Abnormal mismatch repair and other clinicopathologic predictors of poor response to progestin treatment in young women with endometrial complex atypical hyperplasia and well-differentiated endometrial adenocarcinoma: a consecutive case series.

OBJECTIVE: To report the response to progestin therapy in young women with endometrial complex atypical hyperplasia (CAH) or FIGO grade 1 endometrial adenocarcinoma (FIGO 1 EAC) based on clinicopathologic features, including abnormal DNA mismatch repair (MMR) by immunohistochemistry (IHC). DESIGN: Consecutive case series. SETTING: Olive View-UCLA Medical Center in Sylmar, CA, USA, and Cedars-Sinai Medical Center in Los Angeles, CA, USA. POPULATION: Women ≤55 years old with CAH or FIGO 1 EAC. METHODS: Response to progestin therapy in 84 consecutive patients was assessed based on clinicopathologic factors, including age, body mass index (BMI), initial histology, and IHC staining for MMR proteins. MAIN OUTCOME MEASURES: Rates of abnormal MMR protein expression and response to progestin therapy were determined. RESULTS: Six (7%) patients had abnormal IHC staining, of whom five (83%) had FIGO 1 EAC at initial diagnosis. Following progestin treatment, none of the endometrial lesions in patients with abnormal IHC for MMR proteins had resolution of hyperplasia or malignancy, in contrast to 41 (53%) with normal staining (P = 0.028). Age ≤40 years and initial lesion (CAH versus FIGO 1 EAC) were predictors of response to progestin; BMI was not. CONCLUSIONS: In this cohort, 7% of women ≤55 years of age with CAH or FIGO 1 EAC had loss of MMR proteins by IHC. These patients had a higher incidence of invasive cancer and a lower incidence of resolution with progestin therapy. TWEETABLE ABSTRACT: Abnormal MMR protein expression predicts poor response to progestins in young women with CAH or FIGO 1 EAC.

Predictors of invasive adenocarcinoma after conization for cervical adenocarcinoma in situ.

OBJECTIVE: Modified radical hysterectomy has been advocated for the definitive treatment of patients with cervical adenocarcinoma in situ (ACIS) with positive conization margins due to the risk of a co-existing invasive cervical adenocarcinoma (ICA). We sought to identify patients who can be safely managed with an extrafascial hysterectomy based on predictors of invasion in the conization specimen. METHODS: Between 1996 and 2010, we identified 33 patients who had definitive surgical management for cervical ACIS following conization with positive margins and/or positive endocervical curettage (ECC). Demographic and pathologic characteristics were collected by chart review. Statistical analysis was performed using Fisher's exact test. RESULTS: Among 33 patients, 4 (12%) had ICA in the hysterectomy specimen. Predictors of ICA included pathologic suspicion of invasion (PSI) in the conization specimen and positive ECC. In patients with ICA at hysterectomy, PSI and ACIS-positive ECC were found in 75% (p=0.32) and 100% (p=0.09) respectively. When PSI was present and the ECC was positive, the positive predictive value (PPV) for ICA was 33% (2 of 6). When PSI was absent, the negative predictive value (NPV) for ICA was 94% (1 of 16). When both PSI and ECC were negative, the NPV for ICA was 100% (0 of 6). CONCLUSIONS: Women with cervical ACIS have the highest risk for ICA in the setting of positive cone margins, positive ECC, and presence of PSI in the conization specimen. Extrafascial hysterectomy remains a viable option for women with positive cone margins when ECC is negative and PSI is absent.

Sub-Kelvin cooling for two kilopixel bolometer arrays in the PIPER receiver.

The Primordial Inflation Polarization Explorer (PIPER) is a balloon-borne telescope mission to search for inflationary gravitational waves from the early universe. PIPER employs two 32 × 40 arrays of superconducting transition-edge sensors, which operate at 100 mK. An open bucket Dewar of liquid helium maintains the receiver and telescope optics at 1.7 K. We describe the thermal design of the receiver and sub-Kelvin cooling with a continuous adiabatic demagnetization refrigerator (CADR). The CADR operates between 70 and 130 mK and provides ≈10 µW cooling power at 100 mK, nearly five times the loading of the two detector assemblies. We describe electronics and software to robustly control the CADR, overall CADR performance in flightlike integrated receiver testing, and practical considerations for implementation in the balloon float environment.

Scission of DNA at a preselected sequence using a single-strand-specific chemical nuclease.

BACKGROUND: We were interested in developing a protocol for cleaving large DNAs specifically. Previous attempts to develop such methods have failed to work because of high levels of nonspecific background scission. RESULTS: R-loop formation was chosen for sequence-specific targeting, a method of hybridization whereby an RNA displaces a DNA strand of identical sequence in 70% formamide using Watson-Crick base-pairing, leading to a three-stranded structure. R-loops are stabilized in aqueous solution by modifying the bases with chemical reagents. The R-loop was cleaved using a novel nuclease prepared from the Thr48-->Cys mutant of the single-strand-specific M-13 gene V protein (GVP), which was alkylated with 5-(iodoacetamido-beta-alanyl)1,10-phenanthroline. The cleavage products of the pGEM plasmid were cloned in to the pCR 2.1-TOPO vector. Adenovirus 2 DNA (35.8 kb; tenfold larger than the pGEM plasmid) was also cleaved quantitatively at a preselected sequence. CONCLUSIONS: A new method for cleaving duplex DNA at any preselected sequence was developed. The cleavage method relies on the chemical conversion of M-13 GVP into a nuclease, reflecting GVP's specificity for single-stranded DNA. The GVP chimera is the first example of a semisynthetic secondary structure specific nuclease. The chemical nuclease activity of 1,10-phenanthroline-copper is uniquely suited to this technique because it oxidizes the deoxyribose moiety without generating diffusible intermediates, providing clonable DNA fragments. The protocol could be useful in generating large DNA fragments for mapping the contiguity of probes or defining the exon-intron structure of transcription units.

Anal cytology: is there a role for reflex HPV DNA testing?

There is an increased incidence of anal squamous carcinoma and its precursor lesions (anal intraepithelial neoplasia [AIN]) among persons who engage in anal-receptive sex. Analogous to cervical cancer screening, anal Papanicplaou (Pap) smears currently are used to screen these high-risk populations. Human papilloma virus (HPV) has been implicated in anal carcinoma pathogenesis and this study was performed to assess the potential role of HPV DNA testing as an adjunct to anal cytology. We correlated cytological diagnoses and HPV DNA (Digene Hybrid Capture [HC II] assay) in anal specimens collected in SurePath liquid medium from 118 patients; 54.8% of cases diagnosed as atypical squamous cells of undetermined significance (ASC-US) and 87.8% diagnosed as low-grade squamous intraepithelial lesion (LSIL) or above tested positive for high- risk HPV DNA (B+). High-grade SIL (HSIL) was present in 31 of the 51 patients with follow-up. Although a cytological diagnosis of ASC-US or above was a reliable indicator for AIN, cytology frequently did not accurately predict the grade of SIL in subsequent biopsy. Our findings suggest that reflex HPV DNA testing would be helpful in triaging patients diagnosed with ASC-US. However, patients diagnosed with LSIL or above should go directly to ansocopic biopsy.

Localization of p53 protein and human papillomavirus in anogenital squamous lesions: immunohistochemical and in situ hybridization studies in benign, dysplastic, and malignant epithelia.

p53 Protein is a 53-kd nuclear phosphoprotein believed to play an important role in controlling proliferation of neoplastic and normal cells. This "natural tumor suppressor" can be rendered ineffective (or oncogenic) by mutations in the p53 gene or by interactions with proteins synthesized by DNA-transforming viruses, including specific subtypes of human papillomavirus (HPV). We describe the localization of p53 protein in association with HPV in paraffin sections of a spectrum of benign, dysplastic, and malignant anogenital squamous epithelia using immunohistochemical and in situ hybridization techniques. p53 Was detected in 81% of the 48 cases studied. Immunoreactivity for p53 was seen in 83% of the benign and low-grade squamous intraepithelial lesions (SILs), in 73% of the high-grade SILs, and in 86% of the infiltrating squamous carcinomas. In high-grade SILs p53 staining was frequently observed in individual nuclei at various levels of the abnormal epithelium and in the basal layer of the adjacent epithelium, while in squamous metaplasia and low-grade SILs immunostaining for p53 was limited to the basal layer of the epithelium. p53 Was detected in a slightly higher percentage of HPV-positive than HPV-negative epithelia as determined by in situ hybridization. No correlation was observed between p53 immunoreactivity and HPV subtypes. p53 Protein and HPV were detected in anal lesions from a small group of human immunodeficiency virus-positive individuals. Antibodies currently available mainly demonstrate mutant forms of p53 protein that are associated with longer half-lives than the wild-type protein, but demonstration of p53 protein overexpression is not necessarily indicative of malignancy.

Computerized interactive morphometry. An expert system for the diagnosis of lymphoid-rich effusions.

The authors present experimental techniques for the diagnosis of malignant lymphoma and benign lymphocytosis in lymphoid-rich effusions with the use of an inexpensive microcomputer-based video system for computerized interactive morphometry (CIM). Lymphoid cells were randomly selected by a trained observer from real-time images of Papanicolaou-fixed and -stained cytospin smears prepared from pleural, peritoneal, or pericardial effusions. The lymphoid cells were classified by the instrument, based on the size and shape of their nuclear profiles. The morphometric data collected by the instrument were interpreted by a simple rule-based expert system that classified the smears as benign or malignant. One hundred four cases, including 28 malignant lymphomas, 63 benign lymphocytoses, 8 chronic lymphocytic leukemias, and 5 cases with incomplete immunopathologic data, were studied retrospectively. Sixty-three of these effusions had been stained to detect light chain monoclonality. Ninety-one effusions were correctly classified by the expert system. There were four potential false negative diagnoses and one potential false positive diagnosis by the CIM system. Eight effusions from patients with chronic lymphocytic leukemia (CLL) were consistently classified as benign. Although the author's series of patients with a history of CLL is small, their results suggest that CIM is unsuitable for the diagnosis of malignancy in these effusions. If only those effusions from patients with a history of CLL are excluded, the predictive value of a diagnosis of malignant lymphoma was 96.5%, whereas the predictive value of a diagnosis of benign lymphocytosis was 94.0%.

Computerized interactive morphometry and the diagnosis of lymphoid-rich effusions.

An experimental computerized interactive morphometry system for the classification of lymphoid-rich effusions is presented. The relatively inexpensive microcomputer-based system was assembled in our laboratory with commercially available hardware and software. One hundred twenty-two effusions (86 benign lymphocytoses, 26 lymphomas, and 10 chronic lymphocytic leukemias) were studied. Lymphoid cells were selected randomly by the system from real-time images of Papanicolaou-fixed and stained cytospin smears of pleural, peritoneal, and pericardial effusions. Parameters of nuclear shape, area, and optical density were measured automatically. Multiparameter statistical procedures of discriminant classificatory analysis analyzed the distribution of lymphoid nuclear profile integrated optical density to yield three groups of effusions, each with a predictive value of diagnosis of 100%. Neither these statistical procedures nor a simple rule-based expert system accurately classified chronic lymphocytic leukemia effusions based on the distribution of lymphoid nuclear profile area. When chronic lymphocytic leukemia effusions were excluded, however, the predictive values of a diagnosis of lymphoma by statistical analysis and by rule-based expert system were 92.3% and 88.9%, respectively, whereas the predictive values of a diagnosis of benign lymphocytosis were 97.7% and 91.3%, respectively. Potential applications and limitations of this technology for the diagnosis of lymphoid-rich effusions are discussed.

Cytodiagnosis of malignant melanoma. Immunoperoxidase staining with HMB-45 antibody as an aid to diagnosis.

The specificity and sensitivity of HMB-45, an antimelanoma monoclonal antibody, was evaluated in cytologic specimens from extracutaneous melanomas. Melanoma cells in 23 of 25 (92%) cases stained with this antibody. Staining was intense and diffuse in 22 of these melanomas and focal in 1. Amelanotic tumors and tumors with scanty pigment were included. Neither degree of pigmentation, site of primary, cytologic characteristics of tumor, nor source of specimen predicted the immunostaining pattern. Cytopreparations containing benign (6 cases) and malignant (16 cases) cells with which melanoma may be confused constituted the nonmelanoma group. None of the 22 cases in this group stained. Undifferentiated carcinomas, adenocarcinomas, large cell lymphomas, sarcomas, mesothelial hyperplasias, and a benign nerve sheath tumor were included. Effusions, fine-needle aspirates, bronchial material, and imprints were studied. All smears had been fixed and stained by Papanicolaou's method before immunostaining. Excellent cytomorphologic characteristics were preserved, and immunostaining was not affected. HMB-45 antibody is a highly specific and sensitive marker for malignant melanoma in cytologic material.

Keratin and carcinoembryonic antigen in exfoliated mesothelial and malignant cells: an immunoperoxidase study.

Immunoperoxidase technics were used to identify keratin and carcinoembryonic antigen (CEA) in exfoliated cells of fine-needle aspirates and body cavity fluids. Staining was evaluated in cytocentrifuge preparations from 27 malignant and 30 benign cytologic specimens. Most reactive mesothelial cell preparations were strongly positive for keratin and negative or only weakly positive for CEA. Diffuse, peripheral, and perinuclear concentration of staining for keratin was noted in exfoliated reactive mesothelial cells. Positive staining for keratin, predominantly diffuse, was noted in exfoliated cells from 56% of the adenocarcinomas. Sixty-nine per cent of adenocarcinoma preparations were strongly positive for CEA. These findings suggest that keratin proteins are not restricted to squamous cells and that keratin staining does not permit distinction between adenocarcinoma and mesothelial cells in cytologic specimens. Staining for CEA and keratin was compared in cytocentrifuge preparations and histologic sections of 12 adenocarcinomas and 7 lymphomas. In some adenocarcinomas, staining was detected only in cytologic preparations. Possible explanations for these differences are discussed. Variable staining for keratin was observed among exfoliated reactive mesothelial cells, possibly identifying different mesothelial cell populations. All reactive and neoplastic lymphoid cells were negative for keratin and CEA in cytologic and histologic preparations. Immunoperoxidase technics can be applied to rehydrated Papanicolaou-fixed and Papanicolaou-stained cytologic preparations with excellent preservation of cytologic detail.

Diagnosis of malignant lymphoma in effusions from patients with AIDS by gene rearrangement.

Patients infected with human immunodeficiency virus are prone to a wide variety of lymphoproliferative disorders. In these patients the clinical presentation of malignant lymphoma often overlaps with that of benign lymphoid proliferations. Both may include lymphadenopathy, splenomegaly, blood and bone marrow dyscrasias, and lymphocyte-rich effusions. Because benign and malignant lymphocyte-rich effusions, as well as effusions from other malignancies, may contain large cells that resemble immunoblasts or Burkitt's cells, cytomorphologic characteristics alone are unreliable for definitive diagnosis of malignant lymphoma. Usual immunotyping panels using antibodies to B- and T-cell markers frequently fail to demonstrate cell lineage in lymphoma cells of patients with acquired immune deficiency syndrome (AIDS). The authors used gene rearrangement to confirm the diagnosis of malignant lymphoma in effusions from three patients with AIDS when routine cell marker studies failed to demonstrate cell lineage or clonality. Use of biotinylated probes eliminated the need for handling radioactive material and enabled performance of studies in a routine immunohistochemistry laboratory.

Lymphoid-rich effusions. Diagnosis by morphometry using the CAS 200 System.

Lymphoid-rich effusions frequently present diagnostic problems in clinical cytology. In the authors' previous studies, most lymphoid-rich effusions had been correctly classified as benign lymphocytosis or malignant lymphoma by an experimental computerized interactive morphometry system, in which randomly selected lymphoid nuclear profile images were measured in Papanicolaou fixed and stained cytospin smears. The present study used the CAS 200 System and criteria from the previously described rule-based expert system to classify similar preparations of 134 lymphoid rich pleural, peritoneal, and pericardial effusions (90 benign lymphocytoses, 36 malignant lymphomas, and 8 chronic lymphocytic leukemias). A total of 98.9% of the benign lymphocytoses and 88.9% of the malignant lymphomas were correctly classified (predictive values of correct diagnoses 95.7% and 97.3%, respectively). Chronic lymphocytic leukemias could not be distinguished from benign lymphocytoses by nuclear profile areas. Optical density histograms of benign, lymphomatous, and chronic lymphocytic leukemias effusions are described. Advantages and limitations of image analysis and immunocytochemistry are discussed.

Chromogranin as a marker of neuroendocrine cells in cytologic material--an immunocytochemical study.

Cytologic material, including fine-needle aspirations, bronchial brushings, body cavity fluids, and tissue imprints from 39 neuroendocrine and 26 nonneuroendocrine tumors, was stained for chromogranin by the immunoperoxidase technic. Our results suggest that chromogranin is useful in identifying primary as well as metastatic neuroendocrine lesions from a variety of body sites. All lymphoid proliferations and carcinomas, including small-cell anaplastic carcinomas of the lung (oat-cell carcinomas), were negative. Chromogranin appears to be a useful marker in diagnostic cytology. This technic can be applied to routinely prepared cytologic material.

Keratins of different molecular weight in exfoliated mesothelial and adenocarcinoma cells--an aid to cell identification.

Keratin profiles of exfoliated mesothelial and adenocarcinoma cells were determined using antisera to different molecular weight keratins (45, 46, 55, 63 kdaltons) and the immunoperoxidase technic. Most metastatic adenocarcinomas in effusions stained for low (45, 46 kdaltons) and intermediate (55 kdaltons) molecular weight keratins but were negative for 63 kdalton keratin. In contrast, most reactive and malignant mesothelial cells in effusions stained strongly for 63 kdalton keratin and keratins of lower molecular weight. This is the first report of high molecular weight (greater than 60 kdaltons) keratin in exfoliated cells of nonepidermal origin. Differences in staining for 63 kdalton keratin between mesothelial and adenocarcinoma cells may help to distinguish these cells in effusions.

Squamous metaplasia and invasive epidermoid carcinoma of bladder.

Invasive epidermoid carcinoma of the urinary bladder is described in a young woman with a long history of bladder infections and extensive squamous metaplasia without atypia. The literature on squamous metaplasia and leukoplakia progressing to epidermoid carcinoma of the chronic dysuria, bladder infections, and long histories of squamous metaplasia of the bladder should be followed up very closely. If epidermoid carcinoma of the bladder develops, they should undergo radical surgery.

Benign chondrolipomatous tumor of the human female breast.

A benign chondrolipomatous breast tumor occurred in a 66-year-old woman. The preoperative diagnosis, based on mammography and xeroradiography, was fibroadenoma. Grossly, the demarcated lesion resembled a fibroadenoma with islands of cartilage projecting from its cut surface. Histologically, it was composed of benign mature fibrous stroma, fat, breast ducts, and islands of mature cartilage. Most cartilage-containing tumors of the human breast are associated with primary mammary malignant neoplasms. A few of the benign cartilage-containing tumors reported in the literature are discussed. The last case was published in 1909.

Malignant melanoma in effusions: a source of false-negative cytodiagnoses.

Melanoma, a not uncommon tumor, is associated with variable cytomorphology and unpredictable metastatic potential. Although most cytologic diagnoses of malignant melanoma represent metastatic disease, the diagnosis is frequently unsuspected clinically. Three effusions in which cells from metastatic melanomas were not diagnosed are described. Clinical factors that may have contributed to the erroneous cytodiagnoses are illustrated. Cytologic features and adjunctive studies that are helpful in identifying melanoma cells are discussed.

Pseudallescheria: an underdiagnosed fungus?

Pseudallescheria has been identified as one of the "clinically significant emerging mycoses" but has received little attention in the cytology literature. Recognition of this fungus is of particular importance clinically, because unlike most other fungi (including Aspergillus, with which it is most frequently confused), Pseudallescheria is not effectively treated with amphotericin B, the most frequently and often the only antifungal agent administered. Features helpful in the diagnosis of Pseudallescheria in cytologic material are presented.

Optimization of the peritoneal lavage.

Review of 275 consecutive peritoneal lavages and concurrent histologic material from gynecologic operations suggested that cytologic evaluation was clinically indicated for only 60.7% of the lavages, representing 46% of the patients in the study. More than one concurrent lavage was received from 21.6% of all patients in the study, comprising 50% of patients with malignant lavages, 18.7% of patients with benign lavages, and 5.3% of patients for whom cytologic evaluation of peritoneal lavage was not clinically indicated. Malignant cells were diagnosed in 15% of the 167 lavages for which cytologic examination was clinically indicated. In this series of patients, identification of malignant cells in peritoneal lavages did not increase the tumor stage beyond that obtained solely from examination of the concurrent histologic material. There were no false-positive cytologic diagnoses and no lavages in which neoplastic cells were misinterpreted as benign. A significant number of lavages, including several from patients with histologically confirmed peritoneal tumor, were sparsely cellular and/or excessively bloody. It is suggested that although peritoneal lavages might be collected during all gynecologic operations, only specimens from selected cases should be submitted for cytologic evaluation, and greater attention should be given to specimen collection to ensure that only well-preserved and representative material from the peritoneum is submitted for cytologic evaluation.

Pneumocystis carinii in FNA of the thyroid.

We report the diagnosis of Pneumocystis carinii (PC) in a fine-needle aspirate (FNA) from the thyroid of a human immunodeficiency virus infected (HIV+) male receiving aerosolized pentamidine as prophylaxis for Pneumocystis carinii pneumonia (PCP). The clinical diagnosis prior to FNA was multinodular goiter. The patient did not have pulmonary symptoms nor previous diagnosis of PCP at the time of the aspirate diagnosis. Recently, extrapulmonary Pneumocystis carinii (EPC) has been reported with increasing frequency in HIV+ patients receiving prophylactic aerosolized pentamidine. Awareness of extrapulmonary presentations of Pneumocystis carinii infection is a prerequisite for accurate cytologic diagnosis.