Neoplastic Cell Population Research Articles

Metastatic Melanoma of Unknown Primary with Plasmacytoid Morphology Presenting as Acute Liver Failure

Abstract Introduction/Objective Viral hepatitis, drug toxicity, and autoimmune disorders are common etiologies of acute liver failure (ALF). Very rarely, a cause like metastatic melanoma may present as a primary driver of ALF. Malignant melanoma is histologically diverse and plasmacytoid melanoma is an unusual variant that may present as a solitary tumor or metastatic disease. Methods/Case Report A 48-year-old female presented with 2 weeks of right upper quadrant pain, nausea, coffee ground emesis and bleeding per rectum. She had no history of liver disease or alcohol use. On presentation, AST was 83 U/L, ALT was 29U/L, ALP was 159U/L and total bilirubin was 12.7g/dl. Viral hepatitis panel, HIV and chronic liver disease workup was negative. Esophagogastroduodenoscopy and colonoscopy showed no sources of bleeding. She was diagnosed with decompensated cirrhosis, ALF, and acute kidney injury from hepatorenal syndrome. Cirrhosis and multifocal indeterminate T1 hyper/T2 hypo intense lesions (dysplastic or regenerative nodules) were seen on MRI. Liver biopsy revealed metastatic melanoma. Almost entire liver parenchyma on biopsy was replaced by a population of neoplastic cells with plasmacytoid features. Immunostaining was positive for MART-1, HMB45 and SOX-10. Staining for Pan Cytokeratin and CK-7 highlighted the bile ducts and negative staining was seen for CK20, CD3, CD20, PAX-8, GATA-3, CDX-2 and P63. No primary lesion was found. It was deemed unlikely that she would benefit from therapy before complications occur. She passed away in 16 days. Results (if a Case Study enter NA) NA Conclusion In most cases of ALF associated with malignant melanoma, the liver may have a seemingly normal appearance on imaging studies due to the distinctive histology of leukemoid tumor infiltration within the liver sinusoids, resulting in sinusoidal obstruction and subsequent hepatocellular dysfunction. The rapidity of liver failure in these cases is related to the replacement of liver parenchyma by malignant cells. The plasmacytoid morphology is seen in a variety of malignancies and to avoid a potential misdiagnosis of an aggressive malignant melanoma, it is imperative to perform a thorough histopathological evaluation aided with immunohistochemistry. It is important to keep in mind that the potential involvement of malignant tumor cell infiltration may cause ALF and early histological assessment with a liver biopsy may be of paramount significance and may possibly change the course of the disease.

Detection of neoplastic-immune hybrid cells with metastatic properties in uveal melanoma

BackgroundUveal melanoma is the most common non-cutaneous melanoma and is an intraocular malignancy affecting nearly 7,000 individuals per year worldwide. Of these, approximately 50% will progress to metastatic disease for which there are currently no effective curative therapies. Despite advances in molecular profiling and metastatic stratification of uveal melanoma tumors, little is known regarding their underlying biology of metastasis. Our group has identified a disseminated neoplastic cell population characterized by co-expression of immune and melanoma proteins, circulating hybrid cells (hybrids), in patients with uveal melanoma. Compared to circulating tumor cells, which lack expression of immune proteins, hybrids are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression.MethodsTo ascertain mechanisms underlying enhanced hybrid cell dissemination we identified hybrid cells within primary uveal melanoma tumors using single cell RNA sequencing (n = 8) and evaluated their gene expression and predicted ligand-receptor interactions in relation to other melanoma and immune cells within the primary tumor. We then verified expression of upregulated hybrid pathways within patient-matched tumor and peripheral blood hybrids (n = 4) using cyclic immunofluorescence and quantified their protein expression relative to other non-hybrid tumor and disseminated tumor cells.ResultsAmong the top upregulated genes and pathways in hybrid cells were those involved in enhanced cell motility and cytoskeletal rearrangement, immune evasion, and altered cellular metabolism. In patient-matched tumor and peripheral blood, we verified gene expression by examining concordant protein expression for each pathway category: TMSB10 (cell motility), CD74 (immune evasion) and GPX1 (metabolism). Both TMSB10 and GPX1 were expressed on significantly higher numbers of disseminated hybrid cells compared to circulating tumor cells, and CD74 and GPX1 were expressed on more disseminated hybrids than tumor-resident hybrids. Lastly, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting metastasis including GAS6-AXL, CXCL12-CXCR4, LGALS9-P4HB and IGF1-IGFR1.ConclusionThese findings highlight the importance of TMSB10, GPX1 and CD74 for successful hybrid cell dissemination and survival in circulation. Our results contribute to the understanding of uveal melanoma tumor progression and interactions between tumor cells and immune cells in the tumor microenvironment that may promote metastasis.

Association of CD133, ALDH1, CD117 and OCT4 expression with prognosis of patients with cervical cancer.

Cancer stem cells (CSC), a small population of neoplastic cells, are associated with worse prognosis. The aim of this study was to evaluate the expression of ALDH1, CD117, CD133 and OCT4; potential markers of CSC; and their associations with the prognosis of women diagnosed with cervical cancer. This retrospective cohort study included 126 women diagnosed with cervical cancer whose biopsies were analyzed by immunohistochemistry. Median values of marked cells were used to define cutoff points for low and high expression. For specific survival, multivariate analyses showed statistical significance for lymph node metastases (HR 8.15; 95% CI 3.00-22.18) and borderline significance for high CD133 expression (p = 0.058). For overall survival, multivariate analyses showed statistical significance for IIA-IVB staging (HR 4.60; 95% CI 1.46-14.56), lymph node metastases (HR 5.13; 95% CI 12.02-13.03) and high CD133 expression (2.67; 95% CI 1.11-6.43). Considering only women with SCC, the same clinicopathological variables were associated with worse specific and overall survival in univariate analyses. However, higher expression of CD 133 (HR 11.10; 95% CI 2.42-50.94 and 6.00; 95% CI 2.02-17.87) and staging IIA-IVB (HR 5.96; 95% CI 1.30-27.34 and HR 12.47; 95% CI 2.45-63.54) respectively impacted negatively specific and overall survival, as multivariate analyses showed. Secondarily, it was observed that ALDH1 expression was associated with adenocarcinoma and CD117 expression with squamous cells carcinoma. Higher expression of CD133 was associated with worse specific and overall survival, indicating that it could have relevance as a clinical marker and therapeutic target.

Abstract B112: Single-cell profiling of neoplastic cell populations in a KRAS-initiated zebrafish pancreatic cancer model

Abstract While the majority of human pancreatic cancers are classified as ductal adenocarcinomas, the cell of origin of these tumors remains uncertain and many tumors are comprised of dedifferentiated cell types, making it imperative to understand the full spectrum of neoplastic differentiation and how it is regulated. We have developed a new zebrafish model of pancreatic cancer driven by tissue-specific expression of UAS:mK02-KRASG12D under the regulation of a Ptf1a:Gal4/VP16 transcriptional driver. Transgenic zebrafish generate mixed tumors comprised histologically of mixed acinar and ductal elements, providing the opportunity to interrogate specific transcriptional and epigenetic modifiers responsible for regulating neoplastic pancreatic cellular differentiation. Single-cell RNA sequencing of these tumors has confirmed neoplastic cell populations expressing exclusively acinar and ductal markers, as well as distinct clusters expressing markers of both cell lineages. Screening a panel of 21 unique chromatin modifiers and chromatin readers for differential expression in these distinct cell clusters reveals hdac9b to be upregulated in the progenitor-like ductal population. Gain- and loss-of-function genetic studies will be used to determine the functional role of hdac9b in generating distinct neoplastic cell differentiation states. We are also generating pseudo-time trajectory inferences to further determine the directionality of differentiation states in these cells. By generating new insights into regulation of distinct differentiation states in pancreatic cancer, we hope that new strategies for therapeutic manipulation of neoplastic cell differentiation will emerge. Citation Format: Somer M. Matar, Britton C. Goodale, Xiaoying Liu, Annie Katanga, Nicole F. Gallien, Sandra Blazquez, Direna Alonso-Curbelo, Steven D. Leach. Single-cell profiling of neoplastic cell populations in a KRAS-initiated zebrafish pancreatic cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B112.

The Cytokine Storm of Multicentric Castleman Disease.

As described throughout this book, different triggers can elicit a variety of different cytokine storm disorders that share overlapping clinical features (Fig. 31.1). Even within a particular cytokine storm disorder, multiple different triggers can elicit the syndrome. Like HLH, multicentric Castleman disease (MCD) serves as a great example of this as it can be caused by a viral infection, neoplastic cell population, or an unknown cause. Furthermore, the idiopathic subtype of MCD (iMCD) provides one of the first examples of a cytokine storm disorder that could be abrogated with targeted neutralization of a single cytokine when inhibition with the anti-interleukin-6 (IL-6) receptor monoclonal antibody tocilizumab was shown to effectively treat iMCD in the 1990s. Of course, this "iMCD treatment," tocilizumab, has been used in a variety of cytokine storm settings over the last 30+ years.

Macroscopic, cytological and histological diagnosis of proventricular adenocarcinoma in a sulphur-crested cockatoo (Cacatua galerita)

An adult sulphur-crested cockatoo (Cacatua galerita) was unexpectedly found dead and was presented for post-mortem examination. Macroscopically, the proventricular wall was expanded by an infiltrative off-white mass, 10 mm in diameter, with secondary mucosal ulceration. Cytological examination of impression smears revealed a neoplastic epithelial cell population forming occasional well-defined acini with secretory product. Histologically, the neoplastic cells were predominantly arranged in acini and tubules, with fewer in small clusters, supported by a desmoplastic stroma. Neoplastic cells were polygonal, with mostly well-demarcated cell boundaries and a moderate amount of eosinophilic cytoplasm that was multifocally markedly distended by more intensely eosinophilic material. There was an increased nuclear-to-cytoplasmic ratio with moderately sized to large nuclei (approximately 15 μm diameter) with lightly to densely stippled chromatin and single or paired prominent basophilic nucleoli. The mitotic count was 19 in 10 high-power fields (2.37 mm2), with scattered bizarre mitotic figures and atypia including moderate anisocytosis and anisokaryosis and moderate cellular and nuclear pleomorphism. There was frequent intratubular brightly eosinophilic proteinaceous material, which was Alcian blue and periodic acid‒Schiff positive. Proventricular lymphovascular invasion was detected, with metastases within the ventriculus and lungs. The macroscopic, cytological and histological appearance of the neoplasm was consistent with a proventricular adenocarcinoma.

Highly malignant endometrial stromal sarcoma in a cat

An 11-year-old female Persian cat underwent ovariohysterectomy due to dilation of the uterine cavity with irregular thickening of the wall. Macroscopically, the middle and distal regions of the left uterine horn were swollen and the uterine wall was irregularly thickened due to the development of multiple coalescent, variably sized nodules. Microscopically, the nodules had originated in the endometrium and were composed of round to polygonal neoplastic cells arranged in dense sheets or ill-defined fascicles. The neoplastic cells had locally invaded the myometrium and reached the subserosa, with lymphovascular invasion. Immunohistochemically, the neoplastic cell population was partially positive for CD10, an established marker of endometrial stromal sarcoma (ESS) in humans, with focal and diffuse nuclear immunopositivity for oestrogen and progesterone receptors and immunonegativity for desmin and α-smooth muscle actin. Based on these findings, the uterine tumour was diagnosed as ESS and was considered to correspond morphologically to high-grade ESS in humans.

Multiple Myeloma with 1q Gain/Amplification Shows Reduced CD38 Expression Via IL-6R Overexpression

Background: The anti-CD38 monoclonal antibody daratumumab (Dara) significantly improves the outcomes of patients with multiple myeloma (MM). However, chromosome 1q21 gain/amplification (1q+) has been reported to be associated with poor outcomes in patients with relapsed/refractory MM treated with Dara. A recent subgroup analysis of the MAIA study showed that 1q amplification was associated with poor prognosis, even in newly diagnosed patients (Moreau et al. ASH 2022). However, the underlying mechanism of this resistance remains unclear. Aim and Methods: To elucidate the mechanism of Dara resistance in 1q+ MM cells, we focused on CD38 regulation in 1q+ MM. We examined CD38 expression in primary MM cells of 89 patients with newly diagnosed MM. CD38 expression levels were determined as the mean fluorescence intensity (MFI) using flow cytometry (FCM). FCM was performed using the DURAClone RE PC antibody panel on a Navios cytometer, and the data were analyzed using Kaluza analysis software. The CD38 MFI was assessed in the neoplastic plasma cell population (CD38+/CD138+/CD56+ or CD56−/CD19−). The presence of 1q+ was evaluated using interphase fluorescence in situ hybridization (iFISH) in CD138-purified bone marrow plasma cells with a 30% cutoff. Patients with three copies of 1q21 were defined as having a 1q gain, whereas patients with at least four copies were defined as having a 1q amplification. For functional analysis, we employed three human myeloma cell lines (HMCLs) with 1q+ (H929, MOLP8, and MM.1S) and three HMCLs with 1q wild type (WT) (KMS12BM, SKMM2, and NCU-MM1). Results: We found that 1q+ MM cells showed significantly lower CD38 expression than 1q WT MM cells in our primary samples. Furthermore, we found that isolated 1q+ MM without other high-risk cytogenetic abnormalities showed significantly lower CD38 expression than 1q WT MM. We also found that patients with 1q amplification had significantly lower CD38 expression than those with 1q gain. To elucidate the mechanism of CD38 downregulation, we focused on the IL-6 receptor (IL-6R) located on chromosome 1q21 because IL-6-mediated JAK/STAT activation has been shown to regulate CD38 expression. Indeed, as the copy number of 1q increased, the expression of IL-6R also increased in our primary samples. Using published datasets, we also confirmed that 1q21 status is closely associated with IL-6R levels. Furthermore, a significant negative correlation was observed between IL-6R and CD38 expression. Based on these results, we hypothesized that IL-6R overexpression due to 1q gain/amplification contributes to CD38 downregulation in 1q+ MM. To investigate this hypothesis, we first examined the levels of IL-6R in the six HMCLs and verified that 1q+ HMCLs expressed higher levels of IL-6R than 1q WT. We treated these HMCLs with IL-6 and confirmed that only 1q+ HMCLs showed CD38 downregulation. CD38 expression was also downregulated in the primary 1q+ MM samples treated with IL-6 but not in 1q WT samples. Moreover, higher IL-6R expression was observed in 1q+ primary samples than in 1q WT primary samples. IL-6 also led to the robust upregulation of p-STAT3 in HMCLs with 1q+ but not in HMCLs with 1q WT. Particularly, p-STAT3 expression was robustly enhanced in 1q amplification HMCLs that highly expressed IL-6R. Conversely, p-STAT3 expression was minimal in NCU-MM1 cells expressing little or no IL-6R. We also confirmed that IL-6R and STAT3 expressions were significantly and positively correlated in our primary samples and public datasets. Finally, CD38 expression was recovered by treating the 1q+ HMCLs and primary 1q+ MM samples with ruxolitinib (a JAK1/JAK2 inhibitor) or tocilizumab (an anti-IL-6R antibody). Conclusion: Overexpression of IL-6R in 1q+ MM leads to CD38 downregulation through the activation of the JAK/STAT pathway. This may be associated with the reduced efficacy of Dara in patients with 1q+ MM. Ruxolitinib and tocilizumab can potentially restore CD38 expression by suppressing this pathway, thereby enhancing the effects of Dara on 1q+ MM.

Contrasting Features of T-Cell Expansion in Classical Hodgkin Lymphoma: Tumor Microenvironment Vs. Peripheral Blood and before Vs. during Anti-PD1 Treatment

Introduction The Tumor Microenvironment (TME) in classical Hodgkin Lymphoma (HL) contains abundant CD4+ and CD8+ T-cells and only few Hodgkin-Reed-Sternberg cells (HRSC). Despite their low abundance, HRSC comprise the neoplastic cell population that intensively interacts with cells of the TME. Understanding these interactions is crucial to the further development of immune checkpoint blockade (ICB) based treatment options. Clinical trials have shown high efficacy of ICB with anti-PD1 antibodies in relapsed HL and more recently promising results of anti-PD1 antibodies in combination with conventional chemotherapy were reported in the first-line setting. (NIVAHL trial, NCT03004833) In solid cancers, anti-PD1 ICB was shown to revert tumor-induced exhaustion of CD4+ and CD8+ T-cells, thereby enabling T-cell activation and a tumor-directed immune response. Since HL differs in many aspects from non-lymphoid tumors due to e.g. loss of HLA-expression, the exact mechanisms of action of ICB in HL is not fully understood and T-cell expansion after anti-PD1 first-line treatment not yet studied. Methods To characterize T-cell activation at different timepoints and to investigate a possible T-cell mediated immune response in HL, we analyzed T-cell Receptor (TCR) repertoires of the NIVAHL study cohort before (tissue: n=90; blood: n=9) and during anti-PD1 treatment (tissue: n=4; blood: n=9). The final cohort comprised additional TCR repertoires of in-house tissue biopsies of treatment-naïve HL (n=18), relapsed HL after chemotherapy (HL-R; n=18)) and publicly available TCR repertoires of the blood of treatment-naïve HL (n=11), HL-R (n=20) and HL-R patients during anti-PD1 treatment (n=20). (Cader et al. Nat Med. 2020 Sep;26(9):1468-1479) TCR repertoires of in-house reactive lymph nodes (n=8, healthy control) and breast cancer (BC; n=6, positive control) patients served as controls for T-cell activation in the tissue, publicly available TCR repertoires of CMV- (n=22, healthy control) and CMV+ otherwise healthy (n=9, positive control) people as controls in the blood. (De Neuter et al. Genes Immun. 2019 Mar;20(3):255-260) TCR repertoires were sequenced by Adaptive Biotechnologies. For each sample, TCR sequences with the same amino acid sequence were aggregated by the sum of their counts. We computed three measures to describe T-cell expansion: debiased Simpson's Clonality (dSC), Percentage of Singletons (PoS), and clonal expansion. Singletons have recently been defined as TCR sequences detected only once in a given sample. Clonal expansion is computed as the percentage of TCR sequences that increase their frequency in a patient from one timepoint to a following by ≥ 2. Results In tissue biopsies, treatment-naïve HL showed a significantly lower dSC compared to treatment-naïve BC and significantly higher dSC compared to RLN from healthy controls. No significant differences could be observed comparing HL with different treatments (during anti-PD1 vs. after chemotherapy) and treatment-naïve HL vs. HL during anti-PD1 treatment. HL and HL-R biopsies showed a significantly lower clonal expansion of Non-Singletons than BC tumors. In peripheral blood, healthy controls, treatment-naïve HL and HL patients during anti-PD1 treatment showed significantly lower dSC in their CD4+ T-cells compared to CMV+ controls and also compared to HL-R patients. We identified significantly higher dSC in CD8+ T-cells compared to CD4+ T-cells in the blood of HL patients before and during any treatment and in relapsed HL. During anti-PD1 treatment, CD8+ T-cells showed a significantly higher clonal expansion of Non-Singletons than CD4+ T-cells in HL-R and a similar but not significant trend in HL. All observed differences in dSC were significant for the PoS too, but as expected by their respective definitions with an opposing pattern (i.e. dSC high = PoS low). Discussion We did not observe features of intratumoral T-cell expansion in primary HL samples, early during anti-PD1 treatment or at relapse after chemotherapy, suggesting that within the HL tissue, T-cell expansion is hampered. However, patterns of T-cell expansion differed between TME and the peripheral blood, where we observed features of CD8+ T-cell clonal expansion, already prior to any treatment. In summary, our findings suggest a possible anti-tumor immune response of CD8+ T-cells in the peripheral blood of HL patients, that is not found in the TME.

PCNEO, a New Proficiency Testing Program for Flow Cytometric Analysis of Plasma Cell Neoplasms From the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee.

In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program-PCNEO-to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group. To report the results from the first 4 years of the PCNEO program. Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members. A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%-100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%-97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic κ and λ light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%-5.0% clonal plasma cells) and the presence of nonneoplastic plasma cell populations (correctly identified by 91%-96% of participants). Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm.

DUAL PET IMAGING OF NEOPLASTIC AND INflAMMATORY BIOMARKERS IN HIGH GRADE GLIOMA

Abstract AIMS To characterize spatial distributions of 18kDa translocator protein (TSPO) and amino acid uptake as measured through dynamic [11C] (R)-PK11195 and [11C]-methionine PET in high-grade glioma. METHOD Twelve patients with newly diagnosed high-grade glioma underwent dual PET studies. [11C] (R)-PK11195 binding potential (BPND) maps were generated using simplified reference tissue model with grey-matter cerebellar time- activity-curve as tissue input-function. [11C]-methionine uptake was calculated as tumour-to-background ratio (TBR). Volumes of interest (VOIs) were defined on T1W post-contrast MRI as contrast-enhancing (CE) tumour and a ‘peritumoural’ region 5mm from the CE edge. Voxel-wise comparison of the tracers used Pearson’s correlation coeffcient. VOIs were furthermore subdivided into higher/lower TSPO (BPND > 0.17) and higher/lower methionine (TBR > 1.7). RESULTS Within the CE, voxel-wise comparison showed a strong correlation (r > 0.7) in eight subjects, moderate correlation (r > 0.5) in three subjects and weak correlation (r < 0.5) in one subject. Volume (mm3) with high BPND/high TBR accounted for 62±29% whereas high BPND/low TBR accounted for 13±16%. Within the peritumoral area, there was a small reduction in the proportion of subjects in the strong correlation group (6/12) but the pro- portion of voxels with higher TSPO binding and lower methionine uptake was substantially greater (35±16%). CONCLUSIONS Within high-grade glioma, there is a strong overall correlation between TSPO expression and amino acid uptake. However, in the peritumoural region there are areas of elevated TSPO expression without increased methionine uptake which could represent an inflammatory component or a discrete neoplastic cell population that may not be adequately defined with standard imaging biomarkers.

Systemic mastocytosis associated with acute myeloid leukaemia

Bacground: The pathogenesis of systemic mastocytosis with associated haematological neoplasm (SMAHN) is not well understood. Both diagnoses heavily rely on morphological evaluation because SM is rarely suspected in clinical practise. Objective: This case highlighted a possible delay in diagnosis due to underlying conditions or diseases. Case Report: We present a case of a patient with acute myeloid leukaemia (AML), acute myelomonocytic subtype, and concurrent mastocytosis according to World Health Organization (WHO) classification. Due to an extensive accumulation of AML blast cells that obscured the mast cell infiltrates, mastocytosis was not evident at the first diagnosis. Discussion and conclusion: The diagnosis, in this case, was established only in the third bone marrow biopsy after chemotherapy. A high index of suspicion with morphological and immunohistochemical evaluations for neoplastic mast cell populations should be considered. The optimal treatment approach should be chosen based on these two disease entities. Bangladesh Journal of Medical Science Vol. 22 No. 04 October’23 Page : 937-941

Vulval apocrine adenocarcinoma with tumour-associated eosinophilia in a cat

A 10-year-old, spayed female, Domestic Shorthaired cat was referred for surgical removal of a mass on the left vulval fold. An impression smear revealed mixed cell inflammation, with eosinophils predominating focally, and a concurrent bacterial infection, suggesting a primarily inflammatory lesion. However, cytology of a fine-needle aspirate of the mass revealed a neoplastic epithelial cell population, confirmed on histopathology as an apocrine vulval adenocarcinoma with lymphatic invasion and marked tumour-associated tissue eosinophilia. One month after surgical excision of the mass, the cat developed inguinal metastatic lymphadenopathy and chemotherapy was initiated. The patient ultimately developed marked peripheral lymphadenomegaly and was euthanized due to concerns for overall quality of life and comfort. This case highlights that neoplasia should be a consistent differential diagnosis for eosinophilic infiltrates/inflammation. The distinct appearance of the two cytological samples in this case stresses the need for sampling of different sites of a lesion and the importance of not relying on superficial impression smears for clinical management and prognosis.

A Study to Assess the Effect of Structured Teaching Programme on Knowledge of Patients with Acute Myeloid Leukaemia on Chemotherapy Schedule, Side Effects and its Management at a Tertiary Cancer Centre

Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder characterized by a block in differentiation of hematopoiesis, resulting in growth of a clonal population of neoplastic cells or blasts. Chemotherapeutics leads to side effects. To minimize potential toxicities and to maximize the optimal quality of life, nurse can assist the patient by providing instructions and making the care provider equipped with knowledge. The aim of the study was to assess the knowledge regarding chemotherapy, its side effects and its management in patients with AML and to assess the effect of structured teaching programme in experimental and control group. Quantitative, quasi-experimental pretest posttest control group design study was done It was found that there is significant improvement in posttest knowledge score in experimental group (P value -0.000) and number of participants correctly answered to the items in questionnaire increased after the structured teaching program in experimental group.

Phosphorylated STAT3 as a potential diagnostic and predictive biomarker in ALK- ALCL vs. CD30high PTCL, NOS.

The differential diagnosis between ALK-negative anaplastic large cell lymphoma (ALK- ALCL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) with high expression of CD30 (CD30high) are essential. However, no reliable biomarker is available in daily practice except CD30. STAT3 is characteristically activated in ALCL. We aimed to investigate whether the status of STAT3 phosphorylation could help the differential diagnosis. The status of phosphorylation of STAT3 was examined using two antibodies against pSTAT3-Y705 and pSTAT3-S727 by immunohistochemistry in ALK+ ALCL (n=33), ALK- ALCL (n=22) and PTCL, NOS (n=34). Ten PTCL, NOS with diffuse CD30 expression were defined as CD30high PTCL, NOS. Flowcytometric analysis were performed to evaluate the expression of pSTAT3-Y705/S727 in PTCL, NOS (n=3). The median H-scores of pSTAT3-Y705 and S727 were 280 and 260 in ALK+ ALCL, 250 and 240 in ALK- ALCL, and 45 and 75 in CD30high subgroup, respectively. Using H score of 145 as the cutoff value, pSTAT3-S727 alone distinguished between ALK- ALCL and CD30high PTCL, NOS with a sensitivity of 100% and specificity of 83%. Additionally, pSTAT3-S727, but not pSTAT3-Y705, was also expressed by background tumor-infiltrating lymphocytes (S727TILs) in PTCL, NOS. PTCL, NOS patients with high S727TILs H score had a favorable prognosis than those with no TILs (3-year OS rate: 43% vs. 0, p=0.013) or low S727TILs (3-year OS rate: 43% vs. 0, p=0.099). Flowcytometric analysis revealed that of the three patients investigated, two had enhanced pSTAT-S727 signals in neoplastic cell populations, and all three patients were negative for pSTAT3-Y705 expression in both tumor cells and background lymphocytes. pSTAT3-Y705/S727 can be used to help distinguish ALK- ALCL from CD30high PTCL, NOS and pSTAT3-S727 expression by TILs predicts the prognosis of a subset of PTCL, NOS.

Temozolomide: a cytostatic drug that is still important today

TMZ has an advantage over other traditional alkylating agents (carmustine, lomustine, procarbazine), which are highly toxic and have poor patient survival. TMZ circumvents these problems because cytochrome P450 enzymes and the kidneys are not involved in its metabolism, it has predictable side effects (nausea, vomiting, thrombocytopenia, neutropenia), which are usually reversible and only mild to moderate, have been widely described. About half of patients treated with TMZ have high drug resistance induced by activity of O6-methylguanine methyltransferase. Cancer stem cells (CSCs), which are found among the neoplastic cell population, have also been shown to be responsible for resistance to TMZ. Additionally, acquired immunity, induced by TMZ’s epigenetic and genetic alterations, may develop. Currently, there are new therapeutic strategies for GBM based on nanotechnology, which are aimed at improving TMZ treatment (e.g. the use of apolipoprotein), or other techniques (siRNA, which increases the oxygen level in the tumour). Thus, although TMZ was discovered more than three decades ago, this drug will be used to treat not only GBM, but also a large number of neoplastic pathologies. Further research focused on understanding the mechanisms of action and resistance to TMZ is required to improve its clinical application today and in the future. Keywords: alkylating agents, drug resistance, chemotherapy, nanoparticles, cancer, glioblastoma multiforme

GBMdeconvoluteR accurately infers proportions of neoplastic and immune cell populations from bulk glioblastoma transcriptomics data.

Characterizing and quantifying cell types within glioblastoma (GBM) tumors at scale will facilitate a better understanding of the association between the cellular landscape and tumor phenotypes or clinical correlates. We aimed to develop a tool that deconvolutes immune and neoplastic cells within the GBM tumor microenvironment from bulk RNA sequencing data. We developed an IDH wild-type (IDHwt) GBM-specific single immune cell reference consisting of B cells, T-cells, NK-cells, microglia, tumor associated macrophages, monocytes, mast and DC cells. We used this alongside an existing neoplastic single cell-type reference for astrocyte-like, oligodendrocyte- and neuronal progenitor-like and mesenchymal GBM cancer cells to create both marker and gene signature matrix-based deconvolution tools. We applied single-cell resolution imaging mass cytometry (IMC) to ten IDHwt GBM samples, five paired primary and recurrent tumors, to determine which deconvolution approach performed best. Marker-based deconvolution using GBM-tissue specific markers was most accurate for both immune cells and cancer cells, so we packaged this approach as GBMdeconvoluteR. We applied GBMdeconvoluteR to bulk GBM RNAseq data from The Cancer Genome Atlas and recapitulated recent findings from multi-omics single cell studies with regards associations between mesenchymal GBM cancer cells and both lymphoid and myeloid cells. Furthermore, we expanded upon this to show that these associations are stronger in patients with worse prognosis. GBMdeconvoluteR accurately quantifies immune and neoplastic cell proportions in IDHwt GBM bulk RNA sequencing data and is accessible here: https://gbmdeconvoluter.leeds.ac.uk.

What is your diagnosis? Perineal swelling in a dog.

What is your diagnosis? Perineal swelling in a dog.

The Application of the SORE6 Reporter in the Studies of Acute Myeloid Leukemia - a Novel Model for Disease Relapses

Most patients diagnosed with acute myeloid leukemia (AML) suffer from disease relapses within two years after the initial remission. Treatment options for relapsed AML are limited and the clinical outcome associated with this stage of disease is dismal. Partly due to the lack of experimental models, the biology underlying AML relapses is not well understood. We hypothesized that the cancer stem-like (CSL) cells, which represent a minority of the neoplastic cell population, are the major contributors of AML relapses. SORE6, a reporter designed to detect the expression and activity of inducible pluripotent stem cell factors, was used to identify/purify CSL cells in two FLT3-mutated AML cell lines, MOLM-13 and MV4-11. Both cell lines contained ~10% of SORE6+ cells at the steady state, as revealed by their expression of the reporter green fluorescence protein. Compared to SORE6- cells, SORE6+ cells had significantly more spheroid formation in the hanging drop assay (75 vs 35%, p=.006), and were significantly more resistant to Ara-C (inhibitory concentration at 50%, 60 vs 25 nM, p=.0005) and Venetoclax (2.6 vs 8.2 nM, p=.002) in the presence of low-dose Ara-C. By western blots, SORE6+ cells were found to express substantially higher levels of Myc/FLT3/pSTAT5 than SORE6- cells. Myc appeared to be a key driver of SORE6 activity, and the reporter activity was effectively suppressed by Myc pharmacologic inhibitors or shRNA. By subjecting a mixture of bar-coded SORE6- and SORE6+ MOLM-13 cells (in 9:1 ratio to mimic that at the steady state) to the lowest concentration of Ara-C that resulted in no detectable viability in the cell culture, we consistently found evidence of cell regeneration after 8-12 days of undetectable cell viability. Regeneration initially occurred slowly, and the cell growth went into a log phase after 1-2 weeks. Using PCR to detect the relative proportion of bar-coded SORE6- and SORE6+ cells, we found that the regenerated cell population contained a substantially higher proportion of SORE6+ cells, with 64% SORE6+ cells in the initial slow-growth phase. In comparison, the proportion of cells originating from SORE6+ cells increased to an average of 86% during the log-growth phase. Importantly, cells in the log-growth phase were found to have a significantly higher resistance to Ara-C and had significantly more spheroid formation in the hanging drop assay compared to untreated cells. To further validate the concept that CSL cells are the key contributors to the AML relapse, we collected pairs of initially diagnostic and relapsed bone marrow specimens derived from the same patients. By immunocytochemistry, we found that relapsed AML blasts were more frequently positive for Myc, a surrogate marker of SORE6 activity, compared to those in the pre-treatment samples. To conclude, using SORE6, we have developed a technically simple experimental model that capitulates some of the key features of AML relapses. Our model has provided direct evidence that CSL cells are key contributors to the disease relapse in AML. We believe that this in-vitro model is valuable in testing the efficacy of new therapeutic agents against AML, especially those designed to target the CSL cell population.

Selected Articles from This Issue

PD-1T tumor-infiltrating lymphocytes (TIL) are a dysfunctional T-cell population characterized by high PD-1 expression and tumor reactivity. To evaluate their potential as a biomarker for durable clinical benefit to PD-1 blockade, Hummelink and colleagues digitally quantified PD-1T TIL in 120 pretreatment samples from patients with advanced non-small cell lung cancer. PD-1T TIL showed a high sensitivity in predicting clinical benefit and survival to anti-PD-1 and were superior to PD-L1 or TLS as a biomarker. Importantly, the absence of PD-1T TIL reliably identified a patient group without benefit from PD-1 blockade, thereby providing a promising tool to reduce overtreatment.Immune checkpoint inhibitors (ICI) targeting programmed cell death protein 1 and its ligand (PD-1/PD-L1) have revolutionized therapeutic options for renal cell carcinoma (RCC). However, response rates to ICI vary widely (20–70%) in patients with RCC. Predicting responses using conventional tissue-based techniques evaluating PD-L1 expression is of limited value, owing to the dynamic and heterogenous nature of PD-L1 expression. Mulgaonkar and colleagues systematically examined the performance of immunoPET for PD-L1 imaging relative to immunohistochemistry in an RCC tumorgraft platform. This study demonstrates the promising potential of immunoPET for clinical real-time assessment of PD-L1 expression across tumor sites and heterogeneous intratumoral distribution.CSF1R inhibitor treatments have shown to be effective targeted therapies for TGCT. Trials are currently underway to study the effect of discontinuation and retreatment with CSF1R inhibitors. In this study, van IJzendoorn and colleagues investigate the tumor landscape in TGCT using single-cell sequencing and other molecular techniques. They show that the neoplastic cells are not targeted by CSF1R inhibitors, in contrast to what was previously postulated. The authors identify markers for the neoplastic cells and show the lack of an autocrine loop involving CSF1-CSF1R in the neoplastic cells. As a result, CSF1R inhibitor treatments are unlikely to affect the neoplastic cell population in TGCT as is currently assumed. Additionally, this work identifies two new pathways that could be targeted in future trials involving TGCT.The resistance of immunotherapy in renal cancer patients remains unclear despite the high infiltrations of CD8+T cells. Here, Benhamouda and colleagues show that baseline plasma soluble CD27 (sCD27) is associated with resistance to immunotherapy in renal cell carcinoma independently of clinical factors. sCD27 reflects the interaction between CD27 expressed by T lymphocytes and CD70 expressed by tumor cells, which associates with the apoptosis and dysfunction of T lymphocytes. Peripheral blood sCD27 could thus be considered as a marker of T-cell dysfunction due to the CD27-CD70 interaction in the tumor and predict the response to immunotherapy.