68%) and advanced stage (74%). 58 (95%) of the EOC tumors analyzed expressed at least one of the CT antigens evaluated. The mean number of CT antigen expressed was 4.5 (0-17). The most frequently expressed CT antigen was MAGE A4 (65%). Antigen sharing analysis showed the following: 9 tumors shared only one antigen with 62% of the evaluated specimens, while 37 tumors shared 4 or more antigens with 82%. 5 tumors expressed over 10 CT antigens, which were shared with 90% of the tumor panel. CONCLUSION: CT antigens are expressed in 95% of EOC tumor specimens. However, not a single antigen was universally expressed across all samples. The degree of antigen sharing between tumors increased with the total number of antigens expressed. These data suggest a multi-epitope approach for development of immunotherapy for ovarian cancer treatment.

Follicle Depletion Provides a Permissive Environment for Ovarian Carcinogenesis.

We modeled the etiology of postmenopausal biology on ovarian cancer risk using germ cell-deficient white-spotting variant (Wv) mice, incorporating oncogenic mutations. Ovarian cancer incidence is highest in peri- and postmenopausal women, and epidemiological studies have established the impact of reproductive factors on ovarian cancer risk. Menopause as a result of ovarian follicle depletion is thought to contribute to higher cancer risk. As a consequence of follicle depletion, female Wv mice develop ovarian tubular adenomas, a benign epithelial tumor corresponding to surface epithelial invaginations and papillomatosis frequently found in postmenopausal human ovaries. Lineage tracing using MISR2-Cre indicated that the tubular adenomas that developed in Wv mice were largely derived from the MISR2 lineage, which marked only a fraction of ovarian surface and oviduct epithelial cells in wild-type tissues. Deletion of p27, either heterozygous or homozygous, was able to convert the benign tubular adenomas into more proliferative tumors. Restricted deletion of p53 in Wv/Wv mice by either intrabursal injection of adenoviral Cre or inclusion of the MISR2-Cre transgene also resulted in augmented tumor growth. This finding suggests that follicle depletion provides a permissive ovarian environment for oncogenic transformation of epithelial cells, presenting a mechanism for the increased ovarian cancer risk in postmenopausal women.

Metastatic prostatic adenocarcinoma diagnosed in a bronchoalveolar lavage specimen: An unusual presentation of a common tumor.

Metastatic prostatic adenocarcinoma presenting as a primary lung disease is rare. We present a 52-year-old male with a 3-month history of cough, shortness of breath, and weight loss with clinical and radiological findings suggestive of a primary lung disease: Bilateral interstitial and alveolar opacities with blunting of the costophrenic angles, multiple diffuse foci of consolidations and nodules, predominantly subpleural and located in the lower lobes, and diffuse interlobular septal thickening and peribronchial thickening. The patient underwent bronchoscopy and bronchoalveolar lavage (BAL) was obtained. Cytospin smears were diagnostic for a low-grade adenocarcinoma. Clinically, the patient had elevated serum prostate-specific antigen (PSA) levels greater than 5,000 ng/mL. Because of this, immunocytochemistry for PSA was performed which was positive, confirming the diagnosis of metastatic prostatic adenocarcinoma. This unusual case of metastatic adenocarcinoma of the prostate first diagnosed by BAL highlights the significance of available clinical information and the use of immunocytochemistry for proper diagnosis.

GHRH Receptor Expression in Malignant Mixed Müllerian Tumors: A Potentially Targetable Biopredictor.

Malignant mixed Müllerian tumors (MMMTs) are aggressive malignant neoplasms with a high recurrence rate and poor prognosis. Despite advances in adjuvant therapies in recent years, the prognosis of these tumors has not improved. Growth hormone-releasing hormone (GHRH) is produced by a variety of malignant tumors and acts as a growth factor in an autocrine/paracrine manner. Its function requires the presence of its receptors to exert its effects on neoplastic cells. In this study, we evaluated the expression of GHRH receptors (GHRH-R) in a group of MMMTs. Thirty-one examples of MMMTs from endometrium, ovary, uterine tube, and pelvic peritoneum were retrieved from the files of Department of Pathology at the University of Miami, Jackson Memorial Hospital. Immunohistochemistry for GHRH-R was performed on paraffin sections and the staining results were evaluated separately in both epithelial and mesenchymal components of each tumor. The presence of pituitary type growth hormone-releasing hormone receptor mRNA and that of its biologically active splice variant were also evaluated by RT-PCR in 6 of the tumors. Positive immunohistochemical reaction for GHRH-R was detected in 30 tumors (96%). The epithelial and sarcomatous components were positive in 30 (96%), whereas one endometrial tumor was negative in both components. The mRNA for GHRH-R and its splice variant was found in all 6 tested tumors. This study shows that GHRH-R is expressed by the majority of MMMTs in both epithelial and mesenchymal components. This finding could potentially serve as a basis for therapeutic approaches using synthetic peptide antagonists of GHRH-R that have shown significant efficacy with minimal side effects in experimental models.

Unexpected Malignant Diagnosis in Colonic Biopsies: Malignant Transformation of Ovarian Mature Teratomas-Two Case Reports and Review of the Literature.

Colorectal adenocarcinoma is the second cause of cancer-related deaths in the United States. The occurrence of squamous cell carcinoma in the colorectum is extremely unusual. Malignant transformation from mature cystic teratoma of the ovary is a rare event. The most common transformation is squamous cell carcinoma, followed by adenocarcinoma. It occurs more often in elderly patients, who usually present with advance disease. We report two unusual cases of postmenopausal women diagnosed with squamous cell carcinoma in colon biopsies. After surgical resections, the carcinoma was proven to be the result of malignant transformation of ovarian mature cystic teratomas. Since squamous cell carcinoma of the colorectum is extremely rare, the presence of squamous cell carcinoma in a colonic biopsy in a female patient should alert the clinicians to other possible primary sites, as seen in these cases.

HPV in HIV-Infected Women: Implications for Primary Prevention.

BACKGROUND: There is growing evidence that human immunodeficiency virus (HIV)-infected women might have a different human papillomavirus (HPV) type distribution in cervical dysplasia specimens as compared to the general population. This has implications for primary prevention. OBJECTIVE: We aimed to obtain preliminary data on the HPV genotypes prevalent in histological samples of HIV-infected women with cervical intraepithelial neoplasia (CIN) 3/CIS of the cervix in Miami, FL, USA. METHODS: Retrospective data were collected on HIV-infected women referred to the University of Miami-Jackson Memorial Hospital colposcopy clinic between years 2000 and 2008. The histology slides of CIN 3/CIS biopsies underwent pathological review and sections were cut from these archived specimens for HPV DNA extraction. HPV genotyping was then performed using the GeneSquare™ HPV genotyping assay. We report on our first set of 23 samples. RESULTS: Eight high-risk HPV types were detected. Types in decreasing order of frequency were 16, 35, 45, 52, 59, 31, 58, and 56. Most cases had multiple infections. HPV type 16 was the most common (45%) followed by HPV-35 and -45 with equal frequency (40%). No samples contained HPV-18. CONCLUSION: Our preliminary results suggest that cervical dysplasia specimens of HIV-infected women more likely (55%) contain non-16 and -18 high-risk HPV types. We show that this held true for histologically confirmed severe dysplasia and carcinoma-in situ. Epidemiological studies guide vaccine development, therefore HPV type prevalence in CIS and invasive cervical cancer among HIV-infected women should be more rigorously explored to ensure that this highly vulnerable population receives appropriate primary prevention.

High-risk human papillomavirus DNA detected in primary squamous cell carcinoma of urinary bladder.

CONTEXT: We reported previously that more than one-third (37%) of primary bladder squamous cell carcinomas (SCCs) demonstrate diffuse p16 immunoreactivity independent of gender. This observation made us question whether p16 overexpression in bladder carcinoma is due to human papillomavirus (HPV)-dependent mechanisms. OBJECTIVES: To determine whether the presence of high-risk HPV (HR-HPV) DNA could be detected in these tumor cells. DESIGN: Fourteen cases of primary bladder SCC, which were positive for p16 by immunohistochemistry, were probed for the detection of HR-HPV by in situ hybridization and the signal amplification Invader assay. Samples positive for detection of HR-HPV by Invader assay were amplified by using HR-HPV type-specific primers, and amplification products were DNA sequenced. RESULTS: Detection of HR-HPV by the in situ hybridization method was negative in all cases (0 of 14). However, in 3 of 14 cases (21.4%), the presence of HR-HPV DNA was detected with the Cervista HPV HR Invader assay, which was followed by identification of genotype. All positive cases were confirmed by using HR-HPV type-specific amplification followed by DNA sequencing. Identified HR-HPV genotypes included HPV 16 (2 cases) and HPV 35 (1 case). CONCLUSIONS: High-risk HPV DNA is detectable in a subset of primary bladder SCCs. Based on the well-documented carcinogenic potential of HR-HPV, there is a necessity for additional studies to investigate the role of HR-HPV in bladder carcinogenesis.

An immunohistochemical panel to distinguish ovarian from uterine serous papillary carcinomas.

Serous papillary carcinomas (SPCs) share a similar morphology regardless of whether they originate from the ovary or the uterus. Identification of the site of origin of the tumor can be a challenging and a diagnostic dilemma, particularly, in the setting of a pelvic mass or peritoneal carcinomatosis. Recognition of the site of origin influences the staging, management, and prognosis of these malignancies. The purpose of this study is to identify a panel of markers to distinguish the ovarian serous papillary carcinomas (OSPC) from the uterine serous papillary carcinomas (USPC). Formalin-fixed, paraffin-embedded archival tissue from 46 cases of SPCs (33 uterine and 13 ovarian) were stained using antibodies for estrogen receptor (ER), WT1, insulin-like growth factor-II mRNA-binding protein 3 (IMP3), p53, and p16. The OSPC expressed ER (92%), WT1 (100%), IMP3 (92%), p53 (92%), and p16 (92%). The USPCs expressed ER (30%), WT1 (64%), IMP3 (85%), p53 (64%), and p16 (76%). Only ER expression was significantly higher in OSPC compared with USPCs (P<0.001). The combined ER(+)WT1(+) phenotype was present in 92% of the OSPC, whereas only 18% of the USPCs had the same phenotype (P<0.001). Furthermore, 71% of the OSPCs expressed ER(+), p53(+), WT1(+), IMP3(+), and p16(+) immunophenotype, whereas in USPCs, the tumor cells showed immunophenotypic diversity, with only 6% of the USPCs expressing reactivity to all the 5 markers (P<0.001). This study suggests that ER alone or in combination with a limited panel of markers may be used to identify the site of origin of SPCs.

Cortisol and inflammatory processes in ovarian cancer patients following primary treatment: relationships with depression, fatigue, and disability.

Elevations in the pro-inflammatory cytokine interleukin-6 (IL-6) and alterations in the anti-inflammatory hormone cortisol have been reported in a variety of cancers. IL-6 has prognostic significance in ovarian cancer and cortisol has been associated with fatigue, disability, and vegetative depression in ovarian cancer patients prior to surgery. Ovarian cancer patients undergoing primary treatment completed psychological self-report measures and collected salivary cortisol and plasma IL-6 prior to surgery, at 6 months, and at 1 year. Patients included in this study had completed chemotherapy and had no evidence of disease recurrence. At 6 months, patients showed significant reductions in nocturnal cortisol secretion, plasma IL-6, and a more normalized diurnal cortisol rhythm, changes that were maintained at 1 year. The reductions in IL-6 and nocturnal cortisol were associated with declines in self-reported fatigue, vegetative depression, and disability. These findings suggest that primary treatment for ovarian cancer reduces the inflammatory response. Moreover, patients who have not developed recurrent disease by 1 year appear to maintain more normalized levels of cortisol and IL-6. Improvement in fatigue and vegetative depression is associated with the normalization of IL-6 and cortisol, a pattern which may be relevant for improvements in overall quality of life for ovarian cancer patients.

p16INKa immunocytochemistry in fine-needle aspiration cytology smears of metastatic head and neck squamous cell carcinoma.

OBJECTIVES: Patients with head and neck squamous cell carcinoma (HNSCC) may initially present with neck metastases diagnosed by fine-needle aspiration (FNA). In these patients, it is critical to locate the primary site so that targeted therapy can be administered. Nearly a quarter of HNSCC are associated with human papillomavirus (HPV) infection. The great majority of HPV-related primaries originate in the oropharynx. p16INKa (p16) functions as a surrogate marker of HPV infection. We sought to determine if expression of p16 by immunocytochemistry (ICC) in neck metastases could assist in localizing the primary site to the oropharynx. STUDY DESIGN: Diagnostic FNA cytology smears of neck metastases from 90 patients with biopsy-proven primary HNSCC were reviewed. Papanicolaou-stained slides were directly subjected to ICC, using p16 antibody. RESULTS: Twenty-seven (30%) tumors expressed p16 by ICC; 74% of these p16-positive tumors were metastases from oropharynx. There was a significantly higher proportion of p16 expression in patients with primary oropharyngeal carcinoma (47%) versus those whose primary tumor was non-oropharyngeal (15%; p = 0.0013). CONCLUSIONS: p16 expression in FNA cytology smears of metastatic HNSCC is a useful indicator of oropharyngeal origin and can be used to help localize the primary site in cases where this is not clinically evident.

Laryngeal involvement of multiple myeloma.

The objectives of this paper are to discuss a rare cause of laryngeal multiple myeloma, to review unique pathologic findings associated with plasma cell neoplasms, to discuss epidemiology, differential diagnosis, and treatment options for plasma cell neoplasms of the larynx. Laryngeal multiple myeloma, also noted in the literature as "metastatic" multiple myeloma, presenting as a de novo laryngeal mass is extremely rare with few reported cases. Laryngeal involvement of extramedullary tumors is reported to be between 6% and 18% with the epiglottis, glottis, false vocal folds, aryepiglottic folds, and subglottis involved in decreasing the order of frequency. We present the case of a 58-year-old male with a history of IgA smoldering myeloma who presented to a tertiary care laryngological practice with a two-month history of dysphonia, which was found to be laryngeal involvement of multiple myeloma. We review the classification of and differentiation between different plasma cell neoplasms, disease workups, pathologic findings, and treatment options.

Social influences on clinical outcomes of patients with ovarian cancer.

PURPOSE: Previous research has demonstrated relationships of social support with disease-related biomarkers in patients with ovarian cancer. However, the clinical relevance of these findings to patient outcomes has not been established. This prospective study examined how social support relates to long-term survival among consecutive patients with ovarian cancer. We focused on two types of social support: social attachment, a type of emotional social support reflecting connections with others, and instrumental social support reflecting the availability of tangible assistance. PATIENTS AND METHODS: Patients were prospectively recruited during a presurgical clinic visit and completed surveys before surgery. One hundred sixty-eight patients with histologically confirmed epithelial ovarian cancer were observed from the date of surgery until death or December 2010. Clinical information was obtained from medical records. RESULTS: In a Cox regression model, adjusting for disease stage, grade, histology, residual disease, and age, greater social attachment was associated with a lower likelihood of death (hazard ratio [HR], 0.87; 95% CI, 0.77 to 0.98; P = .018). The median survival time for patients with low social attachment categorized on a median split of 15 was 3.35 years (95% CI, 2.56 to 4.15 years). In contrast, by study completion, 59% of patients with high social attachment were still alive after 4.70 years. No significant association was found between instrumental social support and survival, even after adjustment for covariates. CONCLUSION: Social attachment is associated with a survival advantage for patients with ovarian cancer. Clinical implications include the importance of screening for deficits in the social environment and consideration of support activities during adjuvant treatment.

Soy isoflavones in the prevention of menopausal bone loss and menopausal symptoms: a randomized, double-blind trial.

BACKGROUND: Concerns regarding the risk of estrogen replacement have resulted in a significant increase in the use of soy products by menopausal women who, despite the lack of evidence of the efficacy of such products, seek alternatives to menopausal hormone therapy. Our goal was to determine the efficacy of soy isoflavone tablets in preventing bone loss and menopausal symptoms. METHODS: The study design was a single-center, randomized, placebo-controlled, double-blind clinical trial conducted from July 1, 2004, through March 31, 2009. Women aged 45 to 60 years within 5 years of menopause and with a bone mineral density T score of -2.0 or higher in the lumbar spine or total hip were randomly assigned, in equal proportions, to receive daily soy isoflavone tablets, 200 mg, or placebo. The primary outcome was changes in bone mineral density in the lumbar spine, total hip, and femoral neck at the 2-year follow-up. Secondary outcomes included changes in menopausal symptoms, vaginal cytologic characteristics, N -telopeptide of type I bone collagen, lipids, and thyroid function. RESULTS: After 2 years, no significant differences were found between the participants receiving soy tablets (n = 122) and those receiving placebo (n = 126) regarding changes in bone mineral density in the spine (-2.0% and -2.3%, respectively), the total hip (-1.2% and -1.4%, respectively), or the femoral neck (-2.2% and -2.1%, respectively). A significantly larger proportion of participants in the soy group experienced hot flashes and constipation compared with the control group. No significant differences were found between groups in other outcomes. CONCLUSIONS: In this population, the daily administration of tablets containing 200 mg of soy isoflavones for 2 years did not prevent bone loss or menopausal symptoms. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00076050.

Loss of A-type lamin expression compromises nuclear envelope integrity in breast cancer.

Through advances in technology, the genetic basis of cancer has been investigated at the genomic level, and many fundamental questions have begun to be addressed. Among several key unresolved questions in cancer biology, the molecular basis for the link between nuclear deformation and malignancy has not been determined. Another hallmark of human cancer is aneuploidy; however, the causes and consequences of aneuploidy are unanswered and are hotly contested topics. We found that nuclear lamina proteins lamin A/C are absent in a significant fraction (38%) of human breast cancer tissues. Even in lamin A/C-positive breast cancer, lamin A/C expression is heterogeneous or aberrant (such as non-nuclear distribution) in the population of tumor cells, as determined by immunohistology and immunofluorescence microscopy. In most breast cancer cell lines, a significant fraction of the lamin A/C-negative population was observed. To determine the consequences of the loss of lamin A/C, we suppressed their expression by shRNA in non-cancerous primary breast epithelial cells. Down-regulation of lamin A/C in breast epithelial cells led to morphological deformation, resembling that of cancer cells, as observed by immunofluorescence microscopy. The lamin A/C-suppressed breast epithelial cells developed aneuploidy as determined by both flow cytometry and fluorescence in situ hybridization. We conclude that the loss of nuclear envelope structural proteins lamin A/C in breast cancer underlies the two hallmarks of cancer aberrations in nuclear morphology and aneuploidy.

Nuclear envelope structural defects cause chromosomal numerical instability and aneuploidy in ovarian cancer.

BACKGROUND: Despite our substantial understanding of molecular mechanisms and gene mutations involved in cancer, the technical approaches for diagnosis and prognosis of cancer are limited. In routine clinical diagnosis of cancer, the procedure is very basic: nuclear morphology is used as a common assessment of the degree of malignancy, and hence acts as a prognostic and predictive indicator of the disease. Furthermore, though the atypical nuclear morphology of cancer cells is believed to be a consequence of oncogenic signaling, the molecular basis remains unclear. Another common characteristic of human cancer is aneuploidy, but the causes and its role in carcinogenesis are not well established. METHODS: We investigated the expression of the nuclear envelope proteins lamin A/C in ovarian cancer by immunohistochemistry and studied the consequence of lamin A/C suppression using siRNA in primary human ovarian surface epithelial cells in culture. We used immunofluorescence microscopy to analyze nuclear morphology, flow cytometry to analyze cellular DNA content, and fluorescence in situ hybridization to examine cell ploidy of the lamin A/C-suppressed cells. RESULTS: We found that nuclear lamina proteins lamin A/C are often absent (47%) in ovarian cancer cells and tissues. Even in lamin A/C-positive ovarian cancer, the expression is heterogeneous within the population of tumor cells. In most cancer cell lines, a significant fraction of the lamin A/C-negative population was observed to intermix with the lamin A/C-positive cells. Down regulation of lamin A/C in non-cancerous primary ovarian surface epithelial cells led to morphological deformation and development of aneuploidy. The aneuploid cells became growth retarded due to a p53-dependent induction of the cell cycle inhibitor p21. CONCLUSIONS: We conclude that the loss of nuclear envelope structural proteins, such as lamin A/C, may underlie two of the hallmarks of cancer--aberrations in nuclear morphology and aneuploidy.

P16 expression in squamous cell carcinomas of cervix and bladder.

CONTEXT/OBJECTIVE: p16 is a widely used immunohistochemical marker in gynecologic pathology. Strong and diffuse cytoplasmic and nuclear expression of p16 in squamous cell carcinomas (SCC) of the female genital tract is strongly associated with high-risk human papilloma virus infection and neoplasms of cervical origin. However, p16 can be expressed in other neoplasms and in several normal human tissues. Occasionally, SCCs may involve both uterine cervix and urinary bladder. Accurate identification of the site of origin in such cases has therapeutic and prognostic implications. We investigate the potential value of p16 expression in this distinction. DESIGN: We reviewed 74 SCCs, 38 (51%) from urinary bladder and 36 (49%) from uterine cervix obtained between 2003 and 2008. Of the 38 cases of bladder carcinoma, 21 occurred in females and 17 in males. Immunohistochemical analysis for p16 (DAKO M7247, clone 484, dilution of 1:50) expression was done in all cases using the labeled streptavidin-biotin method. RESULT: Strong and diffuse nuclear and cytoplasmic p16 positivity was observed in 45 cases (61%). Of the 38 SCCs of urinary bladder, 14 (37%) expressed p16 (8 males, 6 females). Of the 36 SCCs of uterine cervix, 31 (86%) were positive for p16. CONCLUSIONS: (1) The majority of SCCs of uterine cervix express p16. (2) More than a third of urinary bladder SCCs express p16. (3) SCCs of urinary bladder express p16 independent of gender. (4) p16 immunohistochemical expression alone cannot be used to discriminate between SCCs arising from uterine cervix versus urinary bladder.

A phase I study of 5-fluorouracil/leucovorin and arsenic trioxide for patients with refractory/relapsed colorectal carcinoma.

PURPOSE: This Phase I study was designed to determine a safe combination dose of 5-fluorouracil (5-FU) and arsenic trioxide (ATO) to treat 5-FU-resistant relapsed/refractory colorectal cancer patients. We studied the effect of ATO in the downregulation of thymidylate synthase (TS) in peripheral blood mononuclear cells and in tumor biopsies. EXPERIMENTAL DESIGN: ATO was administered for 5 consecutive days during the first week and twice during weeks 2 to 3 and once on week 4. 5-FU/leucovorin (LV) was administered on days 8, 15, and 22. A modified accelerated titration design was used. 5-FU was dose escalated first followed by a planned dose increase for ATO. RESULTS: No dose-limiting toxicities were seen in seven patients who received 0.15 mg/kg ATO; grade 3 toxicities were as follows: neutropenia 1, diarrhea 1, and bowel obstruction 1. In patients receiving 0.20 mg/kg ATO, grade 3 toxicities were QTc prolongation 1, fatigue 4, alkaline phosphatase elevation 2, diarrhea 2, and peripheral edema 1. TS gene expression in peripheral blood mononuclear cell decreased in all patients. Eight tumors were biopsied, four showed TS downregulation, three showed upregulations, and one did not change. Estimated median progression-free survival and overall survival were 3.1 and 13.9 months, respectively. In patients who showed TS increase or no change versus TS reduction, estimated median progression-free survival was 2.6 versus 7.9 months (P = 0.188) and overall survival was 8.6 versus 11.7 months (P = 0.44), respectively. CONCLUSIONS: Thus, we determined 0.20 mg/kg ATO, 2,600 mg/m(2) 5-FU, and 500 mg/m(2) leucovorin (LV) to be the recommended phase II dose.

ALDH(+)/CD44(+)/CD24(-) expression in cells from body cavity fluids.

BACKGROUND: Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers (CD44(+)/CD24(-)) in stem cells from breast tumors has been reported. This study was undertaken to monitor expression of these markers in cells from body cavity fluids of female patients suspected to have a malignancy. METHODS: Cells from peritoneal and pleural fluids of 100 female patients were examined by diagnostic cytology and analyzed by laser flow cytometry for enhanced ALDH1 expression. Cells from 36 body cavity fluids with ALDH1(bright) fluorescence were then analyzed for the expression of CD44 and CD24 markers. RESULTS: In samples positive for malignancy, ALDH1(bright) cells with both SSC(low) and SSC(high) were seen. In 15 body cavity fluids positive for malignancy, the percentage of ALDH1(bright) cells ranged from 0.26 to 6.34% of the total cells. The percentage of ALDH1(bright) cells with CD44(+)/CD24(-) expression in these samples ranged from 0.02 to 3.66%. ALDH1(bright) cells with CD44(+)/CD24(-) expression were also present in body cavity fluids of patients in whom diagnostic cytology could not detect any malignancy. However, the percentage of ALDH1(bright) and CD44(+)/CD24(-) cells amongst the 21 body cavity fluids with negative cytology was lower than that of samples with malignancy. CONCLUSIONS: Expression of ALDH1(bright) and the CD44(+)/CD24(-) phenotype in body cavity fluids in which diagnostic cytology could not find any malignant cells suggests that this phenotype may not be restricted to the putative breast tumor stem cells. It is possible that only subsets of cells with this phenotype are the putative breast tumor stem cells.

Flow immunocytochemistry of marker expression in cells from body cavity fluids.

Diagnostic cytology based on the examination of cells from body cavity fluids misses approximately 50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy-nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following markers: Ber-EP4, progesterone (PR), MUC4, and thyroid transcription factor-1 (TTF-1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Statistical analysis of 79 samples in which data from cytology, DNA aneuploidy, and expression of at least one of the markers was available showed that by combining data from positive marker expression with that of aneuploidy, the sensitivity was increased from 58.5 to 100%. In contrast, out of the 38 samples designated as non-malignant by diagnostic cytology, nine had aneuploid DNA content and 16 of the diploid samples had a positive marker expression. Specificity was reduced from 74.7 to 31.6% due to the presence of aneuploidy and marker expression in these samples. ALDH1(pos)/CD44(pos)/CD24(neg) expression has been reported to be associated with human breast tumor stem cells. Some of our samples had cells with this phenotype. Flow cytometry offers the advantage of rapid multiparametric analysis of DNA aneuploidy and marker expression in cells from body cavity fluids based on the analysis of a large number of cells without observer bias. By further developing the use of specific markers and aneuploidy, it may be possible to refine flow cytometric analysis for rapid detection of malignant cells in body cavity fluids.