Flow cytometry quantification of tumor-infiltrating lymphocytes to predict the survival of patients with diffuse large B-cell lymphoma.

Introduction: Our previous studies have demonstrated that tumor-infiltrating lymphocytes (TILs), including normal B cells, T cells, and natural killer (NK) cells, in diffuse large B-cell lymphoma (DLBCL) have a significantly favorable impact on the clinical outcomes of patients treated with standard chemoimmunotherapy. In this study, to gain a full overview of the tumor immune microenvironment (TIME), we assembled a flow cytometry cohort of 102 patients diagnosed with DLBCL at the Duke University Medical Center. Methods: We collected diagnostic flow cytometry data, including the proportion of T cells, abnormal B cells, normal B cells, plasma cells, NK cells, monocytes, and granulocytes in fresh biopsy tissues at clinical presentation, and analyzed the correlations with patient survival and between different cell populations. Results: We found that low T cell percentages in all viable cells and low ratios of T cells to abnormal B cells correlated with significantly poorer survival, whereas higher percentages of normal B cells among total B cells (or high ratios of normal B cells to abnormal B cells) and high percentages of NK cells among all viable cells correlated with significantly better survival in patients with DLBCL. After excluding a small number of patients with low T cell percentages, the normal B cell percentage among all B cells, but not T cell percentage among all cells, continued to show a remarkable prognostic effect. Data showed significant positive correlations between T cells and normal B cells, and between granulocytes and monocytes. Furthermore, we constructed a prognostic model based on clinical and flow cytometry factors, which divided the DLBCL cohort into two equal groups with remarkable differences in patient survival and treatment response. Summary: TILs, including normal B cells, T cells, and NK cells, are associated with favorable clinical outcomes in DLBCL, and flow cytometry capable of quantifying the TIME may have additional clinical utility for prognostication.

The over diagnosis of diffuse mesothelioma: An analysis of 311 cases with recommendations for the avoidance of pitfalls.

BACKGROUND: The diagnosis of mesothelioma may be challenging. We investigated a large database of cases in order to determine the frequency with which a diagnosis of mesothelioma was made incorrectly and the most frequent causes of error. DESIGN: A database including more than 4000 consultation cases of histologically confirmed mesothelioma was examined to identify cases in which mesothelioma was diagnosed by at least one pathologist when the available information pointed towards a different diagnosis. RESULTS: There were 311 cases misdiagnosed as mesothelioma. The most common category was metastatic carcinoma to the pleura or peritoneum (129 cases: 73 lung carcinomas, 15 renal cell carcinomas). The next most common category was primary lung cancer (111 cases: 55 sarcomatoid carcinoma, 56 pseudomesotheliomatous carcinoma). The third most common category was primary malignancies arising from or near the serosal membranes (33 cases). The fourth most common category was fibrous pleurisy (38 cases). The most common errors were failure to consider important radiographic information regarding the gross distribution of tumor, lack of awareness or consideration of another malignancy, overreliance on certain immunohistochemical results, and failure to perform certain diagnostic histochemical, immunohistochemical, or ultrastructural studies. CONCLUSIONS: There are a number of diagnostic pitfalls that can lead to the over diagnosis of mesothelioma. Careful attention to clinical and radiographic information as well as performance of appropriate ancillary tests can help to prevent such misdiagnoses. Detailed examples will be presented to assist in the avoidance of these pitfalls with emphasis on the most commonly observed errors.

Diagnostic approach to prevascular (anterior) mediastinal lymphomas: when thoracic pathology meets hematopathology.

Lymphomas are among the most common malignant tumors occurring in the anterior/prevascular mediastinum. Their diagnoses can be challenging in small biopsies, the current most common method of sampling of an anterior mediastinal mass. Because the initial clinical and/or imaging impression may not be that of lymphoma, these specimens may first be evaluated by cytopathologists, surgical pathologists, and thoracic pathologists rather than hematopathologists. Therefore, it is crucial for this group of pathologists to have a practical diagnostic approach to these neoplasms, know their common diagnostic pitfalls, and their main differential diagnoses. This is important because the diagnosis of lymphoma carries significant therapeutic implications (chemotherapy and/or radiotherapy and not surgical resection). Similarly, securing and properly triaging a sample at the time of tissue collection will translate into direct patient benefit since a subset of lymphomas (T-lymphoblastic lymphoma) may present exclusively as an anterior mediastinal mass and the tissue obtained from this site may be the only one available to evaluate prognostic markers and potential targetable molecular alterations. Once a proper initial diagnostic work-up has been performed, a case can be transferred to a hematopathologist for assistance with a refined diagnosis. In this review, we focus on the practical diagnostic approach to the most common prevascular/anterior mediastinal lymphomas with an emphasis on the findings in small biopsies and provide best practice tips for case triage.

Diagnostic Approach to Pulmonary B-Cell Lymphomas in Small Biopsies, with Practical Recommendations to Avoid Misinterpretation.

Pulmonary lymphomas are rare. With the current less invasive approaches used to obtain material for diagnosis, the diagnosis of pulmonary lymphoma is now frequently established in a small biopsy rather than in a resection. Therefore, the diagnosis has become more challenging and requires correlation with the clinico-radiologic presentation and with ancillary studies (immunohistochemistry, flow cytometry, cytogenetics, and/or molecular analysis). Due to the rarity of pulmonary lymphomas, clinical suspicion of a lymphomatous process is low at initial presentation, and material may be only submitted for histopathology. For this reason, herein, we provide recommendations to arrive at the correct diagnosis of the most common lung B-cell lymphomas (marginal zone lymphoma of mucosa-associated lymphoid tissue, diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, lymphomatoid granulomatosis) in the setting of small biopsies, utilizing only immunohistochemistry. The differential diagnosis varies according to the lymphoma subtype and includes reactive conditions, solid tumors, and other hematolymphoid malignancies. Although morphology and immunohistochemistry may be sufficient to establish a diagnosis, in some cases, the best recommendation is to obtain additional tissue via a VATS biopsy/wedge resection with material submitted for flow cytometry, cytogenetics, and/or molecular studies to be able to properly classify a pulmonary lymphoid process.

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22-modified FK520 analog against C. neoformans.

IMPORTANCE: Fungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited, and the development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally active FDA-approved therapy. This research advances the development of much-needed newer antifungal treatment options with novel mechanisms of action.

Primary Cutaneous Anaplastic Large Cell Lymphoma-A Review of Clinical, Morphological, Immunohistochemical, and Molecular Features.

Primary cutaneous anaplastic large cell lymphoma (ALCL) is the second most common cutaneous T-cell lymphoma after mycosis fungoides and belongs to the spectrum of cutaneous CD30+ T-cell lymphoproliferative disorders. Although primary cutaneous ALCL usually presents as a localized nodule or papule with or without ulceration, multifocal lesions may occur in up to 20% of cases. Histologically, primary cutaneous ALCL consists of a diffuse dermal infiltrate of medium to large anaplastic/pleomorphic cells with abundant amphophilic-to-eosinophilic cytoplasm, horseshoe-shaped nuclei, strong and diffuse expression of CD30, and with focal or no epidermotropism. The neoplastic infiltrate may show angiocentric distribution and may extend to the subcutis. Patients with localized or multifocal disease have a similar prognosis with a 10-year overall survival rate of 90%. Approximately 30% of primary cutaneous ALCLs harbor a DUSP22 (6p25.3) gene rearrangement that results in decreased expression of this dual-specific phosphatase, decreased STAT3 activation, and decreased activity of immune and autoimmune-mediated mechanisms regulated by T-cells.

Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22 modified FK520 analog against C. neoformans.

Fungal infections are of mounting global concern, and the current limited treatment arsenal poses challenges when treating such infections. In particular, infections by Cryptococcus neoformans are associated with high mortality, emphasizing the need for novel therapeutic options. Calcineurin is a protein phosphatase that mediates fungal stress responses, and calcineurin inhibition by the natural product FK506 blocks C. neoformans growth at 37°C. Calcineurin is also required for pathogenesis. However, because calcineurin is conserved in humans, and inhibition with FK506 results in immunosuppression, the use of FK506 as an anti-infective agent is precluded. We previously elucidated the structures of multiple fungal calcineurin-FK506-FKBP12 complexes and implicated the C-22 position on FK506 as a key point for differential modification of ligand inhibition of the mammalian versus fungal target proteins. Through in vitro antifungal and immunosuppressive testing of FK520 (a natural analog of FK506) derivatives, we identified JH-FK-08 as a lead candidate for further antifungal development. JH-FK-08 exhibited significantly reduced immunosuppressive activity and both reduced fungal burden and prolonged survival of infected animals. JH-FK-08 exhibited additive activity in combination with fluconazole in vivo . These findings further advance calcineurin inhibition as an antifungal therapeutic approach. Importance: Fungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited and development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally-active FDA approved therapy. This research advances the development of much needed newer antifungal treatment options with novel mechanisms of action.

Liver Metastasis of Thymoma: Case Report and Review of the Literature.

The 2021 "World Health Organization (WHO) Classification of Thoracic Tumours" classifies epithelial tumors of the thymus (thymomas) based on cytomorphology. Thymomas with benign cytomorphology are classified as type A, AB, B1, B2, and B3, while those with malignant cytomorphology are classified as thymic carcinoma. Although all thymomas have malignant potential, extra-thoracic metastasis of thymomas is exceedingly rare and the exact incidence is not known. Literature review demonstrated 39 cases of thymoma with extra-thoracic metastases reported since the publication of the 1999 WHO Classification of Thoracic Tumours. Nine of these cases presented with metastasis to the liver in the setting of concurrent metastasis to other organs, while only three cases metastasized solely to the liver. We herein report a well-documented case of type B1 thymoma with relatively limited stage (IIb) with an isolated, single liver metastasis occurring seven years after primary resection in a patient with concomitant myasthenia gravis. The following report includes a review of the literature, a discussion of thymoma classification and its relevance to prognosis, and an overview of other extra-thoracic metastatic thymoma cases.

Rapidly Progressive Bilateral Vitreoretinal Lymphoma.

A 56-year-old male who presented with unilateral localized sub-retinal lesions suspicious for primary vitreoretinal lymphoma (PVRL) developed florid bilateral ocular involvement and was found to have lesions on MRI of the brain in a five-week period despite the absence of vitreous involvement during the entire course of his disease. His ocular lesions were monitored while on systemic treatment and an excellent clinical response was achieved. His central nervous system (CNS) lesions, however, continued to progress despite chemotherapy and whole-brain radiation. He died 12 months from his time of ocular diagnosis. To our knowledge, this case represents the most rapid progression of PVRL reported in the literature - from unilateral, localized lesions in the sub-retinal space to bilateral ocular involvement and identification of CNS involvement in a five-week period. This case highlights the potential for rapid ocular progression of PVRL stressing the need for early diagnosis. Therefore, we recommend prompt vitreous and, if necessary, sub-retinal biopsy in cases of suspected vitreoretinal lymphoma in addition to neuro-imaging. We emphasize the importance of coordination between pathologists, ophthalmologists, and oncologists for prompt, accurate diagnosis. Delay in diagnosis and treatment can result in rapid intraocular progression and central nervous system spread.

Anti-glioblastoma activity of monensin and its analogs in an organoid model of cancer.

Glioblastoma (GBM) remains the most frequently diagnosed primary malignant brain cancer in adults. Despite recent progress in understanding the biology of GBM, the clinical outcome for patients remains poor, with a median survival of approximately one year after diagnosis. One factor contributing to failure in clinical trials is the fact that traditional models used in GBM drug discovery poorly recapitulate patient tumors. Previous studies have shown that monensin (MON) analogs, namely esters and amides on C-26 were potent towards various types of cancer cell lines. In the present study we have investigated the activity of these molecules in GBM organoids, as well as in a host:tumor organoid model. Using a mini-ring cell viability assay we have identified seven analogs (IC50 = 91.5 ± 54.4-291.7 ± 68.8 nM) more potent than parent MON (IC50 = 612.6 ± 184.4 nM). Five of these compounds induced substantial DNA fragmentation in GBM organoids, suggestive of apoptotic cell death. The most active analog, compound 1, significantly reduced GBM cell migration, induced PARP degradation, diminished phosphorylation of STAT3, Akt and GSK3ß, increased É£H2AX signaling and upregulated expression of the autophagy associated marker LC3-II. To investigate the activity of MON and compound 1 in a tumor microenvironment, we developed human cerebral organoids (COs) from human induced pluripotent stem cells (iPSCs). The COs showed features of early developing brain such as multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1 +), primitive ventricular system (SOX2 +/Ki67 +), intermediate zone (TBR2 +) and cortical plate (MAP2 +). In order to generate host:tumor organoids, we co-cultured RFP-labeled U87MG cells with fully formed COs. Compound 1 and MON reduced U87MG tumor size in the COs after four days of treatment and induced a significant reduction of PARP expression. These findings highlight the therapeutic potential of MON analogs towards GBM and support the application of organoid models in anti-cancer drug discovery.

NUT Carcinoma: Clinicopathologic Features, Molecular Genetics and Epigenetics.

Nuclear protein in testis (NUT) carcinoma is a rare, highly aggressive, poorly differentiated carcinoma occurring mostly in adolescents and young adults. This tumor usually arises from the midline structures of the thorax, head, and neck, and exhibits variable degrees of squamous differentiation. NUT carcinoma is defined by the presence of a NUTM1 (15q14) rearrangement with multiple other genes. In about 70-80% of the cases, NUTM1 is involved in a balanced translocation with the BRD4 gene (19p13.12), leading to a BRD4-NUTM1 fusion oncogene. Other variant rearrangements include BRD3-NUTM1 fusion (~15-20%) and NSD3-NUTM1 fusion (~6%), among others. The diagnosis of NUT carcinoma requires the detection of nuclear expression of the NUT protein by immunohistochemistry. Additional methods for diagnosis include the detection of a NUTM1 rearrangement by fluorescence in situ hybridization or by reverse transcriptase PCR. NUT carcinoma is usually underrecognized due to its rarity and lack of characteristic histological features. Therefore, the goal of this review is to provide relevant recent information regarding the clinicopathologic features of NUT carcinoma, the role of the multiple NUTM1 gene rearrangements in carcinogenesis, and the impact of understanding these underlying molecular mechanisms that may result in the development of possible novel targeted therapies.

Primary histiocytic sarcoma of the clivus with focal extension into central nervous system and neurologic manifestations: First description at an unusual site with an overwhelming and rapid progression.

Histiocytic sarcoma (HS) is a rare malignant neoplasm of macrophage-dendritic cell lineage that can occur at any site. Primary base of skull involvement is exceedingly rare. We present the case of a previously healthy 56-year-old man who complained of headaches and showed localized neurologic symptoms. Magnetic resonance imaging demonstrated a hyperintense and enhancing mass involving the sphenoid bone and the clivus with an extradural component that compressed the distal pons. The differential diagnosis included chordoma or chondrosarcoma. An endoscopic trans-sphenoidal resection was performed. Microscopically, the tumor showed epithelioid and spindle morphology with atypia, mitoses, and necrosis. No osteoid, cartilaginous, or myxoid matrix was identified. By immunohistochemistry, the tumor was positive for CD68 (KP-1) and lysozyme, variably positive for CD4, CD11c, CD14, CD68 (PGM-1), CD45, and CD163, and negative for markers of epithelial, melanocytic, lymphoid, myeloid, muscle, and dendritic cell origin. Expression of PD-L1 by immunohistochemistry and BRAF V600E mutation analysis by PCR were negative. Tumor recurrence developed after radiation treatment with overwhelming progression into a largely infiltrating mass within 2 weeks with clinical deterioration, and the patient died 3 months later. To our knowledge, this represents the first case of primary HS of the clivus reported to date in the English literature, further expanding the spectrum of neoplasms seen at this site as well as the sites where HS can be seen. The overall prognosis of HS in the skull base is poor, with no standard treatment. Further research is warranted to develop effective treatment approaches, which in the future may rely on the expression of checkpoint inhibitors and/or specific molecular markers.

Fluorescent Immunohistochemistry.

Immunofluorescence is an important immunochemical technique that utilizes fluorescence-labeled antibodies to detect specific target antigens. It is used widely in both scientific research and clinical laboratories. Immunofluorescence allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry. However, analysis of samples labeled with fluorescence-labeled antibodies has to be performed using a fluorescence microscope or other type of fluorescence imaging. There are two methods available: direct (primary) and indirect (secondary) immunofluorescence. Here, we describe the principle of immunofluorescence methods as well as the preparation of fresh-frozen and formalin-fixed, paraffin embedded tissues for both direct and indirect immunofluorescence labeling.

Synchronous plasma cell neoplasm and B lymphoblastic leukemia/lymphoma at initial presentation: first report of an unusual association with a good outcome.

A synchronous diagnosis of a plasma cell neoplasm (PCN) and a non-plasma cell hematologic malignancy is very rare. We report what we believe is the first instance of a synchronous PCN and B lymphoblastic leukemia/lymphoma (B-ALL) diagnosed at initial presentation. The patient underwent laboratory evaluation for an underlying plasma cell neoplasm, including immunology studies, bone marrow biopsy, and flow cytometry immunophenotyping. Serum lambda free light chain and serum IgG were elevated, with an IgG lambda M-protein identified by serum protein electrophoresis and immunofixation. The clinical working diagnosis was plasma cell myeloma. Bone marrow biopsy was positive for a composite PCN and B-ALL. The patient received treatment with VDT-PACE chemotherapy followed by autologous stem cell transplant and maintenance therapy with bortezomib/daratumumab and is in complete remission for both diseases 3.5 years after diagnosis. This case not only adds to the known repertoire of hematologic neoplasms that can occur in association to a PCN, but also demonstrates that patients presenting with this rare combination of hematopoietic neoplasms can be effectively treated simultaneously with excellent responses. Additional research is warranted to understand the pathophysiology, to identify potential prognostic factors, and to develop specific therapeutic plans for these patients.

P10s-PADRE vaccine combined with neoadjuvant chemotherapy in ER-positive breast cancer patients induces humoral and cellular immune responses.

Breast cancer patients diagnosed with HR+/HER2- tumors face a persistent risk of distant recurrence long after completion of their treatment. Strategies to induce anti-tumor immune responses could complement standard-of-care therapies for these patients. The current study was performed to examine the feasibility, safety and immunogenicity of adding P10s-PADRE to standard-of-care chemotherapy in HR+/HER2- early-stage breast cancer patients. Twenty-five subjects were treated in a single-arm Phase Ib clinical trial. Five different immunization schedules were considered to evaluate the feasibility of eliciting an immune response. The primary immunogenicity endpoint was antibody titer. The expression of several activation markers on natural killer (NK) cells and serum concentrations of Th1/Th2 cytokines were also examined. The percentage of tumor-infiltrating lymphocytes (TILs) was determined. Antibody response was superior in schedule C where 3 weekly immunizations preceded the first dose of chemotherapy. A significant change in CD16, NKp46 and CD94 expression levels on NK cells and a rise in serum content of IFN-γ was observed after treatment. Schedule C showed an increase in TILs in residual lesions. The combination therapy is safe and immunogenic with treatment schedule C being immunologically promising. Randomized trials focused on long-term survival outcomes are needed to evaluate clinical benefits.

ALK-Negative Anaplastic Large Cell Lymphoma: Current Concepts and Molecular Pathogenesis of a Heterogeneous Group of Large T-Cell Lymphomas.

Anaplastic large cell lymphoma (ALCL) is a subtype of CD30+ large T-cell lymphoma (TCL) that comprises ~2% of all adult non-Hodgkin lymphomas. Based on the presence/absence of the rearrangement and expression of anaplastic lymphoma kinase (ALK), ALCL is divided into ALK+ and ALK-, and both differ clinically and prognostically. This review focuses on the historical points, clinical features, histopathology, differential diagnosis, and relevant cytogenetic and molecular alterations of ALK- ALCL and its subtypes: systemic, primary cutaneous (pc-ALCL), and breast implant-associated (BIA-ALCL). Recent studies have identified recurrent genetic alterations in this TCL. In systemic ALK- ALCL, rearrangements in DUSP22 and TP63 are detected in 30% and 8% of cases, respectively, while the remaining cases are negative for these rearrangements. A similar distribution of these rearrangements is seen in pc-ALCL, whereas none have been detected in BIA-ALCL. Additionally, systemic ALK- ALCL-apart from DUSP22-rearranged cases-harbors JAK1 and/or STAT3 mutations that result in the activation of the JAK/STAT signaling pathway. The JAK1/3 and STAT3 mutations have also been identified in BIA-ALCL but not in pc-ALCL. Although the pathogenesis of these alterations is not fully understood, most of them have prognostic value and open the door to the use of potential targeted therapies for this subtype of TCL.

Breast implant-associated anaplastic large cell lymphoma: clinical follow-up and analysis of sequential pathologic specimens of untreated patients shows persistent or progressive disease.

Breast implant-associated anaplastic large cell lymphoma (ALCL) is a distinctive type of T-cell lymphoma that arises around textured-surface breast implants. In a subset of patients, this disease can involve surrounding tissues, spread to regional lymph nodes, and rarely metastasize to distant sites. The aim of this study was to assess sequential pathologic specimens from patients with breast implant-associated ALCL to better understand the natural history of early-stage disease. To achieve this goal, we searched our files for patients who had breast implant-associated ALCL and who had undergone earlier surgical intervention with assessment of biopsy or cytologic specimens. We then focused on the patient subset in whom a definitive diagnosis was not established, and patients did not receive current standard-of-care therapy at that time. We identified a study group of ten patients with breast implant-associated ALCL in whom pathologic specimens were collected 0.5 to 4 years before a definitive diagnosis was established. A comparison of these serial biopsy specimens showed persistent disease without change in pathologic stage in three patients, progression in five patients, and persistence versus progression in two patients. Eventually, six patients underwent implant removal with complete capsulectomy and four underwent partial capsulectomy. Seven patients also received chemotherapy because of invasive disease, three of whom also received radiation therapy, two brentuximab vedotin after chemotherapy failure, and one allogeneic stem cell transplant. Eight patients achieved complete remission and two had partial remission after definitive therapy. At time of last follow-up, six patients were alive without disease, one had evidence of disease, one died of disease, and two patients died of unrelated cancers. In summary, this analysis of sequential specimens from patients with breast implant-associated ALCL suggests these neoplasms persist or progress over time if not treated with standard-of-care therapy.

Mediastinal Lymphoproliferative Disorders.

Lymphoproliferative disorders comprise 50% to 60% of all mediastinal malignancies in both children and adults. Primary mediastinal involvement is rare (∼5%), whereas secondary mediastinal involvement by systemic disease is more common (10% to 25%). Primary mediastinal disease is defined as involvement by a lymphoproliferative disorder of mediastinal lymph nodes, the thymus, and/or extranodal mediastinal organs without evidence of systemic disease at presentation. In this review, the clinical, radiologic, histopathologic, immunohistochemical, and genetic features of some of the most characteristic mediastinal lymphoproliferative disorders are presented. The entities discussed here include: classic Hodgkin lymphoma with emphasis on nodular sclerosis and mixed cellularity types, and non-Hodgkin lymphomas, including primary mediastinal (thymic) large B-cell lymphoma, mediastinal gray zone lymphoma, mediastinal diffuse large B-cell lymphoma, thymic marginal zone lymphoma, mediastinal plasmacytoma, T-lymphoblastic lymphoma, and anaplastic large cell lymphoma. Although not a malignant process, hyaline vascular Castleman disease is also discussed here as this disorder commonly involves the mediastinum. Despite multiple advances in hematopathology in recent decades, the day-to-day diagnosis of these lesions still requires a morphologic approach and a proper selection of immunohistochemical markers. For this reason, it is crucial for general pathologists to be familiar with these entities and their particular clinicoradiologic presentation.