The Status of Immunohistochemical Studies in Lymphoma Diagnosis

Immunohistochemical studies of formalin fixed paraffin embedded sections today constitute an essential component of the diagnostic process for malignant lymphoma. Lineage related markers are of value in distinguishing various lymphomas from anaplastic large and small cell carcinomas, and from sarcomas. Other more recently available markers are of value in establishing prognosis and, on occasion, in differentiating between neoplastic and reactive proliferations. Meticulous attention to reagent control and technical procedures is essential to all such studies.     

非霍奇金淋巴瘤患者焦虑抑郁情况分析及与病情分期、生活质量的相关性研究

摘要 目的：探讨非霍奇金淋巴瘤（NHL）患者焦虑、抑郁情况,并分析二者与患者病情分期、生活质量的相关性。方法：纳入我院2017年9月～2020年9月收治的NHL患者120例作为NHL组,另选取100例健康志愿者作为对照组,针对两组受试者行焦虑自评量表（SAS）、抑郁自评量表（SDS）评分。根据病情分期将NHL患者分成Ⅰ期组（n=23）、Ⅱ期组（n=40）、Ⅲ期组（n=39）、Ⅳ期组（n=18）,根据患者抑郁、焦虑发生情况分成负面情绪组（n=49）、无负面情绪组（n=71）。利用简明生活质量量表（SF-36）评估生活质量,经Logistic多元回归模型分析NHL患者负面情绪发生的影响因素。结果：NHL组的SDS、SAS评分高于对照组,且Ⅲ期、Ⅳ期组的SDS、SAS评分高于Ⅰ期、Ⅱ期组,Ⅳ期组高于Ⅲ期组（P＜0.05）。负面情绪组躯体功能、躯体疼痛、躯体角色功能、情绪角色功能、心理健康、精力、总体健康评分低于无负面情绪组（P＜0.05）。Pearson线性分析结果显示,SDS、SAS评分与躯体功能、躯体疼痛、躯体角色功能、情绪角色功能、心理健康、精力、总体健康评分呈负相关（P＜0.05）。Logistic多元回归分析结果提示,病情分期Ⅲ～Ⅳ期是NHL患者负面情绪发生的危险因素,受教育年限＞8年、家庭月收入≥5000元是预防负面情绪的保护性因素（P＜0.05）。结论：NHL患者病情分期越高,则焦虑、抑郁越明显,进而降低患者生活质量。     

Effects of Fixation and Tissue Processing on Immunohistochemical Demonstration of Specific Antigens

Identification of biomarkers in archival tissues using immunochemistry is becoming increasingly important for determining the diagnosis and prognosis of tumors, for characterizing preinvasive neoplastic changes in glandular tissues such as prostate, for evaluating the response of tumors and preinvasive neoplastic changes to certain therapies (i.e., as a surrogate intermediate end point), for selecting patients who are candidates for specific therapies (e.g., immunotherapy) and for retrospective studies. For detecting specific biomarkers it is important to understand the limitations imposed by the fixation methods and processing of the tissues. This study was designed to determine the effects of fixation on the detection in archival paraffin blocks of selected antigens postulated to be important in tumor biology. We evaluated the antigens TGFα, p185^erbB-2, broad spectrum keratins, p53, and TAG-72 (B72.3). Fixatives evaluated included standard preparations of neutral buffered formalin, acid formalin, zinc formalin, alcoholic formalin, ethanol, methanol, and Bouin's fixative. We found that in general neutral buffered formalin is the poorest fixative for maintaining antigen recognition by immunohistochemistry and that no single fixative was best for all antigens. The dehydrating (coagulant) fixatives (e.g., ethanol and methanol) preserved immunorecognition of p53 and broad spectrum keratins best while the slow cross-linking fixatives (e.g., unbuffered zinc formalin) were best for demonstrating TGFα and p185^erbB-2. Fixatives other than neutral buffered formalin produced equivalent recognition of the epitope of TAG-72 by B72.3. In formalin fixed archival tissues, only a portion of the antigen signal can be detected by routine immunohistologic methods.     

CNS lymphoma masquerading as stroke: Cases report and literature review

Central nervous system (CNS) involvement in Hodgkins lymphoma (HL) is extremely rare. There are only two reported case series of intracranial involvement of HL. CNS HL can be presented at any point in the course of HL, most mimicking with a prominent neurological symptom. This challenges the diagnosis of CNS involvement and stroke. Here, we report four cases of patients having refractory HL with CNS involvement to garner attention among neurologists for this rare disease presents with stroke symptoms and reviews its disease characteristics, prognosis, and treatment.     

Role of modern radiation therapy in early stage Hodgkin's lymphoma: A young radiation oncologists’ perspective

The role of radiotherapy is well established in combined modality programs for early stage Hodgkin's lymphoma, but still debated with regards to late toxicity issues. Modern radiotherapy prescribing attitudes include lower doses and smaller fields, together with the implementation of sophisticated and dedicated delivery techniques. Aim of this review is to briefly discuss the current role of radiotherapy in this field and the potential future developments. Major trials conducted in recent years in early stage Hodgkin's lymphoma are critically reviewed and discussed with a focus on radiotherapy-related issues and with an attention to current treatment options by a “young” radiation oncologists’ perspective.     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Radiation-induced breast cancer in women with Hodgkin's disease

Aim and background

The aim of this study is to analyze the main clinical and pathologic characteristics of radiation-induced breast carcinomas (BC) following treatment for Hodgkin''s disease (HD) and to identify the risk factors for their induction. To create a mathematical model for the prediction of expected age at which a BC might develop based on the age at treatment for HD.

Materials and methods

Thirty-nine cases of women with BC that developed after treatment for HD in puberty or adolescence were analyzed retrospectively. The median age at initiation of treatment for HD was 12.9 years (9–21). The median age at diagnosis of the second malignancy – breast carcinoma was 32.4 years (22.9–39).

Results

The distribution of patients according to the clinical T stage of breast cancer was as follows: 11 patients with T1 stage BC (28%), 22 with T2 stage (56%) and 6 with stage T3 (16%). Prevalent were tumors localized in the lateral breast quadrants. The observed 5 year survival was 95%.

Conclusion

The risk of solid tumors, especially breast cancer, is high among women with HD disease who were treated with radiotherapy in their childhood. In this article, we propose a specific mathematical age formula which could be used as predictive equation when the age of the treatment for HD is in the range between 9 and 21 years. Systematic screening for breast cancer in these patients would be significantly important for their health and could improve their survival.     

浆母细胞淋巴瘤的诊治进展

浆母细胞淋巴瘤是一种临床罕见、侵袭性极强的B细胞淋巴瘤,多发生在HIV感染的患者,也可见于HIV阴性的免疫功能正常或受抑的人群,常与EB病毒感染相关。由于其特殊的形态学及免疫表型特征、化疗耐药及复发率高的临床特点,诊断及治疗仍极具挑战性,总体预后极差。本文简要总结了PBL在流行病学、病理学特征、临床表现、诊断、治疗及预后因素等方面的进展,期望为PBL的临床诊治提供帮助。     

Incorporating Gravity Model Principles into Disease Mapping

The space‐time variation of disease risk, considering the closeness and availability of care units, is studied by a hierarchical Bayesian model in a gravitational formulation. The pattern of mortality for Hodgkin's lymphoma over the last twenty‐five years in the Tuscany region and the evaluation of the consequences of distance from the nearest radiation‐therapy center are described. Results show a decrease in mortality for Hodgkin's lymphoma and the attraction exercised by each care units.     

Role of endoscopic ultrasound‐guided‐fine needle aspiration biopsy in the diagnosis of lymphoma of the pancreas: A clinicopathological study of nine cases

Objective

Endoscopic ultrasound‐guided‐fine needle aspiration (EUS‐FNA) is an established first‐line procedure in the management of solid and cystic pancreatic masses. Lymphoma is an uncommon diagnosis in EUS‐FNA of the pancreas, and it is more common for such a diagnosis to be because of secondary involvement of the pancreas by a lymphoproliferative disorder than for this to represent isolated primary pancreatic lymphoma (PPL). We present the clinical, EUS and cytological features of these lesions.

Material and methods

After obtaining approval from our Institutional Review Board (IRB), nine cases of lymphoma diagnosed on EUS‐FNA at a tertiary care cancer centre over a period of 8 years from 2008 to 2016 were retrieved from our endoscopy and pathology archives. Rapid onsite evaluation (ROSE) was carried out by a trained cytopathologist in all these cases. Cell blocks were available in seven cases, and immunophenotyping was performed on cell blocks using the immunoperoxidase method. Flow cytometry was performed in two cases.

Results

The most frequent site of involvement was the head of the pancreas (n=5, 55.6%). Four out of nine cases were diagnosed as PPL (44.4%). Five cases were diagnosed as lymphoma secondarily involving the pancreas (55.6%). The most frequent diagnosis was diffuse large B‐cell lymphoma (n=6, 66.7%), followed by Hodgkin's lymphoma (n=2, 22.2%) and peripheral T‐cell lymphoma (n=1, 11.1%).

Conclusion

EUS‐FNA in experienced hands is a valuable diagnostic modality, in conjunction with ROSE, immunohistochemistry and flow cytometry, in the diagnosis and sub‐typing of both primary and secondary pancreatic lymphoma.     

美罗华对CD20 阳性NHL患者血清LDH、beta2- 微球蛋白及预后的影响*

目的：分析美罗华对CD20 阳性非霍奇金淋巴瘤(NHL)患者血清乳酸脱氢酶（LDH）、beta2- 微球蛋白（beta2-MG）水平及预后的影 响。方法：对2004 年1 月-2011 年6 月在我院收治的86 例NHL患者,分别采用CHOP（对照组,n=48）与RCHOP（治疗组,n=48）2 种不同的方案进行化疗,每21 天为一个周期,共6 个周期。比较两组临床疗效、不良反应、LDH和beta2-MG 水平变化及预后。结果： 治疗组的ORR 和DCR 分别为77.1 %和89.6 %,均显著高于对照组56.3 %和72.9 %（P<0.05）;化疗后,两组血清LDH、beta2-MG 均 较化疗前明显下降,而治疗组化疗后血清LDH、beta2-MG 均显著低于对照组(P<0.05);治疗组白细胞减少、恶心呕吐的并发症高于 对照组(P<0.05);化疗后1、2 年生存率两组之间均无显著性差异（P>0.05）,而治疗组化疗后3 年生存率明显升高(P<0.05)。结论：美 罗华联合CHOP方案治疗CD20 阳性NHL患者疗效明显,且耐受性好,可有效调节血清LDH、茁2-MG 水平,提高缓解率,改善患 者预后。     

Dendritic cell populations in colon and mesenteric lymph nodes of patients with Crohn's disease.

Dendritic cells (DCs) are key cells in innate and adaptive immune responses that determine the pathophysiology of Crohn's disease. Intestinal DCs migrate from the mucosa into mesenteric lymph nodes (MLNs). A number of different markers are described to define the DC populations. In this study we have identified the phenotype and localization of intestinal and MLN DCs in patients with Crohn's disease and non-IBD patients based on these markers. We used immunohistochemistry to demonstrate that all markers (S-100, CD83, DC-SIGN, BDCA1-4, and CD1a) showed a different staining pattern varying from localization in T-cell areas of lymph follicles around blood vessels or single cells in the lamina propria and in the MLN in the medullary cords and in the subcapsular sinuses around blood vessels and in the T-cell areas. In conclusion, all different DC markers give variable staining patterns so there is no marker for the DC.     

The thyroid "microsomal" antigen is an epitope on the thyrotropin receptor

Antimicrosomal antibodies are present in the sera of most patients with autoimmune thyroiditis, and Graves' disease. It has, in general, been difficult to separate antimicrosomal activity from that directed against the thyrotropin (TSH) receptor in Graves' IgG preparations. The "microsomal" antigen has been localized to the endoplasmic reticulum and microfollicular aspect of thyrocytes; its structure is however unknown. In an attempt to identify the thyroid microsomal antigen, we studied the interaction of Hashimoto's IgG with high microsomal antibody titre and negative for thyroglobulin with purified thyroid plasma and light microsomal membranes. We allowed Hashimoto's, Graves', and control IgGs to bind to protein blots of thyroid plasma membranes resolved on SDS-PAGE under non-reducing conditions. All seven Hashimoto's IgG at a concentration of 2 mg/ml interacted with an M approximately 197,000 polypeptide corresponding to the TSH holoreceptor. By contrast to Graves' IgG (which were positive at 1 mg/ml), however, this binding was not blocked by pretreatment of the protein blots with TSH. Normal IgGs showed no binding at concentrations of up to 2 mg/ml. Both Hashimoto's and Graves' IgG interacted with TSH-affinity column-purified receptor preparations. Two of the Hashimoto's IgGs induced adenylate cyclase activation in thyroid plasma membranes, three inhibited TSH-stimulated enzyme activation, and two were without effect. Two classes of autoantibodies, other than TSH receptor directed, were encountered; one class raised to antigens common to all seven patients and another class unique to individual patients, eg, Mr 210,000 and Mr 20,000 polypeptides. We propose that the TSH receptor has multiple epitopes (functional domains), and the one to which antimicrosomal antibody bind is likely to be spatially separated from that with which Graves' IgG and TSH interact. Differences in affinity or number of sites allows for the demonstration of Graves' IgG against a background of antimicrosomal antibody.     

Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.     

Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.     

基于体素的形态测量学在阿尔茨海默病及轻度认知功能障碍研究中的应用

阿尔茨海默病(Alzheimer's disease,AD)是发生于老年和老年前期、以进行性认知功能障碍和行为异常为特征的中枢神经系统退行性疾病,是老年痴呆中最常见类型。轻度认知功能障碍(mild cognitive impairment,MCI)是介于正常衰老和痴呆之间的一种中间状态,指有轻度的记忆或认知损伤,但尚未达到痴呆程度的一种状态,日常生活和社会功能不受影响,其中很大一部分患者最终进展为AD。临床诊断AD患者多已达中晚期,为了能早期诊断AD及预测MCI的转归,有关AD的生物学标注物的研究成为近年来的科研热点。AD患者颅脑的大体病理特征为脑萎缩,其萎缩有别于正常老龄化所致的退行性改变,有其自身特点,这种特定形式的萎缩有可能成为AD早期诊断的生物学标志物。基于体素的形态测量学(voxel-based morphometry,VBM)是一种基于像素水平对脑核磁图像进行自动、全面、客观分析的技术,可以定量分析全脑结构、刻画出局部脑区结构特征,是一种较好的脑形态分析工具,广泛用于阿尔茨海默病及轻度认知功能障碍的研究中,本文综述了近年来其研究进展,期望为临床及科研提供参考。     

Studies on the turnover of exogenous mannose-terminal glucocerebrosidase in rat liver lysosomes

We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-β-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner. Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding.     

Role of Immunohistochemistry in Staging Diffuse Large B-cell Lymphoma (DLBCL)

The use of immunohistochemistry (IHC) in staging bone marrow in non-Hodgkin''s lymphoma (NHL) is largely limited to ambiguous cases, particularly those with lymphoid aggregates. Its role in routine clinical practice remains unestablished. This study aimed to determine whether the routine use of IHC in diffuse large B-cell lymphoma (DLBCL) would improve the detection of lymphomatous involvement in the bone marrow. It also sought to determine the impact of IHC on predicting survival compared with routine histological diagnosis using hematoxylin and eosin (H&E), Giemsa, and reticulin staining. The bone marrow trephines of 156 histologically proven DLBCL cases were assessed on routine histology, and IHC using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a), and κ and λ light chains. IHC detected lymphomatous involvement on an additional 11% cases compared with histology alone. Although both routine histology and IHC were good predictors of survival, IHC was better at predicting survival on stepwise multivariate Cox regression analysis. IHC performed routinely on bone marrow trephines has the ability to improve detection of occult lymphoma in experienced hands. Furthermore, it is a better predictor of survival compared with routine histological examination alone. (J Histochem Cytochem 56:893–900, 2008)