Talia M. Saal

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity—defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers—had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

Diagnosing salivary gland tumors (SGTs) through fine-needle aspiration (FNA) biopsies is challenging due to the overlapping cytomorphologic features between benign and malignant tumors. The authors developed an innovative, multiplexed cycling technology for the rapid analyses of single cells obtained from FNA that can facilitate the molecular analyses and diagnosis of SGTs. Antibodies against 29 protein markers associated with 7 SGT subtypes were validated and chemically modified via custom linker-bio-orthogonal probes (FAST). Single-cell homogenates and FNA samples were profiled by FAST cyclic imaging and computational analysis. A prediction model was generated using a training set of 151,926 cells from primary SGTs (N = 26) and validated on a separate cohort (N = 30). Companion biomarker testing, such as neurotrophic tyrosine receptor kinase (NTRK), was also assessed with the FAST technology. The FAST molecular diagnostic assay was able to distinguish between benign and malignant SGTs with an accuracy of 0.86 for single-cell homogenate samples and 0.88 for FNA samples. Profiling of multiple markers as compared to a single marker increased the diagnostic accuracy (0.82 as compared to 0.65-0.74, respectively), independent of the cell number sampled. NTRK expression was also assessed by the FAST assay, highlighting the potential therapeutic application of this technology. Application of the novel multiplexed single-cell technology facilitates rapid biomarker testing from FNA samples at low cost. The customizable and modular FAST-FNA approach has relevance to multiple pathologies and organ systems where cytologic samples are often scarce and/or indeterminate resulting in improved diagnostic workflows and timely therapeutic clinical decision-making.

Introduction Myeloproliferative neoplasms are considered mutually exclusive, and reports of co-occurring mutations are rare. Over the past 10 years, approximately two dozen case reports have been published of co-occurring BCR-ABL positive and CALR positive patients. Here we present the case of a patient who was reported to have myelofibrosis (MF)/myeloproliferative neoplasm (MPN), later developed chronic myeloid leukemia (CML), and ultimately progressed to acute myeloid leukemia (AML). This is unique in the literature reviewed, and may be the first reported case of this overlap syndrome being treated with asciminib. Case presentation A 40-year old male was diagnosed in 2018 with MF/MPN based upon a hypercellular bone marrow biopsy with diffuse reticulin fibrosis. He was negative for JAK2, but was not tested for CALR or MPL mutations. Treatment was ruxolitinib and hydroxyurea. Two years later, the patient was diagnosed with CML after being admitted for recurrent fevers, headache, and shortness of breath. At that time, a type 2 CALR mutation (CALR K385Nfs*47) was noted. He started dasatinib and decitabine, which resulted in a drop from 28.31% BCR-ABL to 0.0196% (Table 1A). However, hyperuricemia and renal failure led to a change in regimen and the patient was started on asciminib and ropeginterferon alfa-2b-njft in January 2022. By December, the patient's BCR-ABL PCR dropped below quantification, but he received a diagnosis of AML based on a bone marrow biopsy which showed 43% blasts. He received cladribine and cytarabine, but was found to have leukemic infiltration of the spinal cord five months later. The patient was referred to hospice and passed away in June 2023. Discussion To our knowledge, this is the first MPN-CML patient reported to have been treated with asciminib. This case illustrates its effectiveness in treating CML, but highlights the need for improved treatments for CALR+ MPN. Moreover, this case illustrates the importance of testing for CALR, MPL, and BCR-ABL in addition to JAK2 immediately when a patient presents with an MPN. It is unknown whether this patient's CALR mutation preceded the BCR-ABL translocation, and there is debate in the literature with respect to mutation chronology. The patient's BCR-ABL burden dropped from 28.31% to 0.0196% while the CALR burden remained around 94% over the same period (Table 1B). Thus, it is unlikely that the BCR-ABL mutation preceded the CALR mutation within the same clone. The patient's >90% CALR allele burden is higher than reported in the other reported cases, where the burden ranged from 10% to 68%. Furthermore, none of the other cases were reported to progress to AML, though follow up data is lacking. The heavy allelic burden in our patient may have been a contributing factor to the development of AML, as has been reported elsewhere. The patient's overall mutational profile is of interest. Type 2 CALR mutations have been reported to have less favorable prognoses than type 1 mutations. In addition to the CALR and BCR-ABL mutations, the patient had ASXL1 A861Hfs*6, EZH2 D730*, and NRAS G12V. All three mutations are associated with unfavorable outcomes in MDS, AML, and/or CML. Genomic instability has been discussed in relation to both CML and JAK2-positive MPN. Our patient's heavy mutational burden suggests CALR-positive neoplasms may also present against a background of inherent genomic instability. Conclusion We reported a case of MPN, followed by CML, and then AML. The patient was positive for CALR and BCR-ABL. He achieved a complete response in CML after targeted treatment, with asciminib consolidating the gains the patient made on earlier therapies. However, he always maintained a high CALR burden. This patient's mutational profile and disease course argue for the importance of early testing for CALR and BCR-ABL in addition to JAK2 and MPL in the setting of an MPN and for further research into the underlying causes of these overlap conditions.

Introduction: For the past several decades the standard of care treatment for frontline therapy for acute myeloid leukemia (AML) in individuals that are eligible for intensive induction therapy has been 7 + 3 (7 days of cytarabine (Ara-C) + 3 days of danuorubicin). Older individuals with AML and those that are ineligible for intensive induction tend to be a more difficult population to treat. Since 2018, venetoclax + hypomethylating agents (HMA) (azacitidine or decitabine) has been considered the standard of care frontline treatment for AML in those who are induction ineligible. Over the past decade there have been significant improvements in genetic and molecular testing that have greatly impacted the development of new frontline treatment therapies for individuals with acute myeloid leukemia (AML) with promising treatment outcomes. Methods: In an effort to quantify the impact that these evolving therapies have had on influencing leukemia treatment practices, a questionnaire was sent to every director/chair of Leukemia at each National Cancer Institute (NCI) designated center. Our two-question questionnaire centered around determining the preferred AML treatment for both patients who are not on clinical trials that are eligible and those that are ineligible for intensive induction therapy. Questions were created based on the 2019 NCCN clinical practice guidelines for AML. The survey was limited to 2 questions so as to elicit higher response rate to the questionnaire. Question #1: What is the preferred induction regimen backbone used at your center - In patients eligible for intense chemo, off-study? 7+3FLAG-IDACLAG-IDAVenetoclax w/ 5-azacytidine or decitabineOther Question #2: What is the preferred induction regimen backbone at your center - In patients who are ineligible for intensive chemo, off-study, and having actionable mutations (IDH or FLT3)? Venetoclax + Hypomethylating agentTargeted agent against IDH or FLT3 withoutvenetoclax +/- hypomethylating agentTargeted agent against IDH or FLT3 withvenetoclax +/- hypomethylating agentOther Results: From our study, we received 31/63 (49.2%) responses and saw that for the intensive induction 90.3% (28/31) respondents selected 7 +3 as the preferred treatment with 3.2% (1/31) choosing FLAG-IDA, with the remaining 6.5% (2/31) choosing “other” ((7 +3) w/ FLT3 inhibitor). Comparatively, for the intensive induction ineligible treatment, 61.3% (19/31) respondents preferred venetoclax + HMAs with 38.7% (12/31) preferring targeted agent w/ venetoclax +/- HMA. Of note, four respondents selected venetoclax + HMA as the preferred treatment, but also preferred aza-ivosidenib for IDH1 mutant AML. Conclusion: Preliminary findings from our survey suggest that results from genetic molecular testing may have a greater influence on the evolution in treatment for the intensive induction ineligible population. As more data comes out regarding combining targeted therapy with more broad-based treatments, clarity will emerge on the most appropriate standard-of-care approach. Additionally, improved turnaround time of testing methodology will likely modify treatment approaches. Our next steps include repeat outreach to obtain 100% response rate and sending a follow-up questionnaire to understand the rational for each specific treatment choice.