Comparative transcriptome reprogramming in oak galls containing asexual or sexual generations of gall wasps.

Oak gall wasps have evolved strategies to manipulate the developmental pathways of their host to induce gall formation. This provides shelter and nutrients for the developing larva. Galls are entirely host tissue; however, the initiation, development, and physical appearance are controlled by the inducer. The underlying molecular mechanisms of gall formation, by which one or a small number of cells are reprogrammed and commit to a novel developmental path, are poorly understood. In this study, we sought a deeper insight into the molecular underpinnings of this process. Oak gall wasps have two generations each year, one sexual, and one asexual. Galls formed by these two generations exhibit a markedly different appearance. We sequenced transcriptomes of both the asexual and sexual generations of Neuroterus quercusbaccarum and Neuroterus numismalis. We then deployed Nanopore sequencing to generate long-read sequences to test the hypothesis that gall wasps introduce DNA insertions to determine gall development. We detected potential genome rearrangements but did not uncover any non-host DNA insertions. Transcriptome analysis revealed that transcriptomes of the sexual generations of distinct species of wasp are more similar than inter-generational comparisons from the same species of wasp. Our results highlight the intricate interplay between the host leaves and gall development, suggesting that season and requirements of the gall structure play a larger role than species in controlling gall development and structure.

An audit to establish the proportion of cervical punch biopsies that meet criteria for adequacy.

This audit was carried out to develop the criteria recommended in various guidelines to establish if a punch biopsy of the cervix is adequate, and to determine if these are met in sufficient proportions of patients. A total of 100 patients who had cervical punch biopsies followed by large loop excision of the transformation zone (LLETZ) specimens were audited and the percentage of punch biopsies that met criteria for adequacy, cited in the criteria or developed from them, was established. Most of the biopsies met the criteria for adequacy, the exception being the presence of an intact squamo-columnar junction (SCJ), which was present in 14%. The highest grade of squamous intraepithelial lesion (SIL) on punch biopsy and LLETZ were most likely to differ if the SCJ was traumatised or not represented, or if the original biopsy were negative or showed ungradable SIL. These results suggest that most of these cervical biopsies originated in the optimal anatomical site on the cervix but were being damaged in an excessive number of cases and that this affected the reliability of the punch biopsy to predict the highest grade of SIL on LLETZ.

Immunohistochemical biomarkers of value in distinguishing primary ovarian carcinoma from gastric carcinoma: a systematic review with statistical meta-analysis.

AIMS: To compare the relative risk of antigen expression being detected immunohistochemically in ovarian and gastric carcinoma aggregated from studies performed for diagnostic purposes, with the relative risks of their expression in all patients in the English literature. METHODS AND RESULTS: Both types of series indicated that cytokeratin (CK) 7 expression was greater and that of CK20 and carcinoembryonic antigen less in ovarian than in gastric carcinoma (P < 0.05). Synthesis of all data available for MUC-2 suggested it was more commonly expressed in ovarian carcinoma, whereas the relative risk in papers that directly compared its expression suggested that it was more common in the gastric carcinoma (P = 0.2, NS). Aggregating all possible data suggested villin was more likely to be expressed in ovarian cancers, whereas studies in which its expression was compared directly in both tumours suggested the opposite. Although statistically significant, patient numbers were small. CONCLUSION: Provided sufficient numbers of cases are studied, analysis of studies comparing antigen expression for diagnostic purposes in tumours from two body sites is likely to be supported in the wider literature. The design of such comparative studies is informed by aggregating data from single tumour studies.

The heart and the oesophagus: intimate relations.

The close anatomical relations of the heart and oesophagus, and the similarity of symptoms attributable to disorders of either organ, often lead to diagnostic difficulty in patients with chest pain. A definitive diagnosis of non-cardiac chest pain attributable to oesophageal reflux or spasm is hampered, both by the need for prolonged ambulatory monitoring of pH, manometry, and endoscopy, and by the common occurrence of asymptomatic reflux and spasm, and the corresponding difficulty in linking an episode of reflux or spasm with an episode of pain. Moreover, some patients with non-cardiac chest pain and normal tests of oesophageal structure and function have centrally mediated hypersensitivity, both within and without the oesophagus. Rather than proceed with investigations, in the absence of symptoms to suggest structural disease of the oesophagus, it would be reasonable to attempt symptomatic treatment with a proton pump inhibitor or an antidepressant.

What is the value of proliferation markers in the normal and neoplastic cervix?

Markers of cellular proliferation have been widely applied in cervical disease and include techniques which are applicable to routinely processed tissue including standard hematoxylin and eosin sections, and sections labelled with antibodies to Ki-67 and PCNA proteins. Flow cytometry and in vivo techniques including labelling with Bromodeoxyuridine (BrdU) and radiolabelled thymidine require more specialized facilities. Increases in the mitotic index and the Ki-67 and PCNA labelling indices, the incidence of aneuploidy together with increases in in vivo labelling with BrdU and radiolabelled thymidine have been demonstrated as the grade of cervical intraepithelial neoplasia (CIN) increases. With respect to invasive tumours increases in these parameters are associated with increased tumour size, stage and improved survival after radiotherapy. At present the major practical application of these markers appears to be in distinguishing between postmenopausal atrophy and CIN lesions on histological sections and, in combination with the Papnet system, in identifying high grade dyskaryosis on blood stained cervical smears. Future development may permit the identification of those patients whose CIN lesion will progress, and who require treatment, to be distinguished from those whose lesions will stay static or regress and who can be followed up cytologically. This promises a more rational use of health care resources. Most of the studies to date have been on small numbers of cases. Meta-analysis of existing studies and large, possibly multicentric, prospective studies are needed to elucidate the value of these markers.

Systematic review and meta-analysis in anatomic pathology: the value of nuclear DNA content in predicting progression in low grade CIN, the significance of the histological subtype on prognosis in cervical carcinoma.

Meta-analysis, though increasingly popular in clinical medicine, has not found acceptance in anatomic pathology. This paper argues that, in combination with a systematic review of the literature, meta-analysis may be usefully applied to pathological research and two examples drawn from gynaecological pathology (the value of nuclear DNA quantitation in predicting progression in low grade cervical intraepithelial neoplasia and the difference in prognosis between squamous cell carcinoma and adenocarcinoma of the cervix) are included to illustrate the methods used and to demonstrate some of the difficulties associated with these techniques.

Distribution of cervical intraepithelial neoplasia: are hysterectomy specimens sampled appropriately?

AIM: To determine whether sampling the anterior and posterior cervical lips in hysterectomy specimens provides the best opportunity for detecting unsuspected cervical intraepithelial neoplasia (CIN). METHODS: The frequency with which CIN was identified at a range of positions on the anterior and posterior lips and left and right lateral aspects of the cervix was assessed in 100 cone biopsy specimens. RESULTS: CIN affected one or other cervical lip in all 100 cases studied and involved the midline positions (12 and 6 o'clock) in 94. The lateral edges of the cervical canal were also involved in 38 cases. CONCLUSION: CIN is more likely to be identified on the anterior and posterior lips than on the lateral aspect of the cervical os. The findings support the continuation of the established practice of taking blocks from the midline in these areas.

How many histological levels should be examined from tissue blocks originating in cone biopsy and large loop excision of the transformation zone specimens of cervix?

AIMS: To establish the value of examining additional histological levels in cone biopsy and large loop excision of the transformation zone (LLETZ) specimens of cervix. METHODS: Three deeper levels were examined from 200 consecutive cone biopsy and LLETZ specimens reported by a single pathologist. RESULTS: Examination of the first deeper level resulted in cervical intraepithelial neoplasia (CIN) being identified for the first time in five cases and in CIN1 being upgraded in five more. Invasive cancer was discovered in two cases that had shown high grade CIN initially. CONCLUSION: Examination of a single further level appears to be sufficient in those patients in whom a specimen is compromised because epithelium including the squamocolumnar junction is missing, or if there is a discrepancy between the histological findings and the preceding colposcopic or cytological history. If invasive disease is suspected on the basis of the cytological, colposcopic, or histological features, one or preferably two further levels should be examined.

Retrospective application of prognostic indices to pancreatitis discovered at necropsy.

Six prognostic indices, which were developed to assess inpatients with acute pancreatitis, were evaluated for possible retrospective application. When applied to a series of 14 cases in whom pancreatitis was first diagnosed at necropsy, the index devised by Jacobs et al was found to be the most useful, because in nine of these cases eight or more of the variables required were available for assessment from the case records. In the other indices evaluated fewer than eight of the required variables were available for retrospective assessment in most cases. Although undiagnosed pancreatitis is probably uncommon as a sole cause of death, the retrospective use of one or more of these indices may help assess the severity of the patient's condition on admission to hospital.

The prognosis in cervical epithelial changes of uncertain significance is similar to that of cervical intraepithelial neoplasia grade 1.

BACKGROUND/AIMS: The term epithelial changes of uncertain significance (ECUS) describes a minor degree of nuclear pleomorphism limited to the basal layers of cervical epithelium in the absence of severe inflammation with associated normal mitoses, koilocytosis, or koilocytosis associated features. This study aimed to investigate the long term prognosis of this lesion. METHODS: Slides from 128 women with low grade cervical abnormalities, accessioned consecutively, were reviewed. RESULTS: In 43 women the initial diagnosis of ECUS was confirmed and in 30 women the initial diagnosis of cervical intraepithelial neoplasia grade 1 (CIN1) was confirmed. Comparison of follow up data from these 73 women revealed a similar prognosis in the two groups in terms of regression to normal, persistence of low grade disease, or progression to high grade CIN. CONCLUSIONS: Low grade cervical disease (ECUS and CIN1) should be managed according to similar treatment protocols.

Distribution of cervical glandular intraepithelial neoplasia: are hysterectomy specimens sampled appropriately?

AIMS: To establish the validity of assuming that by examining midline blocks from the anterior and posterior lips of the cervix, a previously unsuspected cervical intraepithelial glandular neoplasia (CIGN) lesion would be identified. METHODS: The distribution of CIGN in 30 cone biopsy specimens of cervix was examined. RESULTS: Nine low grade and 21 high grade cases were identified involving one or other lip in 29 patients and one or both lateral edges in 20. The distribution of CIGN was unifocal in 20 patients, involved two distinct foci in nine, and three distinct foci in one. Only three patients had a circumferential distribution. Midline disease, either CIGN or squamous cervical intraepithelial neoplasia (CIN), or both, was present in 27 patients. CONCLUSION: These findings suggest that examining the midline blocks from hysterectomy specimens will result in the identification of CIGN lesions in over 90% of patients, either because the CIGN lesion is present in the midline or because an associated squamous CIN lesion will be identified, which will result in the examination of the entire cervix, with the consequent identification of the CIGN lesion.

Follow up of women with borderline cervical smears as defined by national guidelines.

AIM: To determine the proportion of women with abnormalities in cervical smears corresponding to borderline nuclear change, as defined by national guidelines, which return to normal or persist as cytological or histological abnormalities. METHODS: 313 women with borderline nuclear change diagnosed by a single pathologist using the national criteria were followed up for up to two years. RESULTS: On initial follow up, 45% of women had a negative smear or biopsy, 46.5% had a low grade cytological or histological abnormality, and 8.5% had a high grade abnormality. Of 81 patients in whom a second follow up smear or biopsy was available, 47% had no detectable abnormality, 38.5% had low grade lesion, and 14.5% had a high grade lesion. In total, 32 patients (10.2%) had a high grade lesion (defined as moderate or severe dyskaryosis on smear or CINII or CINIII on biopsy) on at least one follow up sample. CONCLUSIONS: The results support the use of the national criteria defining borderline nuclear change in identifying women at increased risk of developing a high grade cervical intraepithelial neoplasia, as identified histologically or cytologically, and highlight the importance of follow up in these patients.

A comparison of three methods of orienting cervical punch biopsies.

AIMS: To compare methods of orienting cervical punch biopsies, since well oriented biopsies are needed to optimise the diagnosis and grading of cervical intraepithelial neoplasia. METHODS: The orientation, the presence and preservation of the squamocolumnar junction, and the presence or absence of the surface layers of the squamous epithelium were compared in 345 cervical biopsies that had either been attached to paper (n = 112), floated directly into 10% formalin (n = 107), or floated into a solution of 10% formalin including 0.05% eosin (n = 126) in the colposcopy clinic. RESULTS: When the specimens were mounted on filter paper before fixation, they were more likely to be optimally oriented, to have a preserved squamocolumnar junction, and to have intact surface epithelium than specimens that were handled using the other methods, even when the specimens floated off the paper in transit. CONCLUSIONS: Cervical biopsy specimens should be mounted on paper before fixation and submission to the laboratory.

The appropriateness of examining the entire cervix histologically in hysterectomy specimens from women with a previous history of cervical intraepithelial neoplasia or dyskaryosis.

BACKGROUND/AIMS: To investigate the necessity of examining the entire cervix in hysterectomy specimens from women with a previous history of cervical intraepithelial neoplasia (CIN) or dyskaryosis. METHODS: The overall frequency with which squamous CIN was encountered in hysterectomy specimens of women with a previous diagnosis of squamous CIN or dyskaryosis was calculated in a sample of 71 women. The frequencies in women with positive or negative smears or biopsies between the initial diagnosis and hysterectomy, and in women with no intervening smear or biopsy, were also estimated. RESULTS: A persistent CIN lesion was identified in 18 patients (13 high grade cases; five low grade cases). A further eight patients who had high grade CIN also had microinvasive disease (seven cases of FIGO stage 1a1; one case of FIGO stage 1a2). Ten of the 20 patients with a positive smear or biopsy, but none of the 16 patients with a negative smear or biopsy between initial diagnosis and hysterectomy, had an abnormality on the hysterectomy specimen. Sixteen of the 35 patients who did not have an interim smear or biopsy had CIN in the hysterectomy cervix. CONCLUSIONS: These results support continuing the practice of examining the entire cervix in hysterectomy specimens from women with a previous cervical abnormality, regardless of its histological or cytological grade, if there has been a positive interim smear or biopsy or if neither investigation has been performed.

Florid cystic endosalpingiosis of the uterus.

A 73 year old woman presented with a right sided adnexal cystic mass. At laparotomy, this proved to be a benign serous ovarian cyst and an aggregation of thin walled subserosal and soft tissue cysts and spongy nodules up to 16 mm in diameter involving the side wall of the uterus and adjacent parametrium. These were removed by total abdominal hysterectomy and bilateral salpingo-oophorectomy. Histologically, the cystic spaces and smaller acini were lined by benign tubo-endometrioid epithelium, with smaller areas typical of serous differentiation and rare microfoci of endocervical-type mucinous epithelium. These features indicated multidirectional Mullerian differentiation in a process that, overall, was consistent with so called florid cystic endosalpingiosis. This lesion is to be distinguished from other benign conditions including multicystic mesothelioma, endometriosis, endocervicosis, florid deep glands of the uterine cervix, and deep Nabothian cysts of the uterine cervix.

Vimentin expression in benign and malignant breast epithelium.

AIMS: To determine vimentin expression in epithelial cells in benign breast disease and malignant breast tumours; to assess the value of vimentin expression as a prognostic indicator in breast carcinoma. METHODS: Frozen and formalin fixed, paraffin wax embedded sections from 78 carcinomas, three phyllodes tumours, 19 fibroadenomas and 19 cases of fibrocystic disease were examined with a monoclonal antibody from the V9 clone. A correlation between vimentin expression and known prognostic indicators was sought in ductal carcinomas. The intracellular localisation of vimentin was examined in benign and malignant lesions. RESULTS: Vimentin expression was identified on frozen section in the cells of ductal (53%), lobular (86%), and mucinous (33%) carcinomas and in the luminal epithelium of fibroadenomas (68%), cases of fibrocystic disease (47%), and a malignant phyllodes tumour. Formalin fixation reduced the percentage of carcinomas and cases of benign disease in which vimentin was detected. This reduction was more pronounced in fibroadenoma and fibrocystic disease than in ductal carcinoma. Associations were identified between vimentin expression as detected on frozen section and tumour grade, size, number of lymph nodes affected, oestrogen receptor content and growth fraction. Only the association with grade was significant (p = 0.045). There was no significant correlation between any of these prognostic variables and vimentin expression on paraffin wax sections. There was no difference in the intracellular localisation of vimentin staining between benign and malignant lesions, or between low and high grade ductal carcinomas. CONCLUSION: There is some loss of vimentin immunoreactivity after formalin fixation. Vimentin expression does not assist in differentiating between benign and malignant breast disease, but is correlated with tumour grade in ductal carcinoma.

Cytokeratin intermediate filament expression in benign and malignant breast disease.

AIM: To carry out a comprehensive study of cytokeratin expression in benign and malignant breast epithelium and breast myoepithelial cells; to examine changes in the cytokeratin profile in malignant and benign epithelium and in carcinomas of increasing histological grade. METHODS: Frozen sections from fibroadenomas (19 cases), fibrocystic disease (19 cases), and infiltrating ductal (68 cases), lobular (seven cases), and mucinous carcinomas (three cases) were examined using a panel of monoclonal antibodies. RESULTS: The luminal epithelium in all fibroadenomas and all cases of fibrocystic disease, as well as tumour cells in most carcinomas, reacted with the specific antibodies to cytokeratins 7, 8, 18, and 19 and to antibodies which included these cytokeratins in their specificities (Cam 5.2, AE1, AE3, RCK102, and LP34). In a few ductal carcinomas none of the tumour cells reacted for cytokeratins 7, 8, or 18. Three ductal carcinomas expressed cytokeratin 14. Only occasional cases expressed cytokeratins 3, 4, 10, and 13. Antibodies which included cytokeratins 5 and 14 in their specificities detected myoepithelial cells less efficiently than antiactin antibodies. CONCLUSION: The cytokeratin profiles in the luminal epithelium in benign breast disease and in tumour cells in most carcinomas are similar in most cases. Some carcinomas, however, are negative for cytokeratins 7, 8, or 18. This may provide a means of predicting the biological behaviour of a histologically borderline lesion.

Vimentin and cytokeratin expression in nodular hyperplasia and carcinoma of the prostate.

AIM: To assess the value of vimentin and cytokeratin (CK) intermediate filament proteins (IFPs) in distinguishing between nodular hyperplasia and carcinoma of the prostate and in predicting prognosis in prostatic cancer. METHODS: Fifteen carcinomas and 49 cases of nodular hyperplasia were studied using frozen sections and monoclonal antibodies to CK and vimentin IFPs. RESULTS: There was no statistically significant difference in vimentin expression between nodular hyperplasia and carcinoma. The luminal epithelium in both also reacted with antibodies which detect CK8, 18 and 19. CK 7 expression was found in 57% of cases of nodular hyperplasia and was not identified in any carcinoma. There was a reaction with antibodies to CK1, 2, 3, 4, 10, 11, and 13 in only a minority of cases. There was no statistically significant difference in vimentin and CK reactivity in high and low grade carcinomas. CONCLUSION: Neither vimentin nor CK expression assists in establishing whether a prostatic lesion is benign or malignant or in predicting the biological behaviour of a prostatic carcinoma.

Ki-67, oestrogen receptor, and progesterone receptor proteins in the human rete ovarii and in endometriosis.

AIM: To examine proliferative activity using the Ki-67 protein, oestrogen receptor protein, and progesterone receptor protein expression in the rete ovarii, and to make comparisons with their expression in endometriosis. METHODS: Immunohistochemistry was used to study the rete ovarii in 24 cases and endometriosis in seven cases, using antibodies to Ki-67 protein (growth fraction (GF) quantified using a point score method) and oestrogen receptor and progesterone receptor (quantified using the H score method). RESULTS: There was no evidence of a significant difference in the Ki-67 protein, oestrogen receptor, and progesterone receptor in the rete ovarii in different phases of the menstrual cycle (proliferative phase: GF = 1.052, oestrogen receptor H score = 13.4, progesterone receptor H score = 15.32; secretory phase: GF = 0.736, oestrogen receptor H score = 7.5, progesterone receptor H score = 1.84). The expression of all three proteins was greater in the foci of endometriosis (GF = 6.99, oestrogen receptor H score = 152.02, progesterone receptor H score = 127.36) than in the rete ovarii (p < 0.0005-0.0008, Mann-Whitney U test). CONCLUSIONS: There is a low rate of cellular proliferation in the rete ovarii and this structure shows less responsiveness to hormone stimulation than foci of endometriosis. These differences may provide a useful tool to distinguish the rete ovarii from endometriosis in cases of diagnostic difficulty.

Jass' classification revisited.

BACKGROUND: In 1986, Jass and colleagues claimed to have improved on Dukes' classification of prognosis for carcinoma of the colon and rectum. To have clinical relevance, such results should be reproducible and confirmed by other institutions. STUDY DESIGN: Retrospective clinicopathologic study of 312 carcinomas of the colon and rectum to determine whether or not Jass' classification is superior to that of Dukes' as assessed by their relative reproducibility and prognostic significance. RESULTS: Dukes' classification had excellent intraobserver and interobserver reproducibility (kappa values of 0.86 and 0.93, respectively). In contrast, the reproducibility of variables assessed by Jass showed only slight to fair agreement (lymphocytic infiltration: intraobserver and interobserver kappa values of 0.08 and 0.05, respectively, growth pattern: intraobserver and interobserver kappa values of 0.37 and 0.41, respectively). Dukes' stage and patient age were the most important prognostic variables on multivariate regression analysis. Tumor differentiation, nuclear polarity, tubule configuration, and lymphocytic infiltration remained significantly related to survival in the presence of Dukes' stage and age. The model which best predicted prognosis was a combination of Dukes' stage, patient age and tumor differentiation. Further addition of the variables assessed by Jass to this model did not significantly improve the prediction of prognosis. CONCLUSIONS: Dukes' classification is of greater prognostic value and more reproducible than the components of Jass' classification. The continued use of Dukes' classification is, therefore, warranted for prognostic and therapeutic decisions in patients with carcinoma of the colon and rectum.