Viable Tumor Cells Research Articles

84P SHR-1316 vs placebo in combination with chemotherapy as perioperative treatment in patients with resectable stage II-III NSCLC: A randomized, double-blind, multicenter, phase Ib/III trial

SHR-1316 is a humanized IgG4 monoclonal PD-L1 antibody which has shown promising antitumor activity in solid tumors. This phase 1b/3 trial evaluates the efficacy and safety of SHR-1316 plus chemotherapy vs placebo plus chemotherapy as perioperative treatment for resectable NSCLC. Here, we reported the results from the phase 1b study. Eligible patients were resectable stage II and III (IIIA and T3N2M0 IIIB) NSCLCs without EGFR/ALK alterations. Patients received neoadjuvant treatment with 3 cycles of intravenous SHR-1316 (20 mg/kg on day 1), nab-paclitaxel (100 mg/m2 on days 1, 8, and 15), and carboplatin (area under the curve 5, 5 mg/mL per min on day 1), of each 21-day cycle, followed by surgical resection. After that, patients further received 16 cycles of SHR-1316 (20 mg/kg on day 1) adjuvant treatment. The primary endpoint was major pathological response (MPR) per blinded independent pathological review (BIPR), defined as ≤10% viable tumor cells in resection specimen. If MPR was >55% then phase 3 trial will be initiated. From Jul 14, 2020 to May 12, 2021, 37 patients received SHR-1316 plus chemotherapy neoadjuvant treatment and 34 patients underwent surgery. As of data cutoff on Nov 26, 2021, 19 (55.9%, 95% CI 39.5-71.1) of the 34 patients underwent surgery achieved MPR per BIPR and 11 (32.4%, 95% CI 19.1-49.2) patients achieved pathological complete response. Of the 37 patients received neoadjuvant treatment, 26 (70.3%, 95% CI 54.2-82.5) patients had an objective response per RECIST v1.1, including 1 with complete response and 25 with partial response. Treatment-related adverse events (AEs) were reported in 37 (100%) patients. 29 (78.4%) patients had grade ≥3 treatment-related AEs and 9 (24.3%) had treatment-related serious AEs. No treatment-related deaths occurred. Grade ≥3 surgery-related AEs within 30 or 90 days after surgery were both reported in 6 (17.6%) of the 34 patients. SHR-1316 in combination with nab-paclitaxel and carboplatin as neoadjuvant therapy showed a high proportion of MPR in resectable NSCLC, and the safety profile was well tolerated. Based on the phase 1b results, the phase 3 trial was initiated and ongoing.

Prognostic impact of inter-metastatic heterogeneity of viable tumour cells in colorectal liver metastases

BackgroundHeterogenous response to neoadjuvant chemotherapy in patients with multiple colorectal liver metastases (CRLM) has been associated with an acquired resistance to systemic therapy. This study evaluated the occurrence of a heterogenous inter-metastatic tumour response with regards to the proportion of viable tumour cells, and its prognostic impact. MethodsA retrospective cohort study was conducted, including all patients with CRLM surgically treated at Karolinska University Hospital, Stockholm, Sweden, from 2013 to 2018. Factors associated with the proportion of viable tumour cells and inter-metastatic heterogeneity were analysed with regression and survival analyses. ResultsOut of 640 surgically treated patients, 405 patients (1357 CRLM), received neoadjuvant chemotherapy. Multiple CRLM were present in 314 patients (78%), out of whom 72 patients (23%) presented with a heterogenous tumour response. The median overall survival (OS) for patients with a heterogenous inter-metastatic tumour response was 36 months, compared to 57 months for patients with a homogenous inter-metastatic tumour response (p < .001). Poor OS in patients receiving preoperative chemotherapy was significantly associated with a heterogenous inter-metastatic tumour response (hazard ratio (HR) 1.68 (1.02–2.78)), right-sided primary tumour (HR 2.01 (1.29–3.43)) and CRLM diameter >5 cm (HR 1.83 (1.06–3.17)). ConclusionOutcome in patients with a heterogenous inter-metastatic tumour response, illustrated by the proportion of viable tumour cells, is inferior to that of patients with a homogenous response. These results suggest that heterogeneity in treatment response is an important marker of aggressive disease and could be of clinical value for decisions on post-operative therapy.

Novel potent azetidine-based compounds irreversibly inhibit Stat3 activation and induce antitumor response against human breast tumor growth in vivo

Signal transducer and activator of transcription (Stat)3 is a valid anticancer therapeutic target. We have discovered a highly potent chemotype that amplifies the Stat3-inhibitory activity of lead compounds to levels previously unseen. The azetidine-based compounds, including H172 (9f) and H182, irreversibly bind to Stat3 and selectively inhibit Stat3 activity (IC50 0.38–0.98 μM) over Stat1 or Stat5 (IC50 > 15.8 μM) in vitro. Mass spectrometry detected the Stat3 cysteine peptides covalently bound to the azetidine compounds, and the key residues, Cys426 and Cys468, essential for the high potency inhibition, were confirmed by site-directed mutagenesis. In triple-negative breast cancer (TNBC) models, treatment with the azetidine compounds inhibited constitutive and ligand-induced Stat3 signaling, and induced loss of viable cells and tumor cell death, compared to no effect on the induction of Janus kinase (JAK)2, Src, epidermal growth factor receptor (EGFR), and other proteins, or weak effects on cells that do not harbor aberrantly-active Stat3. H120 (8e) and H182 as a single agent inhibited growth of TNBC xenografts, and H278 (hydrochloric acid salt of H182) in combination with radiation completely blocked mouse TNBC growth and improved survival in syngeneic models. We identify potent azetidine-based, selective, irreversible Stat3 inhibitors that inhibit TNBC growth in vivo.

Protective and Therapeutic Efficacy of Hesperidin versus Cisplatin against Ehrlich Ascites Carcinoma-Induced Renal Damage in Mice.

This study evaluates the antitumor efficacy of hesperidin (Hesp) versus cisplatin (Cis) in Ehrlich ascites carcinoma (EAC)-bearing mice, as well as its protective effect against Cis-triggered nephrotoxicity. Seventy female mice were allocated into control, Hesp, EAC, Hesp-protected, Hesp-treated, Cis-treated, and Cis+Hesp-treated groups. The inoculation of mice with EAC cells significantly reduced the mean survival time, while significantly increased the body weight, abdominal circumference, ascitic fluid volume, viable tumor cell count, and serum carcinoembryonic antigen, urea and creatinine levels, besides various hematological changes. Additionally, kidney tissue of EAC-bearing mice showed a significant increase in the malondialdehyde level, significant decreases in the reduced glutathione content and catalase activity, marked pathological alterations, and a strong Ki-67 expression with a weak caspase-3 expression in neoplastic cells infiltrating the renal capsule. Conversely, the administration of Hesp and/or Cis to the EAC-bearing mice induced, to various degrees, antitumor responses and alleviated the cytotoxic effects of EAC. In addition to the potent antitumor effect of the concomitant administration of Hesp and Cis, Hesp minimized the renal adverse side effects of Cis. In conclusion, Hesp may open new avenues for safe and effective cancer therapy and could be valuable for enhancing the antitumor potency and minimizing the renal adverse side effects of chemotherapeutic drugs.

Indocyanine Green Labeling of Tumors in the Liver Recurring After Radiofrequency Ablation Enables Complete Resection by Fluorescence-guided Surgery.

Radiofrequency ablation (RFA) is used to treat primary and metastatic tumors in the liver. However, local recurrence after RFA is frequent and subsequent salvage hepatectomy is often ineffective due to difficulty in visualization of tumor margins. In the present retrospective clinical trial, seven patients from the Department of General and Gastro-enterological Surgery, Showa University School of Medicine underwent salvage hepatectomy for recurrent hepatocellular carcinoma (HCC) (n=2), colorectal liver metastasis (n=4) and lung-carcinoid liver metastasis (n=1), after RFA, between 2011 and 2020. Tumors were labeled with indocyanine green (ICG) and resected under fluorescence guidance. Resected specimens were evaluated under fluorescence microscopy as well as by standard histopathological techniques. Pathological findings revealed negative tumor margins in all patients after fluorescence-guided surgery. Six of seven resected tumors had a fluorescent rim, including both HCC and liver metastasis. Fluorescence microscopy demonstrated that viable cancer tumor cells were located only on the inside of the fluorescent rim, and no malignant cells were detected within the fluorescent rim surrounding the tumor. Fluorescence microscopy showed that the tumor margin was secured if the fluorescence signal was completely resected. The present results demonstrate that ICG labeling of liver tumors recurring after RFA enabled complete resection under fluorescence guidance. The present study is the first clinical study to demonstrate that tumor types that generally cannot be completely resected with bright light are fully resectable under fluorescence guidance.

Tissue Contamination During Transportation of Formalin-Fixed, Paraffin-Embedded Blocks.

Tissue carryovers are contaminants of surgical pathology cases in which extraneous tissue is incorporated into tissue blocks. Carryovers occur most frequently at the grossing or embedding stations, but little is published about them. We sought to analyze their transmission during transit to the histology lab. Cassettes of friable donor tissue were mixed with cassettes of spongy recipient tissue in formalin-filled containers and agitated by shipment via pneumatic tube. The tissue cassettes were processed, embedded as blocks, and cut as usual. Liquid samples were prepared from the submission containers as well as from workstation submission containers and histology tissue processor waste. A high rate of contamination (14.9%) was observed under these artificial conditions. Friable donor tissue, including urothelium and colorectal adenocarcinoma, were promiscuous contaminants, as were placental villi. Fluid from submission containers showed viable tumor cells and fragments, which were also present in workstation submission containers and in tissue processor waste fluid. This study implicates liquid transport media as a possible avenue of contamination during submission and transportation of tissue cassettes for histologic processing. Attention should be given to the friability of submitted tissue and physical agitation of the cassettes in transit. Such contaminants may be present in the fluid in tissue submission bins and in tissue processor fluid.

Histopathology and surgical outcome of symptomatic treatment-related changes after gamma knife radiosurgery in patients with brain metastases

A late-onset treatment-related changes (TRCs), which represent radiographic radiation necrosis (RN), frequently occur after stereotactic radiosurgery (SRS) for brain metastases and often need surgical treatment. This study aimed to validate the true pathology and investigate clinical implication of surgically resected TRCs on advanced magnetic resonance imaging (MRI). Retrospective analyses of 86 patients who underwent surgical resection after radiosurgery of brain metastases were performed. Fifty-four patients displayed TRCs on preoperative MRI, comprising pure RN in 19 patients (TRC-RN group) and mixed viable tumor cells in 35 patients (TRC-PD group). Thirty-two patients revealed the consistent diagnosis of progressive disease in both MRI and histopathology (PD–PD group). The TRC-PD group showed larger prescription isodose volume (9.4 cm3) than the TRC-RN (4.06 cm3, p = 0.014) group and a shorter time interval from SRS to preoperative MRI diagnosis (median 4.07 months) than the PD–PD group (median 8.77 months, p = 0.004). Progression-free survival was significantly different among the three groups (p < 0.001), but not between TRC-RN and TRC-PD (post hoc test, p = 1.00), while no difference was observed in overall survival (p = 0.067). Brain metastases featured as TRCs after SRS frequently contained viable tumor cells. However, this histologic heterogeneity had a minor impact on benign local prognosis of TRCs after surgical resection.

Analysis of disease-free survival in CheckMate 274 by PD-L1 combined positive score and tumor proportion score.

491 Background: CheckMate 274 demonstrated a significant improvement in disease-free survival (DFS) with nivolumab (NIVO) versus placebo (PBO) both in the intent-to-treat population (hazard ratio [HR], 0.70; 98.22% confidence interval [CI], 0.55–0.90; P &lt; 0.001) and in patients (pts) with tumor programmed death ligand 1 (PD-L1) expression ≥ 1% assessed by the tumor proportion score (TPS) (HR, 0.55; 98.72% CI, 0.35–0.85; P &lt; 0.001). An exploratory subgroup analysis showed a trend toward a DFS benefit with NIVO in pts with TPS &lt; 1% (0.82; 95% CI, 0.63–1.06). To further characterize the relationship between PD-L1 expression and NIVO efficacy, we report an analysis of DFS based on PD-L1 expression in both tumor and immune cells using the combined positive score (CPS). Methods: CheckMate 274 is a phase 3, randomized, double-blind, multicenter trial of NIVO versus PBO in pts with high-risk muscle-invasive urothelial carcinoma after radical surgery. Pts were randomized 1:1 to NIVO 240 mg or PBO every 2 weeks intravenously for 1 year of adjuvant treatment. The primary endpoints of the study are DFS in the intent-to-treat population and in pts with TPS ≥ 1%. The Dako PD-L1 IHC 28-8 pharmDx assay was used to evaluate TPS. CPS was determined retrospectively from previously stained immunohistochemistry slides using the CPS algorithm. CPS was calculated as the number of both PD-L1 positive tumor and immune cells divided by the number of viable tumor cells in the evaluable tumor area, multiplied by 100; TPS was similarly calculated with the number of PD-L1 positive tumor cells as the numerator. This analysis only included pts with both quantifiable CPS and TPS. Results: Of the 629 pts with quantifiable TPS and CPS, 249 (40%) had TPS ≥ 1% (NIVO, n = 124; PBO, n = 125), 380 (60%) had TPS &lt; 1% (NIVO, n = 191; PBO, n = 189), 557 (89%) had CPS ≥ 1 (NIVO, n = 281; PBO, n = 276), and 72 (11%) had CPS &lt; 1 (NIVO, n = 34; PBO, n = 38). Within TPS &lt; 1% pts, 81% (n = 309) had CPS ≥ 1. The number of pts and the DFS outcomes in pts with TPS ≥ 1% and CPS ≥ 1 are shown in the Table. In pts with TPS &lt; 1% who also had CPS ≥ 1, median DFS (95% CI) was 19.2 (15.6–33.4) months with NIVO versus 10.1 (8.2–19.4) months with PBO. The HR for NIVO versus PBO in these pts was 0.73 (95% CI, 0.54–0.99). Conclusions: This exploratory analysis of PD-L1 expression by CPS showed a higher proportion of pts with CPS ≥ 1 than TPS ≥ 1%, and that most pts with TPS &lt; 1% had CPS ≥ 1. In the CPS ≥ 1 subgroup, median DFS with NIVO was more than double that with placebo. These results support the conclusion that pts with TPS &lt; 1% also benefit from adjuvant NIVO. Clinical trial information: NCT02632409. [Table: see text]

Investigating treatment effects in the microenvironment of renal cell cancer with venous tumor thrombus: Translational analysis of the NAXIVA trial.

368 Background: 10-15% of patients with renal cell carcinoma (RCC) develop venous tumour thrombus (VTT). The presence of VTT makes curative surgery technically challenging, with high peri-operative mortality. VTT contains renal cancer cells that are genetically similar to the parent tumour, but less is known about the non-cancer cell components of the VTT microenvironment. The NAXIVA trial demonstrated that neoadjuvant axitinib can reduce the extent of VTT, with 7/20 patients showing an improvement in VTT level by Mayo classification. This provided the first level II evidence for the use of axitinib in this setting. Non-responders had lower microvessel density and exhausted T cell phenotypes in baseline biopsies. NAXIVA offers a unique opportunity to study the VTT microenvironment and the biological response to axitinib in humans to inform treatment combinations in future trials. Methods: NAXIVA (NCT03494816) was a single arm, multi-centre, UK based phase 2 study. 20 patients with resectable clear cell RCC were treated with up to 8 weeks of axitinib prior to surgery. Translational samples were taken at baseline and at time of surgery. Additional untreated VTT and primary tumour samples were obtained from the ARTIST study. Samples were analysed by immunofluorescence microscopy and RNA sequencing. Results: Untreated VTT has a complex microenvironment. There are established CD31+ microvessels surrounded by SMA+ pericytes and stromal cells. Extensive immune infiltrates are present, including CD8+ and CD4+ T cells, CD68+ macrophages and MPO+ neutrophils. Consistent with previous studies, we find high levels of viable tumour cells in VTT as marked by CA9 and Ki67. Following 8 weeks of axitinib treatment there was a significant reduction in microvessel density in both the primary tumour and VTT, consistent with the mechanism of action of axitinib. This was particularly seen in a CD34+ subset of vessels, which we hypothesise marks younger vessels that may be more sensitive to axitinib. There were no clear changes in T cell or myeloid cell densities. However, Gene Ontology analysis of RNA-seq data showed upregulation of myeloid inflammation signatures upon treatment, which suggests there may be functional changes in the myeloid cells. We are investigating the significance of these genes in more detail. Conclusions: The VTT microenvironment is highly organised and resembles the parent tumour. The complex nature of the VTT microenvironment provides rationale for future trials, potentially combining immunotherapy with axitinib as used in the metastatic setting. The potential increase in myeloid cell activation is significant, as previous studies have shown myeloid activation may alter the effects of immunotherapy. Understanding the interactions between the cells present, and how they change on treatment is therefore critical for the success of these therapies. Clinical trial information: NCT03494816.

Abstract P5-16-13: Safety and efficacy of INT230-6, a potential first-in-class intratumoral therapy, in monotherapy and in combination with pembrolizumab: Results from the IT-01 study [KEYNOTE-A10] in subjects with locally advanced, unresectable and metastatic breast cancer

Abstract Background: INT230-6 is a novel formulation of cisplatin and vinblastine with an amphiphilic cell penetration enhancer that has been shown to enhance dispersion of the drug throughout tumors and allow diffusion into cells when given intratumorally. INT230-6 is being evaluated in monotherapy and in combination with immune checkpoint inhibitors (ICIs) in subjects with various advanced solid tumors, including advanced breast cancer. Methods: This phase 1/2 study evaluated INT230-6 in superficial and deep tumors with INT230-6 Q2W intratumoral injections for 5 doses alone or in combination with 200mg pembrolizumab IV Q3W for 2 years. Total INT230-6 injected in a subject ranged from 0.89 to 649 mL over 5 INT230-6 dosing sessions in each subject, except one subject who had only 2 dosing sessions. Subjects who had completed treatment in dose escalation cohorts were eligible for retreatment; and one subject was retreated with INT230-6 in multiple arms of the study. Advanced breast cancer subjects with an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or below who have failed one or more approved therapies, or have no alternate approved therapy, were enrolled. Subjects must have adequate organ function and measurable disease by RECIST 1.1 criteria including one target tumor for injection. Tumor response using RECIST 1.1 was evaluated at 12 weeks from the first INT230-6 dose and then every 8 weeks. Tumor biopsies were taken prior to INT230-6 dosing on day 0 and on day 28 post dose. Results: 7 advanced triple negative breast cancer subjects (4 monotherapy, 3 pembrolizumab combination) were evaluable as of June 1, 2021. The median age was 56 (range 46-82) years old, with a median of 8 (2, 17) prior systemic therapies for metastatic disease. The intratumoral INT230-6 dose was up to 164 mL (82 mg of CIS, 16.4mg VIN) to tumors in a single dosing session. With INT230-6, 133-200% more volume is injected into the tumor and pharmacokinetics (PK) analysis shows that 95% of INT230-6 active agents remain in the tumor. Accordingly, assessment of tumor response using RECIST principles may be challenging, and even stable disease may represent a large decrease in viable tumor cells as indicated by biopsy evaluations. The most common (&amp;gt;20%) related treatment related adverse events (AE) were localized tumor related pain (71%), nausea (57%), anemia (29%), fatigue (29%), neck pain (29%), and vomiting (29%). AEs were mostly low grade and only one subject experienced grade 3 anemia (13%). There were no related grade 4 or 5 AEs or serious AEs. Disease control rate (DCR), defined as the percent of patients with a complete response, partial response, or stable disease at the first radiologic assessment, was 57%. Median overall survival was 12 months. Pre- and post- biopsy at 28 days after two INT230-6 doses (n= 3 evaluable, monotherapy, and combination with pembrolizumab) showed a 55% decrease in Ki67 and 69% reduction in viable cancer cells. In addition, multiplex immunofluorescence (n= 3 evaluable, combination with pembrolizumab) showed an influx of activated CD4 and CD8 T cells and in some cases a reduction in FoxP3 T-reg cells.. Conclusion: INT230-6 is a potential first-in-class intratumoral therapy for advanced breast cancer being developed in monotherapy and in combination with ICIs. There is a favorable safety profile in this population, similar to the broader metastatic solid tumor population presented elsewhere. There are early signs of cancer cell death in injected tumors and immune activation in heavily pre-treated patients. A Phase 2 expansion cohort of INT230-6 in combination with ICIs is ongoing. In addition, INT230-6 in being studied in a separate randomized Phase 2 neoadjuvant breast cancer study. Citation Format: Philippe Bedard, Lillian L Siu, Jacob Thomas, Diana Hanna, Anthony J Olszanski, Nilofer Azad, Giles Whalen, Matthew Ingham, Syed Mahmood, Lewis H Bender, Ian B Walters, Anthony El-Khoueiry. Safety and efficacy of INT230-6, a potential first-in-class intratumoral therapy, in monotherapy and in combination with pembrolizumab: Results from the IT-01 study [KEYNOTE-A10] in subjects with locally advanced, unresectable and metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-13.

Use of Pralsetinib as Neoadjuvant Therapy for Non-Small Cell Lung Cancer Patient With RET Rearrangement.

RET rearrangements are rare, and occur in 1%-2% of all non-small cell lung cancer (NSCLC) patients. Pralsetinib has a significant anti-tumor effect in patients with advanced NSCLC and a RET rearrangement. Previous studies have confirmed the efficiency of neoadjuvant target therapy for NSCLC. Herein we present a case involving a female patient who was diagnosed with stage IIIA lung adenocarcinoma and harbored a KIF5B-RET rearrangement based on next-generation sequencing. Radiologic downstaging was indicated after pralsetinib treatment. Therefore, a right lower lobectomy and systemic lymphadenectomy were successfully performed. The postoperative pathologic results revealed a response rate of 74% for primary tumor and no residual viable tumor cells were observed in lymph nodes. The tumor, nodes, and metastases (TNM) stage was ypT1cN1M0. The tumor micro-environment (TME) of the primary tumor was also assessed.

Efficacy and toxicity of neoadjuvant immune checkpoint inhibitors in resectable gastric cancer: A meta-analysis and systematic review.

291 Background: Immune checkpoint inhibitors (ICI) have made significant breakthroughs in late-stage gastric cancer. It is an attractive issue whether ICIs also function in the neoadjuvant setting. Methods: A systematic review was performed using combined terms related to “stomach cancer”, “gastric cancer”, “gastroesophageal cancer”, “immune checkpoint inhibitor”, “PD-1”, “PD-L1” and “neoadjuvant” in PubMed and annual meeting of ASCO, ASCO GI, ESMO, and ESMO GI from 2019 to 2021. Complete pathological response (CPR) rates, major pathological response (MPR) rates, R0 resection rates, and side effects were pooled and analyzed. MPR was defined as ≤10% viable tumor cells and included CPR. Study outcomes were pooled using the function METAPHOR in the META package in R.3.6.1. The I2 and P statistics were used to evaluate heterogeneity among studies. Funnel plots were used for publication bias assessment. Results: A total of 13 phase I/II clinical trials, including 332 resectable gastric cancer (T2-4 or N+) patients with neoadjuvant ICI-containing treatments, were collected. The pooled rates of CPR, MPR, and R0 resection were 0.16 (95% credible intervals (CI), 0.12-0.22), 0.36 (95% CI, 0.24-0.51), and 0.97 (95% CI, 0.94-0.99), respectively. As a comparison, outcomes from 25 studies on neoadjuvant chemotherapy were also pooled, with rates of CPR 0.08 (95% CI, 0.06-0.11), MPR 0.22 (95% CI, 0.19-0.26), and R0 resection 0.84 (95% CI, 0.80-0.87). Besides, the overall grade 3 or higher toxicity rates were 0.24 (95% CI 0.03-0.54) vs. 0.28 (95% CI 0.13-0.47) in ICI-based treatment and chemotherapy groups. Stratified by treatment strategies, ICI alone showed the lowest efficacy (CPR 0.07, 95% CI 0.02-0.19 and MPR 0.16, 95% CI 0.03-0.29). Addition of chemotherapy to ICI promoted the CPR (0.15, 95% CI 0.10-0.22) and MPR (0.36, 95% CI 0.22-0.50). Further addition of radiotherapy displayed the highest CPR (0.35, 95% CI 0.21-0.52) and MPR (0.74, 95% CI 0.54-0.93). Only four trials reported the outcomes of dMMR/MSI-H patients, with superior CPR (0.39, 95% CI 0.20-0.62) and MPR (0.82, 95% CI 0.82-1.00), while those of pMMR/MSS patients were 0.05 (95% CI 0.00-0.13) and 0.20 (95% CI 0.20-0.31) respectively, among which 2 trials used ICI only. Conclusions: Neoadjuvant ICI plus chemotherapy/radiotherapy, instead of ICI alone, has better pathological responses and R0 resection rates than chemotherapy. dMMR/MSI-H is a superior biomarker for neoadjuvant ICI therapy for resectable gastric cancer. The value or biomarker of immunotherapy in pMMR/MSS patients remains exploring.[Table: see text]

Tumor Immune-Infiltrate Landscape After Chemo-Radiotherapy in a Case Series of Patients with Non-small Cell Lung Cancer: Pretreatment Predictors and Correlation With Outcome.

IntroductionData on tumor immune-milieu after chemo-radiation (CT-RT) are scarce. Noninvasive tools are needed to improve the treatment of non–small cell lung cancer (NSCLC), especially in the locally advanced (LA) setting.MethodsWe collected a series of superior-sulcus (SS)- patients with NSCLC referred to our Institute (2015-2019), eligible for a preoperative CT-RT. We characterized tumor-infiltrating immune cells (TIICs), determined PD-L1-TPS and the residual viable tumor cells (RVTC). Radiological and metabolic responses were reviewed. We calculated pre-surgery neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR).ResultsEight patients were included. Radiological responses were 6 disease stabilities (SD) and 2 partial responses (PR). Metabolic responses were 4 SD and 4 PR. CD68+-TIICs were correlated with metabolic response and lower RVTC. CD68+-TIICs were associated with higher PLR. Higher PLR values seemed linked with lower RVTC.ConclusionsThese preliminary results could be useful for consolidation treatment selection for patients with LA-NSCLC without evaluable baseline PD-L1 and higher PLR values.

Deeper sections reveal residual tumor cells in rectal cancer specimens diagnosed with pathological complete response following neoadjuvant treatment.

Guidelines and requirements for diagnosing pathological complete response (pCR) in rectal adenocarcinoma following neoadjuvant treatment vary, and there is currently no consensus on the appropriate number of sections to examine per formalin-fixed, paraffin-embedded (FFPE) tissue block. The consequences of systematic use of deeper sections on the diagnostic accuracy and prognosis for patients classified as ypT0 rectal cancer were investigated. In this retrospective study, 23 out of 155 patients who underwent neoadjuvant therapy and surgical resection between 2015 and 2020 were diagnosed with ypT0 rectal cancer. Three additional deeper sections were cut from each FFPE block from the primary tumor site and reviewed for presence of residual tumor cells. Additional sections revealed residual viable tumor cells in seven patients (30.4%), reducing the rate of pCR in the cohort from 14.8 to 10.3%. Of the seven patients, three patients later had local recurrence or distant metastasis during the follow-up period, compared with one patient with no residual tumor cells in deeper sections (p = 0.07). A nonsignificant reduction in disease-free survival (p = 0.08) was observed in the patients with residual tumor. Systematic use of deeper sections in evaluation of tumor regression in rectal cancer reveals the presence of residual tumor cells in a subset of patients originally diagnosed with pCR based on a single section per FFPE block. Although the results are not statistically significant, it cannot be excluded that accurately distinguishing complete from near-complete response may be clinically relevant for prognostic prediction.

Commentary: Cryotherapy and the sebaceous glands.

Sebaceous glands are exocrine epithelial glands secreting protective surface lipids via a holocrine mechanism in which the sebocytes undergo a programmed disintegration. Sebaceous glands are found in the eyelids as the tarsal meibomian glands and the glands of Zeis near eyelash hair follicles.[1] Pathologically, the ophthalmic sebaceous gland lesions range from benign cysts and chalazia to malignant sebaceous gland carcinoma (SGC), which happens to be the most common eyelid malignancy in the Indian population.[2] Cryotherapy has been extensively used in ophthalmology, dermatology, and surgical specialties as a primary or adjunctive treatment for lesions ranging from intraocular tumors, eyelid cysts, skin tags, warts, and acne vulgaris to cervical and hepatic cancers.[3] There are two mechanisms of cryotherapy: (a) freezing-related direct cellular injury and (b) vascular disruption of the lesion. Its role in the treatment of localized basal cell carcinomas of the eyelid has been documented in the literature with a low recurrence rate.[2] The prolonged healing time with secondary intention healing and depigmentation remains a concern with cryotherapy of the skin tumors. However, it helps in preserving the functionality of essential structures like eyelids and the surrounding lacrimal system. The in vivo biological effect of cryotherapy on normal sebaceous glands has been evaluated with the help of coherent anti-Stokes Raman scattering microscopy by Jung et al.[4] They demonstrated that the effect of lipid crystallization had a limited role in causing cellular disruption. Instead, sebocyte loss occurred at temperatures lower than those required for lipid crystallization. In addition, the cryo-treated sebaceous glands began recovering 1–2 weeks after treatment. Ray Jalian et al.[5] also investigated the role of controlled local skin cooling in causing preferential injury to sebaceous glands to understand its mechanism in treating acne vulgaris. They observed that cooling-induced damage led to a 20% reduction in sebum output for 2 weeks, with minimal collateral injury to surrounding tissues. A higher number of freeze–thaw cycles and slower thawing resulted in more significant tissue injury. Immunohistochemistry-based expression of Ki67 (a proliferative marker) and keratin 15 (a progenitor basal cell marker) was not disrupted by cooling. This led them to conclude that cooling-induced damage was temporary and occurred due to disruption of cellular architecture, enzymatic activity, and decreased lipid content. The sebum output recovered after 4 weeks. Though both these studies used a temperature higher than what is used in ophthalmic practice, they did conclusively demonstrate the limited level of cytotoxicity as well as the temporary duration of the effect of cryotherapy on sebaceous glands. The ophthalmic literature describes the use of cryotherapy as an adjunct in addressing the pagetoid spread seen in eyelid SGC.[6] Lisman (1989) first reported the use of cryotherapy to manage the map biopsy-proven sites of pagetoid spread in six cases of sebaceous gland carcinoma, thereby avoiding exenteration.[7] Shields et al.[6] advocated the application of triple freeze cryotherapy on conjunctival edges to eliminate residual tumor cells. On the contrary, Esmaeli et al.[8] reported satisfactory outcomes in 50 patients of SGC without the need for cryotherapy. A survey of practice patterns in Nordic countries revealed that only 27% of respondents offered cryotherapy as an adjunctive treatment for addressing the pagetoid spread in SGC.[9] Thus, the biological effect of cryotherapy on sebaceous glands is limited and reversible. The reassurance provided by triple freeze and slow thaw to the margins may not be reliable in clinical practice. This fact has been highlighted by Alam et al.[10] in the present issue of this journal. They performed a histopathological study on a portion of excised SGC tumor that was subjected to cryotherapy. Greater than 50% of viable tumor cells along with extensive areas of necrosis were observed on microscopy, and the authors concluded that the cell damage induced by cryotherapy is insufficient for achieving adequate tumor control. We believe that an early diagnosis and timely treatment using a wide (~3-mm margin) full-thickness excision, intraoperative frozen section control, and map biopsy for pagetoid spread remains the preferred practice pattern. The adjunctive use of cryotherapy remains doubtful in the SGC of eyelids. Close monitoring and regular follow-up to observe for recurrences are vital in the long-term management of patients.

The effect of direct cell injury inflicted by cryotherapy on eyelid sebaceous gland carcinoma cells: An ex-vivo experimental study.

Purpose:To evaluate the effect of direct cell injury of cryotherapy on eyelid sebaceous gland carcinoma cells by an ex vivo cryotherapy experiment.Methods:It was a prospective interventional case series. Six patients with biopsy-proven nodular sebaceous gland carcinoma were included. After excision of the mass, a thin slice of the mass resembling the thickness of the conjunctiva was shaved off and was oriented over the broad end of a tissue forceps. Cryotherapy was applied to both its anterior and posterior aspects by the triple freeze-thaw technique. The mass was then labeled and sent separately for histopathological evaluation by fixation and staining.Results:A total of six patients with a mean age of 58.2 ± 15.5 years were included. There were four females and two males. The mean duration of the lesion was 21.6 ± 17.51 months. All patients had involvement of the upper eyelid. The patients were clinically staged as T2b (n=2), T1a (n=2), T2c (n=1), and T3a (n=1) respectively. There was no regional lymphadenopathy or metastasis in any of the cases. The experimental cryo-tissue containing the cryo-treated lesion revealed the presence of viable tumor cells (>50%) in all six specimens.Conclusion:The direct cell injury caused by cryotherapy may not be sufficient to kill all the residual sebaceous gland carcinoma cells on the tumor bed.

The Apoptosis Paradox in Cancer.

Cancer growth represents a dysregulated imbalance between cell gain and cell loss, where the rate of proliferating mutant tumour cells exceeds the rate of those that die. Apoptosis, the most renowned form of programmed cell death, operates as a key physiological mechanism that limits cell population expansion, either to maintain tissue homeostasis or to remove potentially harmful cells, such as those that have sustained DNA damage. Paradoxically, high-grade cancers are generally associated with high constitutive levels of apoptosis. In cancer, cell-autonomous apoptosis constitutes a common tumour suppressor mechanism, a property which is exploited in cancer therapy. By contrast, limited apoptosis in the tumour-cell population also has the potential to promote cell survival and resistance to therapy by conditioning the tumour microenvironment (TME)—including phagocytes and viable tumour cells—and engendering pro-oncogenic effects. Notably, the constitutive apoptosis-mediated activation of cells of the innate immune system can help orchestrate a pro-oncogenic TME and may also effect evasion of cancer treatment. Here, we present an overview of the implications of cell death programmes in tumour biology, with particular focus on apoptosis as a process with “double-edged” consequences: on the one hand, being tumour suppressive through deletion of malignant or pre-malignant cells, while, on the other, being tumour progressive through stimulation of reparatory and regenerative responses in the TME.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

Total Variation Constrained Graph Manifold Learning Strategy for Cerenkov Luminescence Tomography.

Harnessing the power and flexibility of radiolabeled molecules, Cerenkov luminescence tomography (CLT) provides a novel technique for non-invasive visualisation and quantification of viable tumour cells in a living organism. However, owing to the photon scattering effect and the ill-posed inverse problem, CLT still suffers from insufficient spatial resolution and shape recovery in various preclinical applications. In this study, we proposed a total variation constrained graph manifold learning (TV-GML) strategy for achieving accurate spatial location, dual-source resolution, and tumour morphology. TV-GML integrates the isotropic total variation term and dynamic graph Laplacian constraint to make a trade-off between edge preservation and piecewise smooth region reconstruction. Meanwhile, the tetrahedral mesh-Cartesian grid pair method based on the k-nearest neighbour, and the adaptive and composite Barzilai-Borwein method, were proposed to ensure global super linear convergence of the solution of TV-GML. The comparison results of both simulation experiments and in vivo experiments further indicated that TV-GML achieved superior reconstruction performance in terms of location accuracy, dual-source resolution, shape recovery capability, robustness, and in vivo practicability. Significance: We believe that this novel method will be beneficial to the application of CLT for quantitative analysis and morphological observation of various preclinical applications and facilitate the development of the theory of solving inverse problem.

Neoadjuvant Chemotherapy for High-risk Intrahepatic Cholangiocarcinoma – Does Pathologic Response Mean Better Outcomes?

Introduction: We analyzed our experience with neoadjuvant chemotherapy (NAC) for patients with high-risk intrahepatic cholangiocarcinoma (IHC) focusing on the significance of pathologic response as a predictor of survival. Methods: A single-institution, prospectively maintained database was analyzed for patients with IHC who were treated with chemotherapy prior to definitive resection from 2006-2019. The tumor specimens were assessed for treatment response and survival outcomes were analyzed. Major pathologic response was defined as < 50% viable tumor cells. Results: Our study includes 45 patients who received NAC followed by hepatectomy for IHC, 40 of which (89%) were considered stage III at time of diagnosis. Indications for NAC consisted of nodal involvement or poor performance status in 38 patients (84%) and initially unresectable disease in 7 patients (16%). NAC regimens included: gemcitabine and cisplatin (64%); gemcitabine, cisplatin and nab-paclitaxel (27%); gemcitabine and carboplatin (4%); and FOLFOX (4%). For the entire study cohort, median overall survival (OS) was 45 months and recurrence free survival (RFS) was 9.7 months. 39% of patients had major response, including 2 with complete response, and 61% had minor response. Pathologic response was not associated with median RFS (11 months major response vs. 14 months minor response, p=0.7) or OS (44 months major response vs. 75 months minor response, p=0.9) (Figure 1). Conclusions: Pathologic response to NAC is not associated with improved survival. Still, OS following NAC for these high-risk patients with IHC is encouraging. Future studies should focus on objective biomarkers of response that may better prognosticate survival.