Puja K. Puri

CNS location of uterine-related neurons revealed by trans-synaptic tracing with pseudorabies virus and their relation to estrogen receptor-immunoreactive neurons

Retrograde, transneuronal tracing with Barthas strain of pseudorabies virus was used in rats to identify spinal cord, brainstem and hypothalamic loci of uterine-related neurons that could function in the regulation of uterine activity. Based on the premise that estrogen might influence such uterine-related neurons, the existence of estrogen receptors in neurons in these same loci was examined. Viral injections were made into the uterine cervix, body and cervical end of the uterine horns, and the rats allowed to survive for four to six days. After four days, mainly the spinal cord, medulla and pons contained virus-infected neurons. After longer survival times, progressively higher levels of the neuraxis contained viral-labeled neurons, so that by six days hypothalamic uterine-related neurons were identified. First-order virus-infected neurons were visualized by immunohistochemistry in the pelvic paracervical parasympathetic ganglia and in inferior mesenteric sympathetic ganglia. Preganglionic and putative interneurons were labeled in the lumbosacral spinal cord and thoracic spinal cord mainly in the lateral horn area (sacral parasympathetic nucleus and intermediolateral nucleus), lateral aspect of the dorsal horn, intermediate gray, lamina X and dorsal gray commissural area. In the brainstem, labeling was most evident and consistent in the nucleus tractus solitarius, ventrolateral medulla, raphe magnus and pallidus nuclei, parapyramidal area, A5 cell group, Barringtons nucleus of the pons and periaqueductal gray of the midbrain. In the hypothalamus, virus-infected neurons were most marked in the paraventricular nucleus, with fewer in the medial preoptic area and ventromedial hypothalamic nucleus. Estrogen receptor-immunoreactive neurons were most often present among the virus-labeled uterine-related neurons of the spinal cord, nucleus tractus solitarius, ventrolateral medulla, periaqueductal gray, medial preoptic area and ventromedial hypothalamic nucleus. These results identify a multisynaptic pathway of neurons whose eventual output is involved in uterine functions, whose distribution is similar to that revealed by pseudorabies virus tracing from other visceral organs, and which are often mixed among estrogen-responsive neurons.

Hydroxychloroquine-induced hyperpigmentation: the staining pattern

We report two cases of hydroxychloroquine‐induced hyperpigmentation presenting in a 50‐year‐old Caucasian female (case 1) and a 78‐year‐old female (case 2), both receiving 400 mg per day. Case 1 had an arthritis predominant undifferentiated connective tissue disease, which was treated with hydroxychloroquine for 4–5 years. She presented with a mottled, reticulated macular gray pigmentation involving the upper back and shoulders. Case 2 had a history of systemic lupus erythematosus and rheumatoid arthritis, treated with hydroxychloroquine for 1.5 years. She presented to the hospital for treatment of constrictive cardiomyopathy and was noted to have a blue macular pigmentation involving the right temple. The biopsies from both patients showed superficial dermal, yellow‐brown, non‐refractile and coarsely granular pigment deposition. A Fontana‐Masson stain highlighted some of these granules, while the Perl’s iron stain was negative. Rare, previous reports of hyperpigmentation indicate the presence of both melanin and hemosiderin in patients being treated with antimalarial medication. To our knowledge, this staining pattern for hydroxychloroquine has not been previously reported in the literature and supports that hydroxychloroquine, in addition to chloroquine, binds to melanin.

Accurate identification of proliferative index in melanocytic neoplasms with Melan‐A/Ki‐67 double stain

To the Editor, Accurately classifying melanocytic neoplasms can sometimes be extremely difficult for pathologists. In addition to clinical information and hematoxylin and eosin stained slides, immunohistochemical stains may aid in the diagnosis.1 For example, HMB-45 is used in evaluating melanocytic neoplasms. In conventional compound nevi, it highlights junctional and superficial dermal nests with loss of expression as the lesion progresses deeper in the dermis.2 Conversely, in melanoma, the melanocytes can stain superficial and deep melanocytes in a patchy1 or diffuse pattern.2 Melan-A is another immunohistochemical marker that can also be used to highlight the distribution of junctional and dermal melanocytes, clearly highlighting the presence of confluent melanocytes and demonstrating pagetoid scatter.3 Additionally, the proliferative activity of the melanocytic lesion can be assessed with Ki-67.1,2 The lesion is proliferating rapidly if there is increased staining in the deeper portion of the lesion. However, other cells such as lymphocytes, histiocytes and epithelial cells can also display proliferative activity, thus contributing to the difficulty in establishing the best diagnosis. Two color immunohistochemistry dual labeling with antibody cocktails is currently available for detection of more than one antigen in the same tissue slide. One must chose an antibody which targets antigens in different compartments of the cell, as well as use different chromogens or detection systems in order to visualize the antibodies.4 For instance, Ki-67 is a nuclear marker and has been paired with other cytoplasmic markers such as CD315 and cytokeratins6 to assess proliferative activity in specific cell-lines. Similarly, we developed a double staining technique using Melan-A and Ki-67 to evaluate proliferative activity of melanocytic neoplasms. Modification of previously described immunohistochemical methods was performed on current and archived cases.7,8 Formalin-fixed tissue samples were processed and embedded in paraffin. Fourmicrometer sections were cut, placed on positivecharged slides, air dried and then heated in a 65◦C oven for 30 minutes. After removal of paraffin with xylene and clearing with alcohol, the slides were placed in hydrogen peroxide and methanol to quench endogenous peroxidase activity. Subsequently, the sections were hydrated and washed with deionized water. The Melan-A and Ki-67 immunohistochemistry required heat-induced epitope retrieval pretreatment. The slides were then placed in preheated Dako Target Retrieval Solution at a pH of 6.1 (Dako USA, Carpinteria, CA). The slides were heated for 20 minutes in a 100◦C water bath.9 The solutions and slides were cooled for 20 minutes, washed in deionized water and then placed in Tris-buffered saline (TBS) at a pH of 7.5. Tissue sections were incubated for 1 hour at room temperature with a primary antibody cocktail of mouse monoclonal Melan-A, clone A103 (Dako USA, Carpinteria, CA, diluted 1:100) and rabbit polyclonal Ki-67 (Biocare Medical, Concord, CA, diluted 1:50). Following incubation with the primary antibody, the slides were rinsed with TBS. Mach 2, kit number 1 (Biocare Medical) containing a cocktail of alkaline phosphatase conjugated anti-mouse and horseradish peroxidase conjugated anti-rabbit was used to label the bound primary antibodies. Diaminobenzidine (DAB) was used to visualize the bound Ki-67 antibody (brown). Following the application of DAB, the slides were washed with TBS and Vulcan Fast Red (Biocare Medical) was applied to the tissue sections to show the bound Melan-A antibody (red). Hematoxylin counterstain was applied, followed by rapid dehydration with alcohol, clearing with xylene and permanent cover slipping. We retrospectively searched for cases of melanocytic lesions where a combination MelanA/Ki-67 immunohistochemical stain was used at Duke University Medical Center from 2007 to 2008.

CD4/CD8 double negative pagetoid reticulosis: a case report and literature review

Pagetoid reticulosis is an indolent primary cutaneous T‐cell lymphoma. It typically presents as a solitary and slowly growing patch or plaque on the extremity, histologically characterized by an acanthotic epidermis infiltrated with atypical lymphocytes. Here, we present histological, immunophenotypical and molecular findings of a 29‐year‐old Jamaican man with bilateral wrist plaques. Histology showed marked acanthosis, hyperkeratosis and an intraepidermal infiltration consisting of large atypical lymphocytes. Immunohistochemical stains showed CD3 and CD5 positive T cells with significant loss of CD7, double negative CD4 and CD8 and strong positive CD30. Molecular analysis showed a monoclonal T‐cell receptor (TCR) gamma gene rearrangement. Review of the literature confirms that the immunophenotype of pagetoid reticulosis is variable with decreasing frequency of CD8+ cytotoxic/suppressor T cell, CD4+ helper T cell and least commonly CD4/CD8 double negative phenotypes. Although CD4/CD8 double negative phenotype appears to be associated with higher proliferation index, it does not appear to confer prognostic significance.

Immunohistochemical markers in fibrohistiocytic lesions: factor XIIIa, CD34, S-100 and p75.

Background:The distinction between dermatofibroma (DF), dermatofibrosarcoma protuberans (DFSP), and other benign and malignant cutaneous spindle cell lesions frequently requires immunohistochemical staining. CD34 and factor XIIIa are the most commonly used immunostains; however, they may exhibit aberrant expression and introduce the potential for misdiagnosis. There is some data supporting that p75 and S100A6 may be additional helpful immunohistochemical markers. Methods:We undertook a large case series examining the use of CD34 and factor XIIIa as well as p75 and S100A6 in DF, cellular DF, DFSP, indeterminate fibrohistiocytic lesion, and scar. Results:As expected, CD34 stained DFSP, although it was usually negative in DF. Factor XIIIa was generally positive in DF and negative in DFSP. There were exceptions in both cases of DF and DFSP. S100A6 was routinely negative in all entities studied. P75 was negative in all cases except DFSP, approximately half of which showed weak and/or patchy positivity. Conclusions:We conclude that to date, CD34 and factor XIIIa remain the most reliable immunohistochemical markers for DF and DFSP.

Scleroderma and IgG4-related disease.

Abstract:IgG4-related disease is a syndrome which involves lymphoplasmacytic infiltrates and soft tissue sclerosis, elevated serum IgG4 titer, and increased IgG4-positive plasma cells in a variety of tissues. Scleroderma is also characterized by fibrosis and lymphoplasmacytic infiltrates. To our knowledge, the presence of IgG4-positive cells has not been well characterized in scleroderma. A retrospective review of scleroderma and related disorders (calcinosis, raynauds syndrome, esophageal dysmotility, sclerodactyly, telangiectasia (CREST) syndrome, progressive systemic sclerosis, morphea) was performed. Thirty-four cases of scleroderma and related disorders were identified; IgG4-positive and IgG-positive plasma cells were counted in 10 HPF and an IgG4:IgG ratio determined. A cutoff ratio of 0.3 was used to define significant elevation. Three of the scleroderma cases had IgG4:IgG greater than 0. Only 1 case had a significant elevation. Of the 3 cases with elevated ratio, IgG4-positive cells ranged from 2 to 64 (median = 14), with an IgG4:IgG ranging from 0.06 to 0.34 (median = 0.22). Similar results were produced with the other sclerosing disorders. These results suggest that scleroderma is not part of the IgG4-related disease spectrum.

Cutaneous metastatic cholangiocarcinoma: a report of three cases and review of the literature

Cutaneous metastasis from cholangiocarcinoma is an extremely rare event. Herein, we present three cases with review of the literature. Case 1 is that of a young female with scalp metastasis. Cases 2 and 3 involve cutaneous metastasis to the sites of prior biliary drains, one occurring in a young female with a history of multiple biliary surgeries and one in a male with a history of sclerosing cholangitis. Review of the literature shows that the presentation of cutaneous metastases from cholangiocarcinoma can vary in terms of anatomic location and clinical features. The pathological and immunohistochemical profile of metastatic cholangiocarcinoma can be non‐specific, and accurate diagnosis relies in part on clinical correlation. In summary, metastatic disease should always be included in the differential diagnosis of cutaneous lesions in patients with known malignancy.

Expression of focal TTF‐1 expression in a case of CK7/CK20‐positive Merkel cell carcinoma

To the Editor, Although rare cases of cytokeratin (CK)7/CK20positive Merkel cell carcinoma (MCC) have been reported,1 we report an unusual case of CK7/CK20positive MCC that also showed focal thyroid transcription factor-1 (TTF-1) immunoreactivity. An 85-year-old male presented with a past medical history of non-melanocytic skin cancer. He had a leiomyosarcoma on the left vertex of the scalp, as well as multiple basal cell carcinomas and squamous cell carcinomas involving the scalp, face and upper trunk. Subsequently, he underwent four additional biopsies of the scalp. The lesion of interest presented as a dermal nodule in the right frontal scalp. Histopathologic examination of the right frontal scalp nodule showed dermal sheets and nests of cells with stippled chromatin and a high mitotic rate, consistent with a neuroendocrine carcinoma (Fig. 1 A,B). Immunohistochemical stains showed the tumor to be diffusely positive for neuron-specific enolase (NSE) and synaptophysin (Fig. 2 A,B). The majority of the tumor cells expressed CK7 in a perinuclear dotlike pattern (Fig. 2C). CK20 was weakly positive in a cytoplasmic pattern in approximately 10% of tumor cells (Fig. 2D). Cells expressing TTF-1 were seen in clusters and constituted <5% of the tumor (Fig. 2E). A neurofilament stain was positive in approximately 30% of the tumor cells. Chromogranin was negative. The presence of CK7 positivity and focal TTF-1 positivity is somewhat unusual. The patient was evaluated by a thoracic surgeon and a radiation oncologist. Primary extracutaneous neuroendocrine carcinoma was excluded with systemic work-up and radiology, as a positron emission tomography (PET) scan was negative, and a computed tomography (CT) scan was also negative. A

Multicentric Reticulohistiocytosis A Unique Case With Pulmonary Fibrosis

BACKGROUND Multicentric reticulohistiocytosis (MRH) is a rare disease of uncertain etiology that most commonly presents as a papulonodular cutaneous eruption accompanied by erosive polyarthritis. Although MRH is considered a systemic disorder in that it targets skin and joints, involvement of thoracic and visceral organs is uncommon. OBSERVATIONS A woman presented with diffuse cutaneous nodules, and skin biopsy findings revealed classic features of MRH. However, she also manifested severe pulmonary symptoms. A lung biopsy specimen showed prominent histiocytic infiltrates exhibiting the same characteristic morphologic features as those seen in her skin. Furthermore, the lung biopsy findings were significant for a pattern of usual interstitial pneumonia accompanied by notable lymphoid aggregates, a pattern of interstitial lung disease typical of systemic autoimmune and inflammatory conditions. CONCLUSIONS These findings are notable because a histiocytic pulmonary infiltrate suggestive of direct pulmonary involvement by MRH is a rare event. In addition, presentation of MRH in the setting of usual interstitial pneumonia is unique. These observations document a new clinical and histopathologic presentation of MRH that is significant for expanding the idea of MRH as a systemic disease while supporting the notion that MRH is promoted by an inflammatory milieu.

Trichothiodystrophy with Dysmyelination and Central Osteosclerosis

SUMMARY: Trichothiodystrophy (TTD) is a rare group of autosomal recessive disorders of DNA repair unified by the presence of sulfur-deficient brittle hair. We report a 3-year-old boy with classic clinical features of TTD, including ichthyosis, alopecia, developmental delay, and tiger-tail banding of the hair shaft on polarizing microscopy. Brain MR imaging showed both diffuse dysmyelination and osteosclerosis, findings that, in combination, may be specific for TTD.