ANATOMIC PATHOLOGY

 Dabska tumor (Endovascular papillary angioendothelioma) of testis: a case report with brief review of literature

 Alka Bhatia, Ritambra Nada, Yashwant Kumar, Prema Menon

 Abstract

The Dabska tumor also known as Endovascular papillary angioendothelioma is a rare type of hemangioendothelioma characterized by intraluminal papillary endothelial structures. Most of these are superficial in location but occurrence in deeper tissues is also known. Authors describe case report of testicular Dabska tumor in a child presenting as inguinal hernia. To the best of our knowledge this is the first case report describing the occurrence of this rare entity in testis.

 Background

The Dabska tumor is a rare, low-grade angiosarcoma that often affects the skin and subcutaneous tissues of children. Microscopically, it is characterized by anastomosing vascular channels, some of which contain papillary projections or tuft-like structures sometimes resembling renal glomeruli. These vascular channels as well as papillations are lined by cuboidal endothelial cells and are often flanked by dense hyalinized zones containing lymphocytes. The hyalinized material is thought to represent the basement membrane material possibly synthesized by tumor cells. Although rare cases of Dabska tumors have been reported in deeper locations, there is no case report describing the occurrence of this entity in testis. We therefore report first case of Dabska tumor arising in testis of a 1 year, 10 month old child who first presented as inguinal hernia. 

Case Report

A 1 year 10 month old male child was referred to our pediatric surgery department for pain and tenderness in a scrotal swelling for past 2 days. The swelling was diagnosed as non-obstructive inguinal hernia at the referral centre at 2 months of age. As it was not progressing the parents were advised to wait till the age of one year. There was no history of any trauma to genitalia. On examination the scrotum was swollen on left side and left testis was not palpable separately. The right testis was normally palpable in scrotal sac.  Ultrasonographic examination of the swelling showed a markedly enlarged testis with a heterogenous echotexture.  A possibility of testicular tumor was suggested and Fine Needle Aspiration Cytology (FNAC) / biopsy were advised. The FNAC showed few clusters of spindle cells and some cells with prominent nucleoli and vacuolated cytoplasm. However no definite opinion was given. A biopsy was also done which was reported as infarction of testis with extensive vascular proliferation. Subsequently a high left inguinal orchidectomy was carried out.  Intraoperatively the hernial sac contained clear fluid and no bowel loops were seen. The specimen was sent for histopathology.

 

The gross examination revealed a tumor replacing the entire testis; however shape of the testicle was maintained.  The tumor measured 4cm in diameter. Outer surface was smooth and brownish in colour.  The cut surface was also reddish brown with areas of hemorrhage. Though the tumor was solid, prominent cystic change and necrosis were seen in the central portion.  Microscopic examination showed a vascular tumor composed of anastomosing, irregular vascular channels forming papillae in some areas. These papillae had hyalinized cores and were lined by atypical endothelial cells. The vascular channels were also lined by plump cuboidal endothelial cells with focal hobnailed appearance and lymphocytic sprinkling. Frequent intracytoplasmic vacuolations and many mitotic figures were identified. The background showed large areas of hemorrhage and infarction. Few residual seminiferous tubules were found entrapped within the tumor tissue.  Immunohistochemically, the tumor cells showed strong positivity for CD34. The spermatic cord was normal. 

Discussion

The Dabska tumor was first described in 1969 by Maria Dabska in skin and subcutaneous tissues in children and named as malignant endovascular papillary angioendothelioma. She described 6 cases in her series. Since then approximately 30 cases have been described in the literature. Of these 18 were children and 12 were adults. The age of the patients ranged from birth to 83 years and there was no sex predilection. Besides skin and subcutaneous tissue, the tumor has been described in other deeper locations like spleen, soft tissues, bone, and tongue. Generally it presents as a slow-growing, painless, usually intradermal nodule that grows to 2-3 cm in diameter. In our case this tumor presented as inguinal hernia and after surgery was found to be a solid mass with cystic change. This kind of presentation has not been described in any of the previous studies. At low power, Dabska tumor appears similar to cavernous lymphangiomas. The cuboidal or hobnail endothelial cells lining the vascular structures are characterized by a high nuclear cytoplasmic ratio and an apically placed nucleus that produces a surface bulge, accounting for the term “hobnail” or “matchstick”. Individual endothelial cells range from cuboidal to tall and cylindrical with vacuolated cytoplasm and hyperchromatic eccentric nuclei on their luminal border. Mitotic figures may be seen, as in our case. Many intraluminal lymphocytes may also be evident, often attached to the endothelial cells. All these features were noted in our case. Immunohistochemically the tumor cells are positive for Von-Willebrand factor, CD31, CD34 and vascular endothelial growth factor receptor-3 (VEGFR-3). The staining for CD34 and VEGFR-3 is most intense out of these. In the present case strong positivity for CD34 immunostaining was observed in tumor cells. Ultrastructural examination demonstrates tumor cells with irregular nuclei, abundant perinuclear cytoplasmic filaments, and many pinocytotic vesicles. Weibel-Palade bodies may be evident. The hyaline globules consist of electron-dense basement membrane material.

 

Along with a closely related tumor, the retiform hemangioendothelioma it comes under the category of Hobnail hemangioendothelioma, a subtype of hemangioendothelioma. Pathophysiologcally it has been viewed as a distinct intralymphatic neoplasia and renamed as papillary intralymphatic angioendothelioma. These tumors are known to occur in previously existing benign vascular lesions like cavernous hemangiomas. In our case no cavernous hemangioma like areas could be found even after extensive sampling.

 

Orchidectomy is the treatment of choice. Prognostically these tumors are low-grade lesions with a capacity to extend to regional lymph nodes.1 Three of the original 6 cases were locally aggressive, with tumor invasion into deeper structures, including bone, musculature, fascia, and/or tendons. One of Dabska's original 6 Dabska tumor patients ultimately died of widespread pulmonary metastases. Therefore, Dabska tumor though believed to have a favorable prognosis, can be locally invasive with a potential to metastasize. In the present case there was no evidence of any nodal involvement or distant metastasis and the child was doing well on last follow up after 1 year of surgery.

 

The case describes the first occurrence of this entity in testis with an unusual presentation in the form of inguinal hernia. It also highlights the difficulty in diagnosing such tumors on FNAC or small biopsy specimens as it was mistaken for an infarct on biopsy.

Diagnostic Pathology 2006, 1:12 ,1746-1596

 

My Approach To The Interpretation Of Endometrial Biopsies And Curettings

[MY APPROACH] 

McCluggage, W G

Abstract

A major proportion of the workload in many histopathology laboratories is accounted for by endometrial biopsies, either curettage specimens or outpatient biopsy specimens. The increasing use of pipelle and other methods of biopsy not necessitating general anaesthesia has resulted in greater numbers of specimens with scant tissue, resulting in problems in assessing adequacy and in interpreting artefactual changes, some of which appear more common with outpatient biopsies. In this review, the criteria for adequacy and common artefacts in endometrial biopsies, as well as the interpretation of endometrial biopsies in general, are discussed, concentrating on areas that cause problems for pathologists. An adequate clinical history, including knowledge of the age, menstrual history and menopausal status, and information on the use of exogenous hormones and tamoxifen, is necessary for the pathologist to critically evaluate endometrial biopsies. Topics such as endometritis, endometrial polyps, changes that are induced by hormones and tamoxifen within the endometrium, endometrial metaplasias and hyperplasias, atypical polypoid adenomyoma, adenofibroma, adenosarcoma, histological types of endometrial carcinoma and grading of endometrial carcinomas are discussed with regard to endometrial biopsy specimens rather than hysterectomy specimens. The value of ancillary techniques, especially immunohistochemistry, is discussed where appropriate.

APA, atypical polypoid adenomyoma; EIC, endometrial intraepithelial carcinoma; EIN, endometrial intraepithelial neoplasia; WHO, World Health Organization

In many histopathology laboratories, endometrial specimens account for a major proportion of the workload. Most specimens are taken because of abnormal uterine bleeding or other related symptoms, and the pathologist is expected to exclude an endometrial cancer or a precancerous lesion. In some cases, a benign cause for abnormal uterine bleeding is identified, such as endometritis or endometrial polyp. In this review, I will outline my approach to the interpretation of endometrial biopsy specimens, especially concentrating on areas which, in my experience, create difficulties for pathologists. Endometrial biopsy specimens are now rarely taken to date the endometrium and to assess whether ovulation has occurred, as serum measurements of various hormones give equivalent or more information. In this review, dating of the endometrium will not be discussed, as this has been dealt with in detail recently. Similarly, pure mesenchymal lesions, benign or malignant, may be identified on endometrial biopsy and these will not be discussed further.

CLINICAL HISTORY

In evaluating an endometrial biopsy specimen, an adequate clinical history is important, including the age of the patient and the reason for the biopsy. The menopausal status as well as the date of onset of the last menstrual period and the length of the menstrual cycle in premenopausal women should be provided. In many cases of postmenopausal bleeding, the patient is not actually postmenopausal but rather is perimenopausal, with a prolonged interval between periods. This results in the clinician and the patient assuming that the woman is postmenopausal. Before biopsy, many women with abnormal uterine bleeding are already taking exogenous hormones, especially progestogenic compounds, to control the bleeding, and this information is not always conveyed to the pathologist. Other women may be taking hormone replacement therapy or contraceptives. These hormonal compounds may alter the morphological appearance of the endometrium and a knowledge that these, and other relevant drugs such as tamoxifen, are being taken is of paramount importance to the pathologist.

CRITERIA FOR ADEQUACY OF ENDOMETRIAL BIOPSY SPECIMENS

Previously, endometrial biopsy specimens were largely obtained by dilatation and curettage carried out under general anaesthesia. Most endometrial specimens are now taken at outpatients by pipelle or other techniques, with the result that many biopsy specimens contain scant, or even no, endometrial tissue. Paradoxically, superficial endometrial biopsy specimens with scant tissue often take longer to assess than intact biopsy specimens with an appreciable amount of tissue. The pathologist is faced with making a decision on whether the biopsy specimen is adequate. A recent study showed that there is considerable disagreement among specialist gynaecological pathologists about what constitutes an adequate endometrial biopsy specimen. It has been shown that in a postmenopausal woman with an atrophic endometrium and no focal lesion on ultrasound scan, the presence of scant endometrial tissue in biopsies from outpatients is the norm and there is little chance of missing relevant pathology. Furthermore, the classification of a biopsy specimen as inadequate may have medicolegal and clinical implications. For example, some clinicians routinely conduct a repeat biopsy when an endometrial specimen has been classified as inadequate. It is my policy in reporting endometrial specimens that a biopsy specimen (from either outpatient clinic or curettage) is classified as inadequate only if no endometrial tissue is present. If there is any endometrial tissue, no matter how little, I do not categorise the specimen as inadequate. Instead, I use the term unassessable for those biopsies where minimal endometrial tissue is present and state that, although there is no hyperplasia, malignancy or any other specific diagnostic lesion, the tissue cannot be assessed. The presence of even a minimal amount of endometrial tissue provides presumptive evidence that the endometrial cavity has been entered, although theoretically endometrial-type glands with or without stroma can be derived from tuboendometrial metaplasia or endometriosis within the cervix. It is emphasised that biopsy specimens with scant tissue, which usually comprise only superficial strips of endometrial glands, should be examined carefully under high power to look for mitotic activity (abnormal in truly postmenopausal women) and atypia. If intact tissue, comprising glands and stroma, is present then this can be typed, although with a comment that only a limited amount of tissue is available for examination.

The author has suggested algorithm for assessing the adequacy of an endometrial biopsy specimen. The interpretation of the relevance of an unassessable specimen or scant specimen rests with the clinician. As discussed, this is the norm with an atrophic endometrium and no focal lesion on ultrasound scan, but not a reason for repeating the biopsy. With a thickened endometrium, a focal lesion or a strong clinical suspicion of major pathology, however, a scant specimen may be an indication of the need for repeat biopsy. Generally, this should comprise a formal curetting rather than an outpatient biopsy.

DIAGNOSTIC ALGORITHM FOR EXAMINATION OF ENDOMETRIAL BIOPSIES

Tissues not derived from the endometrium are commonly represented in endometrial biopsies. These should be identified, evaluated and mentioned in the pathology report. Cervical tissue, either cervical glandular or squamous epithelium with or without stroma, may be present and should be examined to exclude major pathology. The presence of adipose tissue or even intestinal mucosa may indicate uterine perforation, which should be conveyed to the clinician. In cases of uterine perforation, mesothelial cells or inflammatory cells may be mixed in with the adipose tissue. This may reflect the presence of underlying pelvic or peritoneal abnormalities, which has resulted in a reactive mesothelial proliferation that has fixed the uterus and made perforation more likely. Adipose tissue in an endometrial biopsy specimen may rarely be derived from a uterine lipoleiomyoma or lipoma. Other tissues that may be present in an endometrial biopsy specimen include myometrial smooth muscle and tissue derived from the lower uterine segment or isthmus. Tissue from the lower uterine segment may morphologically be confused with an endometrial polyp, as the stroma has a fibrous appearance and the glands are often few in number. The absence of thick-walled stromal blood vessels and the characteristic admixture of mucinous endocervical epithelium (usually present on the surface) and ciliated tubal-type epithelium (usually in the deeper glands) should, however, suggest an origin from the lower uterine segment.

After assessing tissue that is not derived from the endometrium, the endometrial tissue present (if any) should be examined. It is useful to give some indication of the amount of endometrial tissue represented and the criteria for adequacy have already been discussed. Next, the endometrium should be typed with reference to the age of the patient, the date of onset of the last menstrual period and any hormonal treatment. The morphological features of a cyclical and atrophic endometrium will not be detailed, but a few salient points are mentioned in the following sentences. Caution should be exercised in diagnosing a proliferative endometrium in the absence of mitoses, although these may be few in number. Stromal and glandular mitoses are commonly found in a proliferative endometrium. An atrophic endometrium, which may or may not be an indication of the postmenopausal state (atrophy is also characteristic of some hormonal agents), may be confused with a proliferative endometrium, as the glands commonly have a tubular appearance and there may be apparent nuclear stratification. A further point of confusion is that not all areas of the endometrium respond at the same rate to endogenous or exogenous hormones—for example, some areas may show proliferative features but others exhibit early secretory activity. Difficulty arises when an endometrium from a truly postmenopausal woman (as stated previously, some women with postmenopausal bleeding are not truly postmenopausal, but rather are perimenopausal, with irregular cycles) shows proliferative activity without features of hyperplasia. My approach is to state in the report that there is continuing proliferative activity suggesting ongoing oestrogenic stimulation, although there are no features of hyperplasia or malignancy. Endometrial proliferative activity may occur with uterine prolapse and in endometrial polyps in postmenopausal women.

ARTEFACTS IN ENDOMETRIAL BIOPSY SPECIMENS

Several common artefacts are observed in endometrial biopsy specimens, which have received scant attention in the literature. Some of these may be misinterpreted as endometrial hyperplasia or even as carcinoma if not appreciated to be artefactual. Telescoping refers to glands within glands and is commonly seen. Artefactual crowding and compression of glands may result in consideration of a complex endometrial hyperplasia). With this artefact, the glands become moulded together and often there is tearing of the tissue around the glands, which is a clue to the artefactual nature. An artefact that is especially common with, but not exclusive to, biopsy specimens from outpatients is the presence of superficial strips of endometrium with a pseudopapillary architecture). This may result in consideration of a wide range of papillary lesions, benign and malignant, which occur in the endometrium. Such superficial strips of papillary endometrium, which are generally atrophic, should be examined under high power to look for proliferative activity and nuclear atypia. Crushed endometrial glands and stroma may be extremely cellular and can cause concern. As with other tissues, crushed areas should not be viewed in isolation.

ENDOMETRITIS

Endometritis may result in symptoms of abnormal uterine bleeding and the pathologist should always exclude this. Lymphocytes, including natural killer cells and lymphoid aggregates, are a normal component of the endometrium, and polymorphs are characteristic of the premenstrual and menstrual phases. The presence of plasma cells is widely regarded as the most useful criterion for a diagnosis of endometritis, although these are often admixed with other inflammatory cells, both acute and chronic, and may be a minor component of the inflammatory cell infiltrate. In endometritis, the inflammatory cell infiltrate may be focal and plasma cells just beneath the surface glands are usually most easily identified. Other morphological features that alert the pathologist to a possible endometritis may also exist. These include an endometrium that exhibits a disturbance in maturation—for example, focal areas that are out of cycle with other areas, stromal oedema and, characteristically, a spindle-cell alteration of the stroma, especially around glands. A degree of architectural complexity and cytological atypia may also be seen if the inflammatory cell infiltrate is marked, resulting in potential overdiagnosis of a hyperplasia or carcinoma. Problems in recognising plasma cells may occur, especially on histological sections that are less than optimal. Endometrial stromal cells may have a plasmacytoid appearance with eccentric nuclei, and the pathologist should be certain that classic plasma cells are present. It is emphasised that in the absence of the other morphological features of endometritis described earlier, an exhaustive search for plasma cells is not justified. Occasional stromal plasma cells may be identified in an otherwise normal endometrium, and in these circumstances a diagnosis of endometritis should not be made. Plasma cells may be present in the stroma of endometrial polyps and also in association with an endometrial malignancy. A variant of endometritis, termed “focal necrotising endometritis, has been described and is characterised by an inflammatory cell infiltrate comprising lymphocytes, neutrophils and histiocytes without plasma cells. Granulomatous endometritis raises the possibility of sarcoidosis, tuberculosis and other granulomatous diseases.

Ancillary studies may aid in the diagnosis of endometritis, although they are not generally necessary. Histochemical staining with methyl green pyronin may be used to identify plasma cells, as can immunohistochemical analysis or in situ hybridisation for [kappa] and [lambda] light chains. Plasma cell markers VS38 (CD38) and syndecan (CD138) have also been used to identify plasma cells, although in my experience endometrial glandular and stromal cells are diffusely positive with VS38, making identification of plasma cells impossible. By contrast, syndecan is more useful, as most stromal cells are negative, although endometrial glands are positive. Antibodies against B and T lymphoid cells may also be of value. In the normal endometrium, most lymphocytes are T cells and natural killer cells (granulated lymphocytes). B lymphoid cells are rare, accounting for fewer than 1% of all endometrial leucocytes, and are found mainly in lymphoid aggregates in the basal cell layer as well as in scattered individual cells more superficially. A recent study showed that in cases of endometritis, the number of T lymphocytes and natural killer cells did not differ from controls. A substantially increased number of B cells, however, was identified in cases of endometritis and these were observed in unusual locations, such as beneath the surface epithelium, intraepithelially and within glandular lumina. This may be of value in diagnosing endometritis on small biopsy specimens in which the superficial endometrium is preferentially sampled.

ENDOMETRIAL POLYPS

Polyps are a common cause of abnormal bleeding in premenopausal and postmenopausal women. The pathological diagnosis is generally straightforward if the gynaecologist is aware of the presence of a polyp, has conveyed this information to the pathologist and has removed the polyp intact. The gynaecologist may believe that a polyp is present, but histological examination shows a cyclical endometrium, often secretory in type, reflecting the fact that an abundant secretory endometrium may have a polypoid appearance. Often, the gynaecologist is not aware of the presence of a polyp. In such instances, fragments of polyp are often admixed with fragments of non-polypoid endometrium, making the diagnosis difficult, as the features can be subtle. When a biopsy is carried out for abnormal uterine bleeding, the pathologist should always consider the possibility of a polyp. On examination under low power, the initial clue to the presence of a polyp is often the admixture of fragments of a normal cyclical endometrium and fragments that are morphologically different. The stroma is generally more fibrous than in the surrounding endometrium, although this is not invariable. Other morphological features commonly found in polyps include collections of thick-walled stromal blood vessels, glandular architectural abnormality (often in the form of dilated glands with unusual shapes and focal crowding) and various epithelial metaplasias. The glands within a polyp often show proliferative activity, even when the surrounding endometrium does not. The following points on endometrial polyps are worthy of mention:

1. Proliferative activity is common in endometrial polyps, even in postmenopausal women. This is of no relevance, although I generally include a comment to this effect in the pathology report. In such circumstances, it is useful if non-polypoid endometrium is also present, and whether this also shows proliferative activity needs to be mentioned. If no non-polypoid endometrium is represented, I comment, stating that proliferative activity may occur in, and be confined to, endometrial polyps in postmenopausal women, but that non-polypoid endometrium is not represented.

2. A diagnosis of simple hyperplasia should not be made in the case of an endometrial polyp, as a glandular architecture reminiscent of simple hyperplasia is a normal feature within a polyp. Focal mild glandular crowding is not uncommon and in my opinion does not warrant a diagnosis of complex endometrial hyperplasia. With considerable glandular crowding, however, a diagnosis of complex hyperplasia should be made. This may be confined to the polyp or also include the non-polypoid endometrium. As discussed earlier, the histological features of any non-polypoid endometrium represented should be described and, if none is present, this should be mentioned, and the report should state that complex hyperplasia may be confined to the polyp or also include the non-polypoid endometrium. In such circumstances, the gynaecologist should probably conduct a repeat biopsy of the endometrium. If atypical hyperplasia is present in an endometrial polyp, this should be reported and the surrounding non-polypoid endometrium thoroughly evaluated; this often necessitates repeat biopsy.

3. Carcinomas, so-called polyp cancers, may arise in endometrial polyps. Obviously, if a cancer is identified in an endometrial polyp removed by biopsy, it is impossible to ascertain, without full evaluation of the surrounding endometrium, whether the cancer has arisen in or has secondarily involved the polyp. Polyp cancers may be endometrioid in type, but serous proliferations, serous carcinoma or its precursor lesion endometrial intraepithelial carcinoma (EIC), have a particular propensity to arise in or be associated with otherwise benign endometrial polyps. Endometrial polyps should be carefully examined to look for small foci of serous carcinoma or EIC. This is discussed more fully later.

4. Endometrial polyps are particularly common in association with tamoxifen. This is also discussed later.

 

OTHER BENIGN ENDOMETRIAL LESIONS

It is useful to have a checklist of benign lesions other than those listed earlier, including granulomas, placental site nodules and the various forms of epithelial and stromal metaplasias. These metaplasias will not be discussed in detail, as they have been reviewed recently. The following are a few salient points regarding endometrial epithelial metaplasias:

1. Epithelial metaplasias, especially squamous or mucinous in type, may coexist with hyperplasia or a carcinoma. In case of florid epithelial metaplasia, a hyperplastic or malignant process should be looked for and excluded. This may be problematic when there is florid squamous metaplasia of the keratinising or morular type. Assessing the glandular architecture and cytology may be difficult, as the squamous elements can be so extensive that the underlying glandular component is almost totally obliterated. Such florid examples of squamous metaplasia, which may occur in association with obstruction, so-called ichthyosis uteri, may also result in consideration of a well-differentiated squamous carcinoma. Indeed, in the setting of chronic obstruction, squamous carcinoma of the endometrium may develop from florid squamous metaplasia.

2. Ciliated cells are common in a normal cyclical endometrium. I would reserve a diagnosis of ciliated metaplasia for cases characterised by extensive ciliation and where the cells have abundant eosinophilic cytoplasm, resulting in an appearance similar to the epithelium of the normal fallopian tube. Ciliated cells are normal in the lower uterine segment and should not be interpreted as ciliated metaplasia.

3. Papillary syncytial metaplasia, although termed metaplasia, is actually a reparative response occurring after breakdown. When florid, this may be suggestive of a serous carcinoma or an EIC. In this circumstance, attention should be paid to the background endometrium, which, in papillary syncytial metaplasia, usually shows features of breakdown. In problematic cases, p53 immunohistochemistry (discussed below) may be of value.

4. Many endometrial epithelial metaplasias occur without obvious causation. Epithelial metaplasias are, however, especially likely to be seen in endometrial polyps and in association with hormonal preparations.

5. With florid mucinous metaplasia, exclusion of a well-differentiated mucinous adenocarcinoma can be extremely difficult, if not impossible, on endometrial biopsy, as some mucinous adenocarcinomas of the endometrium are cytologically bland, even those which show myometrial infiltration. Mucinous proliferations of the endometrium, which are identified on biopsy, can be subdivided into categories based on the degree of architectural complexity, which correspond to an increasing risk of the presence of mucinous adenocarcinoma in subsequent hysterectomy.

6. Benign papillary proliferations (not strictly metaplasias) with fibrovascular cores are rarely seen, especially on the surface of endometrial polyps. These papillary proliferations may be architecturally complex and are often associated with epithelial metaplasias. These are benign proliferations, and in this situation the presence of architectural complexity does not signify a hyperplastic process.

7. With some epithelial metaplasias for example, clear-cell metaplasia, papillary syncytial metaplasia and benign papillary proliferation the differential diagnosis may be between a metaplastic process and a type-2 endometrial cancer or an EIC. In such instances, immunohistochemical analysis may be of value, in that EIC and type-2 cancers usually show diffuse intense nuclear p53 staining, whereas oestrogen receptor is generally negative or weakly positive. By contrast, most metaplastic processes are oestrogen receptor positive and exhibit a weak heterogeneous pattern of p53 staining. Many exceptions exist, however, with some clear-cell carcinomas in particular being p53 negative or weakly positive.

8. The surface of some endometrial carcinomas, of either the mucinous or endometrioid type, has an appearance that may closely mimic cervical microglandular hyperplasia or various forms of metaplasia such as mucinous metaplasia or papillary syncytial metaplasia. These superficial areas are especially likely to be sampled on biopsy and may pose problems in diagnosis.

 

EFFECTS OF HORMONES ON THE ENDOMETRIUM

Hormones have varying effects on the endometrium and it is essential that the clinician supplies details to the pathologist regarding any hormone treatment. Such information is not always provided. With any unusual, non-cyclical appearance of the endometrium, the pathologist should consider exogenous hormones. Some hormone preparations, especially those that contain both oestrogen and progestogen (most modern hormone replacement treatment regimens), characteristically result in a weak or poorly developed secretory endometrium, whereas with other preparations the endometrium is atrophic. Progestogen-only compounds result in a characteristic morphological appearance with atrophic or weak secretory-type glands set in an expanded stroma that exhibits varying degrees of pseudodecidualisation. This pseudodecidualisation is often most prominent just beneath the surface glands and is usually accompanied by an inflammatory cell infiltrate, largely comprising natural killer cells. Similar appearances occur with the Mirena coil, an intrauterine device containing progestogen, which is widely used. This often produces a polypoid surface and other morphological features, including reactive atypia of the surface glands, epithelial metaplasias, deposition of stromal haemosiderin, calcification, necrosis and stromal myxoid change.

EFFECTS OF TAMOXIFEN ON THE ENDOMETRIUM

Tamoxifen is widely used as adjuvant therapy in the management of breast cancer. The use of tamoxifen as a prophylactic agent in patients with a family history of breast cancer is now being investigated. The effects of tamoxifen on the endometrium has been the subject of several reviews. Although the efficacy of tamoxifen in breast cancer is due to its anti-oestrogenic properties, tamoxifen may exert a proliferative effect on the endometrium, resulting in the lesions described later in this article. Endometrial samples from women taking tamoxifen tend to be scanty, as tamoxifen may result in fibrosis of the endometrial stroma, making evaluation by biopsy difficult. The fibrosis can result in cystic dilatation of endometrial glands on an obstructive basis and this can be seen on hysteroscopy.

The most common endometrial lesions seen in association with tamoxifen are benign polyps, which may be single or multiple. In some instances, the whole of the endometrium has a polypoid appearance and it may be difficult to discern individual polyps. Tamoxifen-associated endometrial polyps do not have pathognomonic histological features, although they are often larger than sporadic polyps and are more likely to exhibit epithelial metaplasias, staghorn glands and stromal condensation around glands, reminiscent of a cambium layer. In some cases, this stromal condensation may result in consideration of a low-grade adenosarcoma. Tamoxifen-associated polyps should be extensively sampled and examined carefully under the microscope, as small carcinomas, of both the endometrioid and non-endometrioid type, may occur. Indeed, as discussed previously, there is a peculiar tendency for serous carcinoma and EIC to arise within endometrial polyps, whether sporadic or associated with tamoxifen.

Tamoxifen may result in the full spectrum of endometrial hyperplasias and in the development of endometrial cancers, both endometrioid and non-endometrioid. As women with breast cancer are more likely to develop endometrial carcinoma due to common risk factors, such as high socioeconomic status, low parity or hyperoestrogenic states, it is difficult to ascertain whether a true association exists between tamoxifen and the development of endometrial cancer. Most epidemiological studies, however, suggest that tamoxifen is associated with an increased risk of developing endometrial cancer, which is two to three times that in patients with breast cancer who are not taking tamoxifen. Clearly, the risk increases with increasing duration and dosage of tamoxifen. As the effects of tamoxifen on the endometrium are believed to be due to oestrogenic activity, it is expected that most endometrial cancers should be endometrioid in type, as these neoplasms are hormone receptor positive. This is, however, controversial, with some studies suggesting that type-2 cancers, such as serous carcinoma, are proportionally more common in association with tamoxifen. Indeed, it has been suggested that carcinosarcomas (in reality, metaplastic carcinomas often associated with an epithelial component of type-2 carcinoma are particularly likely to be associated with tamoxifen, and adenosarcomas have also been reported. Other studies, however, have found most tamoxifen-associated endometrial neoplasms to be low-grade endometrioid adenocarcinomas.

ENDOMETRIAL HYPERPLASIA

In my experience, various aspects related to endometrial hyperplasia commonly create problems for pathologists. In the following sections, I discuss some of these problematic areas.

Exclusion of benign mimics

Before diagnosing an endometrial hyperplasia, it is important to exclude the many benign mimics. The potential benign mimics of endometrial hyperplasia are listed in box 1. Most of these show an increase in the normal gland to stroma ratio, which is a defining feature of complex endometrial hyperplasia and is present in most cases of atypical hyperplasia (complex atypical hyperplasia). One of the most common lesions to be misdiagnosed as a hyperplasia is an endometrial polyp, especially when this is removed piecemeal, and when the gynaecologist is not aware of the presence of a polyp and the suggestion of this is not conveyed to the pathologist. The morphological features of endometrial polyps, as well as several of the other potential benign mimics of endometrial hyperplasia, have been discussed. A secretory endometrium (and Arias–Stella effect endometrium) often shows an increase in the gland to stroma ratio and may be misdiagnosed as an endometrial hyperplasia, especially when subnuclear vacuolation is not obvious. In general, secretory activity is rare in endometrial hyperplasias, although this does occur, especially when hormone treatment has already been instigated. Cystic atrophy is distinguished from simple hyperplasia by the atrophic appearance of the glands, including the lack of proliferative activity. In general, mitotic activity should be identified before diagnosing an endometrial hyperplasia.

Classification of endometrial hyperplasia

Endometrial hyperplasias should be classified according to the 1994 World Health Organization (WHO) scheme. Older terms such as cystic hyperplasia and adenomatous hyperplasia should be avoided. The term dysplasia should not be used with regard to the endometrium. The 1994 WHO classification was based on a seminal study of endometrial hyperplasia, and divides hyperplasias into simple and complex forms depending on the glandular architecture. In simple hyperplasia, the normal gland to stroma ratio is largely maintained, although there may be a slight increase. In complex hyperplasia, there is an increase in the gland to stroma ratio. Simple and complex hyperplasias are further divided into atypical and non-atypical categories on the basis of the presence or absence of nuclear atypia. Assessment of nuclear atypia is extremely subjective, resulting in marked interobserver variation, even among specialist gynaecological pathologists, in the classification of endometrial hyperplasia. The 1994 WHO classification results in four categories of endometrial hyperplasia simple non-atypical, complex non-atypical, simple atypical and complex atypical. In practice, atypia usually occurs in endometria with complex architecture and it is uncommon to diagnose simple atypical hyperplasia, although this rarely occurs. As a result, many pathologists use three categories of endometrial hyperplasia simple, complex and atypical. All these categories have an increased risk of developing an endometrioid-type endometrial adenocarcinoma, although this is extremely low in simple and complex endometrial hyperplasias (approximately 1% and 3%, respectively), which are usually self-limiting lesions that regress; although once the lesions are diagnosed, hormonal treatment is usually instigated. The risk of developing an endometrioid adenocarcinoma after a diagnosis of atypical hyperplasia is difficult to ascertain, as, once atypical endometrial hyperplasia is diagnosed, it is treated either hormonally or more usually surgically. With atypical hyperplasia, however, there is a considerable risk of transformation into an endometrioid adenocarcinoma, probably of the order of 25-40%.  Additionally, an endometrioid adenocarcinoma may already be present and not sampled by biopsy. In my experience, many cases diagnosed as atypical endometrial hyperplasias represent well-differentiated endometrioid adenocarcinoma.

Morphological features of endometrial hyperplasia

In a simple hyperplasia, the normal gland to stroma ratio is maintained or there is slight increase. The endometrium shows proliferative activity, with cystically dilated glands of irregular sizes and shapes. Some glands may exhibit out-pouchings, infoldings and budding. No nuclear atypia is seen, the nuclei being oval and maintaining their orientation to the underlying basement membrane. A major problem is the distinction between simple endometrial hyperplasia and disordered proliferative endometrium, a term widely used, although the histological features are not well characterised. The morphological features of these overlap and the distinction is poorly reproducible. In my practice, I require to see large numbers of dilated glands to diagnose simple hyperplasia, whereas if only occasional dilated glands are present, I diagnose this as disordered proliferative endometrium, especially if the patient is perimenopausal. Disordered proliferative endometrium is common in the perimenopausal years because of anovulatory cycles. In any case, the management of simple endometrial hyperplasia and disordered proliferative endometrium is usually identical, in the form of progestogenic compounds. Furthermore, a continuum exists between disordered proliferative endometrium and simple hyperplasia. In essence, these are both benign conditions related to prolonged oestrogenic stimulation, with little risk of transformation into endometrioid-type adenocarcinoma.

In complex hyperplasia, there is an increase in the gland to stroma ratio with glandular crowding. The glands are often closely packed, although some stroma usually remains between individual glands. The glands show proliferative activity and, by definition, there is no nuclear atypia. Simple hyperplasia is usually a generalised condition, whereas most, but not all, cases of complex hyperplasia are focal, often occurring on a background of simple hyperplasia.

In atypical hyperplasia, there is, by definition, nuclear atypia. Assessing nuclear atypia is one of the most subjective and problematic areas in gynaecological pathology, but this assessment is crucial in the management of endometrial hyperplasia, as surgery is generally undertaken for atypical hyperplasia, whereas hormone treatment is common for non-atypical hyperplasia, although there are many exceptions. Nuclear atypia may be subtle and in its evaluation it is useful to compare the cytology of the atypical glands with that of the residual normal endometrial glands. In atypical hyperplasia, the nuclear changes are often accompanied by cytoplasmic changes, such that the cells have more abundant, often eosinophilic, cytoplasm. Cytoplasmic changes, including increased cytoplasmic eosinophilia, may also be seen in various endometrial metaplasias, such as ciliated metaplasia. Nuclear features in atypical hyperplasia, which, as stated earlier, are often subtle, include nuclear stratification, loss of polarity, nuclear rounding and the presence of nucleoli. In fact, rather than overt nuclear atypia, it is often the differing nuclear features between abnormal and normal glands that is characteristic. This is reflected in a well-publicised suggested alternative terminology, proposed by Mutter and coworkers (discussed later in this review), for the classification of precursor lesions of endometrioid-type endometrial adenocarcinoma. In this classification, the term endometrial intraepithelial neoplasia (EIN) is used and rather than true nuclear atypia, cytological demarcation between normal and abnormal areas is evaluated. It should be emphasised that the term EIN refers to the precursor lesion of endometrioid adenocarcinoma and is different from EIC, the precursor of serous adenocarcinoma, which is discussed in detail later in this review.

Distinction between atypical hyperplasia and grade-1 endometrioid adenocarcinoma

It may be difficult, or even impossible, in a small biopsy specimen to distinguish between an atypical hyperplasia at the upper end of the spectrum and a grade-1 endometrioid adenocarcinoma. This is not surprising, as these two lesions are part of a spectrum without clearly defined boundaries. As a reflection of this continuum, it is acceptable in an endometrial biopsy to render a diagnosis of at least atypical hyperplasia, cannot exclude grade-1 endometrioid adenocarcinoma. It has been suggested that in biopsy specimens, these two lesions should be combined under the designation endometrioid neoplasia. Usually, the management of atypical hyperplasia and grade-1 endometrioid adenocarcinoma is identical, comprising hysterectomy. In a young woman with, for example, polycystic ovary syndrome with atypical hyperplasia or grade-1 endometrioid adenocarcinoma, and who wishes to retain her fertility, conservative management in the form of progestogenic compounds may be undertaken after MRI, to exclude myoinvasive disease. If such treatment is undertaken, then regular endometrial biopsies should be carried out, for example, at 3-monthly intervals. In these circumstances, successful pregnancies have ensued, although there is a high risk of subsequent recurrence of hyperplasia or carcinoma. On the other hand, it is desirable to distinguish between atypical hyperplasia and grade-1 endometrioid adenocarcinoma, as many clinicians are reluctant to manage grade-1 adenocarcinoma conservatively. Moreover, if a diagnosis of adenocarcinoma is made on endometrial biopsy, the patient is more likely to undergo full staging surgery, including peritoneal washings, and can be entered into clinical trials.

Several features assist in distinguishing between atypical hyperplasia and grade-1 endometrioid adenocarcinoma. A desmoplastic stromal response is strong supportive evidence of an adenocarcinoma, but this is identified only in a minority of biopsies of endometrial carcinoma. A solid back-to-back glandular architecture with complete exclusion of stroma is suggestive of adenocarcinoma, as in most atypical hyperplasias a little stroma remains between glands. Bridging between adjacent glands, resulting in a cribriform pattern, as well as the presence of luminal necrosis with polymorph infiltration, suggests an adenocarcinoma. A surface papillary pattern, maze-like meandering glands and solid non-squamoid areas also suggest an adenocarcinoma. As stated earlier, however, atypical hyperplasia and grade-1 endometrioid adenocarcinoma form part of a spectrum without sharp demarcation.

SUGGESTED ALTERNATIVE TERMINOLOGY FOR CLASSIFICATION OF PRECURSOR LESIONS OF ENDOMETRIOID ADENOCARCINOMA

EIN terminology has been extensively championed by Mutter and coworkers. A recent review in this journal has summarised the arguments and I will only briefly discuss them. EIN classification is based on the integration of morphological, molecular genetic, morphometric and prognostic data. Briefly, five criteria must be fulfilled for a diagnosis of EIN:

1. Area of glands exceeding that of stroma (volume percentage stroma <55%)

2. Cytology differing between architecturally crowded focus and background—that is, cytological demarcation

3. Maximum linear dimension of the lesion exceeding 1 mm

4. Exclusion of benign mimics

5. Exclusion of carcinoma.

It is emphasised that, although the criteria were derived by using morphometry and molecular studies, EIN is diagnosed on standard morphological examination.

Proponents of the EIN scheme regard most, but not all, cases that would be diagnosed as simple or complex non-atypical hyperplasia by using the WHO system as a response of the endometrium to oestrogen excess, and they do not use the term hyperplasia.

Opponents of the EIN system point out that implementation of this will require retraining pathologists and clinicians who will be confused by yet another classification of endometrial hyperplasia. As nuclear atypia per se is not a prerequisite for a diagnosis of EIN, some cases that would be classified as complex non-atypical hyperplasia by using the WHO scheme may be categorised as EIN. My own view is that we should continue to use the 1994 WHO classification until further evidence emerges regarding the reproducibility, practicalities and prognostic implications of the EIN system.

ATYPICAL POLYPOID ADENOMYOMA

Only a few comments will be made regarding atypical polypoid adenomyoma (APA), concentrating on those issues that are likely to pertain to endometrial biopsy specimens. APA usually occurs in young women and most commonly affects the lower uterine segment. Although APA is a polypoid lesion, this may be removed piecemeal. Histologically, architecturally complex glands are set in an abundant stroma that usually exhibits smooth-muscle differentiation, although in some cases smooth-muscle features are not obvious, the stroma having a more fibrous appearance, and the term atypical polypoid adenomyofibroma has been used. Associated cytological atypia is usually mild or moderate. Squamous metaplasia, in the form of morules, sometimes with central necrosis, is often prominent. The main alternative diagnostic consideration on endometrial biopsy is likely to be an endometrioid adenocarcinoma showing myometrial infiltration. In APA, however, the smooth muscle stroma is generally more cellular and disorganised than normal myometrium. Furthermore, in a biopsy specimen from an endometrioid adenocarcinoma, it would be extremely unusual to see only myoinvasive disease in the absence of free tumour fragments.

ADENOFIBROMA AND ADENOSARCOMA

Adenofibroma, with a benign stromal component, and adenosarcoma, with a malignant one, are two of the mixed epithelial and mesenchymal (mixed Mullerian) tumours of the uterus. In both, the epithelial element is benign. Carcinosarcomas (malignant epithelial and mesenchymal components) will not be discussed, as these have been recently reviewed. Both adenofibroma and adenosarcoma are rare neoplasms that are rarely seen in endometrial biopsy specimens, but adenosarcoma is much more common. Some authorities do not recognise adenofibroma, but prefer to regard all the lesions in the adenofibroma–adenosarcoma spectrum as adenosarcomas, albeit some with a low-grade malignant stromal component. The management of adenofibroma and adenosarcoma is identical, comprising hysterectomy. In most cases, this is curative, unless the sarcomatous component is high grade or there is sarcomatous overgrowth or myometrial infiltration. In a small percentage of cases without these features, there is vaginal recurrence after hysterectomy. Adenofibroma and adenosarcoma are polypoid lesions, the adenosarcoma sometimes forming multiple polyps, and may rarely be identified on endometrial biopsy. As the malignant stromal component in adenosarcoma is sometimes subtle and low grade, adenosarcoma may present as recurrent endometrial polyps, with the correct diagnosis being made only after examination of multiple biopsies. Depending on what areas have been sampled on biopsy, adenosarcoma may also be initially diagnosed as a pure mesenchymal lesion. Morphologically, adenosarcoma is usually composed of leaf-like papillary projections, with an architecture reminiscent of phyllodes tumour of the breast. The epithelial element, which lines the papillary projections, is benign and may be simple cuboidal or columnar, mucinous, ciliated or even squamous in type. One of the most useful clues suggesting a diagnosis of adenosarcoma is the increased cellularity of the stroma around the epithelial elements, resulting in a cambium layer. In these areas, the stromal cells are often mildly atypical and it is here that mitotic figures are usually identified, although these may be sparse and may vary in number from area to area. According to the recent WHO classification of tumours of the breast and female genital organs, a mitotic count of >=1 HPF is necessary for a diagnosis of adenosarcoma. In practice, however, with a mitotic count of less than this and if the characteristic leaf-like architecture and cambium layer are present, many would diagnose adenosarcoma rather than adenofibroma. I agree with this view and reiterate that hysterectomy is the management of choice for an adenofibroma, as the lesion should be seen in its entirety to exclude the presence of areas diagnostic of adenosarcoma. Rarely, sex cord-like foci may be present in the stromal component of an adenosarcoma, similar to those seen in endometrial stromal sarcomas. Heterologous elements may also be present.

ENDOMETRIAL CARCINOMA

Issues relevant only to the diagnosis of endometrial carcinoma on biopsy specimens will be considered. In 1983, Bokhman first proposed that there were two major variants of endometrial cancer—namely type 1 (the prototype of which is endometrioid carcinoma) and type 2 (the prototype of which is serous carcinoma). It is imperative that an endometrial cancer is both typed and graded, if appropriate, on an endometrial biopsy (the term endometrial adenocarcinoma does not suffice, as endometrial adenocarcinomas may be of several different morphological subtypes). This is especially so in countries with a well-developed gynaecological oncology service, such as the UK, where different referral patterns exist for cancers of type 1 and type 2. For example, many endometrioid adenocarcinomas (grade 1 and grade 2) are dealt with in a cancer unit, whereas type-2 cancers, including serous and clear-cell carcinoma, are usually managed at a cancer centre. More extensive surgical staging may be undertaken with a type-2 cancer—for example, omentectomy and lymphadenectomy may be carried out. Similarly, in many regions, grade-3 endometrioid carcinomas are managed in a gynaecological oncology cancer centre. It is controversial whether serous carcinoma (and other type-2 carcinomas such as clear cell) should be graded, and most take the view that these are by definition grade-3 tumours. I believe that it is useful to state this on the pathology report, especially if dealing with general gynaecologists who are not routinely occupied with the management of gynaecological neoplasms. All endometrioid carcinomas (and other type-1 carcinomas such as mucinous carcinoma) should be graded by using the revised 1988 International Federation of Gynecology and Obstetrics grading system. In this system, for an architectural grade-1 or grade-2 tumour to be upgraded to a grade-2 or grade-3 neoplasm, respectively, high-grade (grade 3) nuclei should be present. Grade-3 nuclei are defined as enlarged and pleomorphic, with abnormal coarsely clumped chromatin and large irregular nucleoli. In assessing the proportion of solid and non-solid elements, squamous foci should be ignored. Assessing whether solid areas are squamous or non-squamous is often difficult, as squamous foci may exhibit a variety of patterns, including a clear cell and spindle cell morphology.

Differentiating between an endometrioid and a serous carcinoma may be problematic on occasion. An endometrioid carcinoma with a papillary growth pattern may be misdiagnosed as a serous carcinoma and, conversely, serous carcinoma with a glandular growth pattern and little or no papillary formation may be mistaken for an endometrioid carcinoma. A diagnosis of a papillary adenocarcinoma should not be made without specification of the morphological type. Architecturally well-differentiated endometrioid carcinomas usually have low-grade nuclei; and in an architecturally well-differentiated neoplasm with grade-3 nuclei and without papillary formation, a glandular variant of serous carcinoma should be considered. In the distinction between an endometrioid and a serous carcinoma, immunohistochemical analysis may be of value. Endometrioid adenocarcinomas, especially when grade 1 or grade 2, are usually oestrogen receptor positive and p53 negative. Conversely, serous carcinomas usually show diffuse nuclear p53 reactivity and are oestrogen receptor negative. Many exceptions exist, however, with occasional serous carcinomas being p53 negative and a considerable proportion exhibiting oestrogen receptor positivity, albeit often focal. Conversely, some endometrioid adenocarcinomas are p53 positive, although grade-1 and grade-2 neoplasms only rarely exhibit the diffuse strong nuclear reactivity that is characteristic of serous carcinoma. Mixed endometrioid and serous carcinomas are not uncommon. Staining with oestrogen receptor and p53 may also be of value in identifying small foci of EIC in a polypoid or non-polypoid endometrium or in suggesting a diagnosis of serous neoplasia when only small fragments of tissue are present in an endometrial biopsy specimen.

As referred to previously, serous carcinoma and its presumed precursor lesion EIC (also variously termed endometrial carcinoma in situ or surface serous carcinoma) have a marked propensity to arise in or to be associated with otherwise benign endometrial polyps. I use the term presumed precursor lesion, as it is possible that EIC, defined as the replacement of residual atrophic glands by cells with markedly atypical nuclei that are characteristic of serous neoplasia without endometrial stromal, myometrial or vascular invasion, actually represents a growth pattern of serous carcinoma and not a precursor lesion. Endometrial polyps, especially when large and occurring in elderly patients, should be carefully scrutinised for small serous proliferations.

A commonly encountered problem in endometrial carcinoma is in determining the nature of clear-cell areas. This may indicate a pure clear-cell carcinoma or a component of clear-cell carcinoma, a variant of type-2 endometrial cancer usually with markedly atypical nuclei, sometimes with a hobnail pattern. Occasional clear-cell carcinomas, however, have relatively bland nuclei. Negative staining for oestrogen receptor and diffuse p53 reactivity favour a clear-cell carcinoma, although a significant proportion of clear-cell carcinomas do not show this immunophenotype. Clear cells are not uncommon in endometrioid adenocarcinomas. This may represent a non-specific change in clear cells, a secretory variant of endometrioid carcinoma or clear-cell squamous areas, Clearing of the cytoplasm in an endometrioid adenocarcinoma may also be a result of prior progestogen treatment.

 

Potential benign mimics of endometrial hyperplasia

 

Artefacts

Cystic atrophy

Lower uterine segment endotrium

Disordered proliferative endometrium

Secretory endometrium or Arias Stella effect

Benign papillary proliferations

Endometritis

Endometrial polyps

 

Take-home messages

In the evaluation of an endometrial biopsy specimen, an adequate clinical history is important, including details regarding the use of exogenous hormones. In a postmenopausal woman with an atrophic endometrium and no focal lesion on ultrasound scan, the presence of scant endometrial tissue in an outpatient biopsy is the norm. When an endometrial biopsy is carried out for abnormal uterine bleeding, the pathologist should always consider the possibility of a polyp. Serous proliferations, either serous carcinoma or endometrial intraepithelial carcinoma, have a propensity to arise in endometrial polyps. All endometrial cancers should be typed and, if appropriate, graded, even for small biopsy specimens.

Journal of Clinical Pathology, 59(8), August 2006, pp 801-812

 

Frozen section for parotid surgery: should it become routine?

 Ali A. Arabi Mianroodi, Elizabeth A. Sigston and Neil A. Vallance

Background:  The role of fine-needle aspiration (FNA) in salivary gland lesions as a preoperative diagnosis has always been under scrutiny. Several studies have shown that frozen section (FS) is accurate for pathological diagnosis and decision-making during the surgery. This study has been carried out to assess the accuracy of FNA and FS in parotid surgeries.

Methods:  All parotid lesions removed between July 1998 and June 2003 by the Department of Otolaryngology, Head and Neck unit at Monash Medical Centre were reviewed. FNA, FS and definitive pathology were collected and discrepancies were identified.

Results:  Eighty-five parotid tumours had been removed. Thirty-three were malignant in which squamous cell carcinoma was the most common. FS was able to differentiate benign tumours from malignant with 100% accuracy (30 cases). FS was able to alter the surgical decisions in six cases. FNA had been carried out in 62 cases, with sensitivity and specificity of 77.2 and 90%, respectively.

Conclusion:  Frozen section in parotid surgery is accurate, inexpensive and may add important information that alters management and improves the outcome. The use of FS routinely in parotid surgery is recommended.

ANZ Journal of Surgery, Volume 76 Page 736  - August 2006

Glycogen Pseudoground Glass Change in Hepatocytes

Wisell J, Boitnott J, Haas M et al

Ground glass cytoplasmic change in hepatocytes is typically associated with chronic hepatitis B infection. We report 12 cases of glycogen pseudoground glass change that closely mimics hepatitis B inclusions. Nine individuals were immunosuppressed secondary to liver or kidney transplant (N=3), bone marrow transplant (N=2), HIV infection (N=2), kidney dialysis (N=1), or chronic inflammatory bowel disease (N=1). Medication history was available in 10 individuals and all were on multiple medications (range 2 to 33). Histologically, the pseudoground glass change was identical to the ground glass change seen in chronic hepatitis B infection, with distinct, circumscribed, gray-glassy inclusions surrounded by a rim of cytoplasm. The background livers showed mild or no inflammation and mild or no fibrosis. All cases were negative for chronic hepatitis B infection. The pseudoground glass change was PAS positive and diastase sensitive. Electron microscopy of the inclusions showed glycogen in 3/3 cases. No evidence for viral particles or significant endoplasmic reticulum proliferation was seen. Three cases had follow-up biopsies (1, 1, and 36 mo), and the pseudoground glass was persistent in 2 cases and showed partial resolution in 1 case (1 mo biopsy interval). Authors conclude that glycogen pseudoground glass change is typically seen in immunosuppressed individuals on numerous medications. The changes are generally seen in the background of mild chronic hepatitis with mild or no fibrosis. Glycogen pseudoground glass change can resolve, but may also persist for years.

Am J Surg Pathol. 2006 Sep;30(9):1085-1090.

  

MICROBIOLOGY 

Viable Bacteria Present within Oral Squamous Cell Carcinoma Tissue 

Samuel J. Hooper, St John Crean, Michael A. O et al

 

Despite increasing interest in the possible relationships between bacteria and the different stages of cancer development, the association of bacteria with cancer of the oral cavity has yet to be adequately examined. With that in mind, the primary objective of this study was to identify any bacterial species within oral squamous cell carcinoma tissue using a standard microbiological culture approach. At the time of surgery, a 1-cm3 portion of tissue was harvested from deep within the tumor mass using a fresh blade for each cut. Whenever possible, “superficial” portions from the mucosa overlying the tumor and nontumorous control specimens from at least 5 cm away from the primary tumor site were also obtained. Surface contamination was eliminated by immersion in Betadine and washing with phosphate-buffered saline. Each specimen was aseptically macerated and cultured on nonselective media under both aerobic and anaerobic conditions. Isolates were identified by 16S rRNA gene sequencing. Twenty deep-tissue specimens, 19 with corresponding superficial tissues and 12 with control tissues, were successfully processed. A diversity of bacterial taxa were isolated and identified, including several putatively novel species. Most isolates were found to be saccharolytic and acid-tolerant species. Notably, some species were isolated only from either the tumorous or nontumorous tissue type, indicating a degree of restriction. Successful surface decontamination of the specimens indicates that the bacteria detected were from within the tissue. A diversity of bacterial groups have been isolated from within oral squamous cell carcinoma tissue. The significance of these bacteria within the tumor warrants further study.

 

Oral cancer is the sixth most common malignancy worldwide and is particularly prevalent in developing countries, such as in Southeast Asia, where up to 40 percent of all malignancies are located within the oral cavity. More than 90% of cancers in the mouth are squamous cell carcinomas (SCCs) originating from the oral mucosa. With an average allstage, 5-year survival rate for oral cancer of less than 50%, the annual mortality figures are comparable to those of carcinoma of the cervix and malignant melanoma. There is evidence that the incidence of cancer of the oral cavity is increasing in many parts of the world, including the United Kingdom. In the past, oral cancer predominantly affected men in their sixth or seventh decade. However, more recently the male-to-female ratio has reduced dramatically and there has been a striking increase in the number of cases in those under the age of 45.

It has been reported that the majority of cases of head and neck cancer can be related to tobacco use and heavy alcohol consumption. Other possible risk factors include viral infections, poor oral hygiene, and infection with Candida species. The latter microorganisms are associated with some forms of leukoplakic lesions, the presence of which has long been recognized as an independent risk factor for carcinoma. The involvement of other microorganisms, particularly bacteria, has not been studied to any great extent, although recently there has been increasing evidence to suggest that closer study of this issue may be warranted. A number of bacterial species have been associated with different cancers following either epidemiological or laboratory-based studies. For example, Chlamydia trachomatis infection has been associated with an increased risk for the development of invasive cervical carcinoma. Bacteremia and endocarditis due to Streptococcus bovis have likewise been linked with malignancies in the colon, and Helicobacter pylori infection has long since been considered a causative agent of both gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphomas. Moreover, several mechanisms by which different bacteria may play a role in cancer development have been proposed, for instance, through the induction of chronic inflammation, by interference, either directly or indirectly, with eukaryotic cell cycle and signaling pathways, or via the metabolism of potentially carcinogenic substances. The latter mechanism is of relevance in the oral cavity, where the local microflora may promote carcinogenesis by converting ethanol into its carcinogenic derivative, acetaldehyde. Following the ingestion of alcohol, salivary bacteria have been shown to produce levels of acetaldehyde that can induce DNA damage, mutagenesis, and secondary hyperproliferation of the epithelium. Interestingly, microbial acetaldehyde production is increased in heavy drinkers and smokers, offering a possible explanation for these risk factors. The association of bacteria with oral tumors is of increasing interest. In a study of intraoral carcinomas, Nagy et al. demonstrated a difference in the microflora associated with the surface of tumors in comparison to control sites. More recently it has also been reported that patients with oral SCC (OSCC) tend to possess significantly raised concentrations of certain bacteria in their saliva. This apparent alteration of the oral microflora in OSCC cases is of particular interest because of its potential application as a diagnostic tool to predict oral cancer. In order to demonstrate a role for bacteria in the development of oral cancer, the first step must be to identify such organisms within tumor specimens. Furthermore, sufficient attention must be given to the elimination from any tissues tested of the microbes that occur naturally on the surfaces of the tumors. In addition, salivary contamination of the sample must be prevented during subsequent handling. The presence of Streptococcus anginosus DNA in oropharyngeal tumors has been reported following studies using specific PCR primers (. However, this molecular approach was limited to a single group of bacteria, and no inferences can be made regarding the viability and therefore potential activity of the species detected. The aim of this investigation was to determine whether viable bacteria are present within the tissue of oral squamous cell carcinoma. A prerequisite of this was the development of a robust method for the elimination of surface microbial contamination from specimens of tumor tissue.

 

MATERIALS AND METHODS 

Sample collection. Tissue specimens were obtained from 16 male and 4 female OSCC patients with an average age of 66.9 years. Ethical approval for the study was granted by the South Wales Local Ethics Research Committee, and subjects agreed to participate with their informed consent. Tumors were removed surgically, and specimens from the resected OSCC were harvested under aseptic conditions. The technique involved the surgeon rescrubbing and placing the specimen on a separate sterile surface. With a new blade for each cut, a 1-cm3 specimen was removed without compromising the pathological margins. This specimen was further divided into a “deep tissue” specimen (consisting entirely of tissue from within the tumor mass) and a “superficial” specimen (consisting of tissue from within and from the surface of the tumor). Whenever possible, a control specimen consisting of nontumorous tissue harvested at least 5 cm away from the primary tumor site was also obtained. Specimens were aseptically transferred to the laboratory in separate vials of transport medium, used to maintain the viability of any bacterial cells present. Transport medium was comprised of tryptone (1% wt/vol; Oxoid), yeast extract (0.5% wt/vol; Oxoid), glucose (0.1% wt/vol; BDH Ltd.), cysteine hydrochloride (0.1% wt/vol; BDH Ltd.), sodium hydroxide (50 mM; Sigma), and horse serum (2% vol/vol; TCS Ltd.) and was sterilized by filtration using a 0.2-_m filter. All subsequent handling of the specimens was carried out using aseptic technique on surfaces cleaned with Hycolin phenolic disinfectant (2% vol/vol; Bilaurand Labs Ltd.). Tissue specimens were placed in Betadine antiseptic solution (Seton Healthcare Group plc) for 3 min to disinfect the surface of each. Subsequently, tissues were vortexed in multiple 500-_l aliquots of phosphate-buffered saline (PBS) to encourage the removal of any bacteria on the tissue surface. Final washes were retained and analyzed by both culture-dependent and cultureindependent techniques to determine whether surface decontamination was successful. Specimens were aseptically bisected. Half of each was placed in Tris-EDTA buffer (pH 7.4) and stored at _80°C for molecular analysis. The remainder was subjected to immediate culture-dependent analysis.

Bacterial culture. Tissue specimens were aseptically macerated with disposable scalpels and vortexed for 30 seconds in PBS (500 _l), and the neat suspensions were used to make 10-fold (10_1) and 100-fold (10_2) dilutions. Neat suspensions (50 _l) were each spread onto blood agar (BA), fastidious anaerobe agar (FAA) (BA and FAA supplemented with 5% defibrinated sheep blood; TCS Biosciences Ltd.), and Sabouraud’s agar (Lab MTM; International Diagnostics Group plc). The dilutions and final PBS washes of the specimens (see above) were spread onto BA and FAA. BA and Sabouraud’s agar plates were incubated aerobically at 37°C for 48 h. FAA plates were incubated in an anaerobic cabinet at 36°C for 96 h. Following incubation, all colony types on the plates were subcultured for purity, the cellular morphologies of Gram-stained smears were recorded, and isolates were stored at _80°C using Microbank cryovials (Pro-Lab Diagnostics) prior to identification by 16S rRNA gene sequence analysis.

Molecular identification of isolates. Genomic DNA was extracted from suspensions of each of the bacterial isolates using a commercial kit (Puregene; Gentra Systems) according to the manufacturer’s instructions (extraction protocol for 1 ml gram-positive bacteria;). PCR amplification of 16S rRNA genes was performed in a reaction volume of 50 _l, consisting of 0.2 mM each deoxynucleoside triphosphate, 2.0 mM MgCl2, 1.5 U Taq DNA polymerase, 5 _l 10_ PCR buffer (all reagents from Promega), 5 _l (between 50 fg and 50 ng) of extracted DNA as template, and 0.5 _M each primer. The primer pair used were 27F (5_-GTGCTGCAGAGAGTTTGATCCTGGCTCAG-3_) and 1492R (5_- ACGGATCCTACGGGTACCTTGTTACGACTT-3_), specific for the domain Bacteria (6) (synthesized by Invitrogen). A touchdown protocol was used whereby in the first cycle, denaturation was performed at 94°C for 6 min, primer annealing was performed at 65°C for 1 min, and extension was performed at 72°C for 2 min. In subsequent cycles, denaturation was performed for 1 min and the annealing temperature was decreased by 2°C each cycle for 11 cycles, after which 25 cycles were carried out under the same conditions. In the final cycle, extension was performed for 12 min. Contamination controls (tissue surface washes) were also subjected to PCR amplification; negative results indicated successful decontamination. The 16S rRNA gene PCR products were partially sequenced using the 357F primer (5_-CTCCTACGGGAGGCAGCAG-3_) (12), ABI Prism BigDye Terminator cycle sequencing ready reaction kits (Perkin-Elmer), and an automated DNA sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems). This gave reliable sequences of at least 500 nucleotides in length, which were compared to all GenBank DNA sequence entries using the FASTA sequence homology search program  (http://www.ebi.ac.uk/services/index.html). Whenever this sequence was insufficient to provide a conclusive identification, PCR products were further sequenced using the 27F and 1492R primers to give a sequence of at least 1,200 nucleotides in length.

Nucleotide sequence accession numbers. Sequences AY880043 through AY880059 were deposited into GenBank.

 RESULTS

The results of the PCR and both aerobic and prolonged anaerobic cultures of surface washings were routinely negative, indicating that the surface decontamination protocol used was successful. Bacteria were isolated from all specimens: 20 deep tissue tumor specimens, 19 corresponding superficial specimens, and the nontumorous control tissues that were obtained from 12 of the 20 patients. The species identities are summarized in Table 1. An average of 6 isolates was cultured from each specimen with a total of 90 distinct species or phylotypes. The isolates were mostly bacterial, but also included isolates of Candida albicans from three patients and a single isolate of a Saccharomyces species. A wide range of bacteria was seen, including a number of taxa with low 16S rRNA gene sequence homology to anything previously existing in the public databanks. Complete 16S rRNA gene sequences were obtained for these seemingly novel taxa and deposited with GenBank; nucleotide accession numbers are given in Table 1. For all patients studied, the superficial tissues yielded the same isolates found in the corresponding deep-tissue specimens, with additional species in most cases. Comparison of the proportions of each sample type positive for each species revealed 17 organisms for which the difference in proportion between tumorous and nontumorous tissues was at least 10%, where “tumorous” tissues represented both the deep tumor and superficial samples combined.

 

DISCUSSION

To our knowledge this is the first time that viable bacteria have been detected within the tissue of oral squamous cell carcinoma. Immersion in Betadine was required to eliminate any viable bacteria on the surface that may have been present due to salivary or instrument contamination during surgery. A diversity of species were isolated. However, it is important to remember that this is not a comprehensive analysis of the microflora; it has been well established that approximately 50% of oral bacteria are not cultivable by standard culture techniques as used in this study. Nevertheless, cultivation of bacteria remains a useful diagnostic tool for the detection and identification of viable organisms, including many known pathogenic species. The majority of the taxa detected have previously been isolated from the oral cavity. However, there were a small number of exceptions. For example, some species previously only reported as isolates from environmental sources were detected, including Dietzia psychralcaliphila and Gordonia sputi. A number of isolates were detected which, although not typically regarded as common members of the oral microflora, have been isolated from the oral cavity previously and/or are known to be human pathogens, for example, those belonging to the genera Micrococcus, Propionibacterium, Streptomyces, Bacillus, Enterococcus, Exiguobacterium, Staphylococcus, and Pseudomonas. As the successful surface decontamination indicates that the bacteria isolated from the samples were not carried over from other sources, such as saliva, it is interesting to note that so many species, including ones not commonly regarded as pathogenic, were present in the tumor tissue. This may support the emerging findings that many different bacterial species are capable of infecting oral tissue and invading epithelial cells. For instance, it is of some interest to note that recent reports find streptococci to be a major component of this epithelial intracellular microflora, as several members of this genus were isolated from within both tumorous and nontumorous samples. The detection of Streptococcus anginosus in these tissues is also particularly noteworthy because it supports the findings from previous studies in which S. anginosus DNA was detected in head and neck carcinoma by PCR. Additionally, the presence of Streptococcus mitis/Streptococcus oralis in both the nontumorous and tumorous samples is consistent with the previously reported detection of these microorganisms in esophageal carcinoma and control tissues by molecular means. The great diversity of species isolated together with the relatively low number of patients and specimens used in this study make it difficult to apply statistical analyses to draw conclusions regarding bacterial specificity. However, a number of interesting trends are apparent from the results. In all cases, the superficial tissues yielded exactly the same isolates as the corresponding deep-tissue specimens plus, in most cases, additional species. This may indicate a degree of restriction of bacteria in the deeper tumor tissue in comparison to the overlying mucosal sites. Perhaps not all species coming from the oral cavity and invading the mucosa can invade or survive in the tumorous tissue. Several species detected in the nontumorous control tissue were not detected in the tumor tissues, and vice versa. For instance, Exiguobacterium oxidotolerans, Prevotella melaninogenica, Staphylococcus aureus, Veillonella parvula, and species of Bacteria and Micrococcus were isolated only from tumorous specimens and not at all from nontumorous ones. Conversely, Moraxella osloensis, Prevotella veroralis, and species of Actinomyces were grown only from nontumorous tissues. This could indicate that, while bacteria are present within all the oral mucosal tissues, there are potentially significant differences between the microfloras within tumorous compared to nontumorous mucosae. Although we were unable to apply full hypothesis-testing statistics to support this finding, the observed alteration in microflora composition may perhaps be similar to the reported differences in the proportions of salivary bacteria that can theoretically be used as a predictor for OSCC. The simple descriptive parameters used in this study suggest six candidate species, isolated from over 10% more of the amalgamated tumorous than nontumorous samples, that may prove to be significantly associated with OSCC tissue. Similarly, there were 11 taxa that were in proportionally at least 10% more of the control samples than the tumorous tissues, which may represent the species that are most likely not to be associated with OSCC. It is interesting to note that the majority of species isolated were saccharolytic and acid tolerant. For instance, yeasts, actinomycetes, bifidobacteria, lactobacilli, streptococci, and veillonellae,  all of which were detected in this study, are known to produce short-chain organic acids from carbohydrates and consequently to lower the pH of their local environment. Asaccharolytic or weakly fermentative species isolated from these specimens, such as Fusobacterium and Prevotella species, have also been shown to be capable of survival at relatively low pHs. The microenvironment of solid tumors is typically hypoxic, with an acidic extracellular pH, and so it is not surprising that there might be a degree of selectivity in favor of acid tolerance. To what degree the presence of acidproducing bacteria within tumors contributes to the acidic microenvironment is as yet unknown.

Based on the 16S rRNA gene sequence data, several of the cultivated isolates appear to represent species either not previously characterized or without standing in the current nomenclature. There were 7 different phylotypes (from a total of 10 isolates) that failed to demonstrate significant homology (99% sequence match) to any GenBank entry. Given the current wide-ranging nature of the sequence databases for known bacteria, it is possible that these represent “novel” species. Interestingly, an additional nine isolates showed significant sequence matches to existing sequences representing taxa that are unnamed and have seemingly not been cultured or characterized before. Four specimens contained a Prevotella species with a significant sequence match to “oral clone BE073,” an uncultured putative species detected in purified crevicular epithelial cells. One tumor tissue specimen yielded an isolate matching “Scardovia genomospecies C1,” a taxon recently detected in the microflora of dental caries. A Streptococcus species was detected from one superficial specimen that matched the “Streptococcus sp. oral strain T4-E3” phylotype, previously detected by molecular means on the surface of the tongue dorsa. Isolates were cultivated from two superficial tissues that sequence-matched “Bifidobacterium sp. oral strain H6-M4,” another taxon cloned from the tongue dorsa microflora. The 16S sequence for these isolates also matched the proposed species “Bifidobacterium urinalis,” although this nomenclature is as yet unpublished and has no current standing. It will be of great interest to characterize these isolates further. The implications of the presence of a diversity of viable bacteria deep within the tissue of squamous cell carcinoma are unclear. Although it has been shown in animal models that bacteria injected intravenously may seed to tumor tissue, the types of bacteria isolated and the fact that the composition of the deep tissue microflora was similar to, but less species rich than, the overlying mucosa tend to imply a local origin for the bacteria detected within the tumor. The apparent differences between the microflora of the tumor and control tissues suggest a degree of bacterial specificity that merits further study. For instance, it would be useful in future studies to include an assessment of microbial load to determine the numerical predominance of selected species. As evidence that bacteria are involved in the development of many different cancers increases, it is interesting to speculate that the species isolated from the tumor tissue may play a role in the carcinogenic process, a concept worthy of further investigation.

Journal of Clinical Microbiology p.1719-1725, May 2006

 

Value of semen culture in the diagnosis of chronic bacterial prostatitis: A simplified method

Budia A, Luis Palmero J, Broseta E, Tejadillos S et al

Objective. To investigate the role of semen cultures versus segmented urine cultures for the diagnosis of bacterial chronic prostatitis.

Material and methods. We retrospectively examined 895 patients (age range 17-67 years) who met the consensus criteria for clinical chronic prostatitis/chronic pelvic pain syndrome, 50.1% of whom had dysuria and/or perineal discomfort, 37.4% infertility of unknown etiology and 12.5% erectile dysfunction. Segmented urine cultures, including expressed prostatic secretions (EPSs) and semen culture, were performed in all patients. Results. Of the 895 patients, 182 had significant positive cultures for Gram-negative microorganisms (Escherichia coli was the commonest specimen isolated: 70.4% of cases) and 283 had significant positive cultures for Gram-positive microorganisms. We compared the culture yield in EPS and/or the urine voided after prostatic massage (VB3) sample (four-glass method) with that of the semen sample. In the Gram-negative group, 32 patients were diagnosed by means of semen culture (negative EPS and/or VB3 sample) and in only five cases was a positive diagnosis made despite a negative semen culture (positive EPS and/or VB3 sample). In the remaining subjects, diagnosis was performed with the aid of both EPS/VB3 sample and semen (both of which were positive). In the Gram-positive group, there was significant growth of such microorganisms in semen in every case considered positive, but in only 46 cases was diagnosis achieved via EPS and/or VB3 sample. A diagnosis of chronic prostatitis by Gram-positive microorganisms in these patients was only considered when the same microorganism was retrieved in repeated cultures without previous treatment. Only three cases met such criteria (all of whom had negative EPSs). To evaluate the diagnostic efficiency of the semen and EPS samples, we analyzed their sensitivity and specificity, obtaining higher sensitivity in semen than EPS samples for significant Gram-negative cultures: 97% vs 82.4%. In significant Gram-positive cultures, the sensitivity of semen samples was 100%, compared to only 16.1% for EPS.

Conclusions. A semen sample has higher sensitivity than an EPS for the diagnosis of bacterial chronic prostatitis. In our clinical work-up, first-void urine and a semen culture are considered the only tests necessary to diagnose chronic prostatitis.

Scand J Urol Nephrol. 2006;40(4):326-31.