Randomized open-label controlled study of cancer vaccine OSE2101 versus chemotherapy in HLA-A2-positive patients with advanced non-small-cell lung cancer with resistance to immunotherapy: ATALANTE-1.

BACKGROUND: Patients with advanced non-small-cell lung cancer (NSCLC) treated with immune checkpoint blockers (ICBs) ultimately progress either rapidly (primary resistance) or after durable benefit (secondary resistance). The cancer vaccine OSE2101 may invigorate antitumor-specific immune responses after ICB failure. The objective of ATALANTE-1 was to evaluate its efficacy and safety in these patients. PATIENTS AND METHODS: ATALANTE-1 was a two-step open-label study to evaluate the efficacy and safety of OSE2101 compared to standard-of-care (SoC) chemotherapy (CT). Patients with human leukocyte antigen (HLA)-A2-positive advanced NSCLC without actionable alterations, failing sequential or concurrent CT and ICB were randomized (2 : 1) to OSE2101 or SoC (docetaxel or pemetrexed). Primary endpoint was overall survival (OS). Interim OS futility analysis was planned as per Fleming design. In April 2020 at the time of interim analysis, a decision was taken to prematurely stop the accrual due to coronavirus disease 2019 (COVID-19). Final analysis was carried out in all patients and in the subgroup of patients with ICB secondary resistance defined as failure after ICB monotherapy second line ≥12 weeks. RESULTS: Two hundred and nineteen patients were randomized (139 OSE2101, 80 SoC); 118 had secondary resistance to sequential ICB. Overall, median OS non-significantly favored OSE2101 over SoC {hazard ratio (HR) [95% confidence interval (CI)] 0.86 [0.62-1.19], P = 0.36}. In the secondary resistance subgroup, OSE2101 significantly improved median OS versus SoC [11.1 versus 7.5 months; HR (95% CI) 0.59 (0.38-0.91), P = 0.017], and significantly improved post-progression survival (HR 0.46, P = 0.004), time to Eastern Cooperative Oncology Group (ECOG) performance status deterioration (HR 0.43, P = 0.006) and Quality of Life Questionnaire Core 30 (QLQ-C30) global health status compared to SoC (P = 0.045). Six-month disease control rates and progression-free survival were similar between groups. Grade ≥3 adverse effects occurred in 11.4% of patients with OSE2101 and 35.1% in SoC (P = 0.002). CONCLUSIONS: In HLA-A2-positive patients with advanced NSCLC and secondary resistance to immunotherapy, OSE2101 increased survival with better safety compared to CT. Further evaluation in this population is warranted.

IFCT-1502 CLINIVO: real-world evidence of long-term survival with nivolumab in a nationwide cohort of patients with advanced non-small-cell lung cancer.

BACKGROUND: Immunotherapy using inhibitors targeting immune checkpoint programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) is currently the standard of care in patients with advanced non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: We carried out a nationwide cohort retrospective study of consecutive patients with advanced, refractory NSCLC who received nivolumab as second to later lines of treatment as part of the expanded access program. Key objectives were to assess the efficacy and safety of nivolumab and the efficacy of first post-nivolumab treatment. RESULTS: Nine hundred and two patients were enrolled: 317 (35%) with squamous cell carcinoma and 585 (65%) with non-squamous cell carcinoma. Median age was 64 years; there were 630 (70%) men, 795 (88%) smokers, 723 (81%) patients with an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0/1, 197 (22%) patients with brain metastases, and 212 (27%) with liver metastases. Best response was partial response for 16.2% and stable disease (SD) for 30.5%. Progression-free survival and overall survival (OS) rates at 2, 3, and 5 years were 8% and 25%, 6% and 16%, and 4% and 10%, respectively. At multivariate analysis, ECOG PS ≥2 [hazard ratio (HR) = 2.13, 95% confidence interval (95% CI) 1.78-2.55, P < 0.001], squamous histology (HR = 1.17, 95% CI 1.01-1.36, P = 0.04), and presence of central nervous system metastases (HR = 1.29, 95% CI 1.08-1.54, P = 0.005) were significantly associated with lower OS. Four hundred and ninety-two patients received at least one treatment after discontinuation of nivolumab, consisting of systemic therapies in 450 (91%). Radiation therapy was delivered to 118 (24%) patients. CONCLUSION: The CLINIVO cohort represents the largest real-world evidence cohort with the use of immune checkpoint inhibitor in advanced, metastatic NSCLC after failure of first-line chemotherapy, with long-term follow-up and analysis of subsequent therapies. Our data confirm the efficacy of nivolumab in a cohort larger than that reported in landmark clinical trials and identify prognostic factors, which reinforces the need for accurate selection of patients for treatment with immune checkpoint inhibitors. Our data indicate that oligoprogression is frequent after nivolumab exposure and provide a unique insight into the long-term survival.

Homozygous deletions inactivate DCC, but not MADH4/DPC4/SMAD4, in a subset of pancreatic and biliary cancers.

Loss of heterozygosity (LOH) of chromosome arm 18q is frequent in gastrointestinal cancers. Over 90% of pancreatic carcinomas have 18q LOH. Bi-allelic inactivation of the MADH4/DPC4/SMAD4 gene at 18q21.1 is seen in about half of pancreatic carcinomas with 18q LOH. In the remaining tumors with 18q LOH, MADH4 is not mutated and its expression is unaffected, and no alterations in MADH2/SMAD2, a MADH4-related gene at 18q12.3, have been found. A controversial candidate tumor-suppressor gene at 18q21.2 is DCC (deleted in colorectal carcinoma), which encodes a netrin-1 receptor component with functions in cell migration and apoptosis. Reduced or absent DCC expression has been observed in many cancers, but few somatic mutations that would clearly inactivate DCC function have been reported. We studied a panel of 115 pancreatic and 14 biliary cancers for homozygous deletions of DCC exons and flanking 18q regions. Seven homozygous deletions were seen in the region that includes the DCC gene. In two tumors, the deletions inactivate DCC but not MADH4. A physical and transcript map of the deleted regions was constructed, and DCC was the only known gene affected by all seven deletions. These data are the strongest mutational evidence presented yet in support of the hypothesis that DCC or another gene in the region distal to MADH4 is inactivated, playing a causal role in cancer development.

Genomic FHIT analysis in RER+ and RER- adenocarcinomas of the pancreas.

Alterations of the candidate tumor suppressor gene FHIT have been reported in multiple tumor types, including pancreatic carcinoma. The mechanism of FHIT genomic inactivation is unusual, most frequently occurring by homozygous deletion, whereas only rare cases have missense mutations. Altered (shortened) transcripts and reduced protein expression are reported, but a genetic basis for these is often inapparent. We studied FHIT genomic alterations of pancreatic carcinomas. Loss of heterozygosity (LOH) was found in 41% of 93 carcinomas without microsatellite instability (RER(-)), but no mutations were found by genomic sequencing. Homozygous deletions inside the FRA3B fragile site were found in four RER(-) tumors, but only two affected the FHIT coding region. In contrast, FHIT alterations were found in the three RER(+) pancreatic carcinomas screened; two had FHIT homozygous deletions affecting exon 5 and the third had a heterozygous missense mutation (H76N). The excess occurrence of homozygous deletions at this site in RER(+) pancreatic cancers is statistically significant (P < 0.01). Since homozygous deletions have not previously been reported in RER(+) carcinomas at any genomic site, an extremely high rate of site-specific deletion must exist within the FRA3B-related FHIT gene. Consequently, the paucity of documented inactivating point mutations cannot be used to judge the presence or absence of putative FHIT-related selective pressures that act during tumorigenesis of RER(-) neoplasia. Nonetheless, the identification of a heterozygous mutation as the sole sequence abnormality might raise doubt as to whether strong selective pressures are afforded by FHIT genomic inactivation in this tumor type. Genes Chromosomes Cancer 27:239-243, 2000.

Antigenic phenotyping of lymphoid cells and B cell gene rearrangement in type B gastritis and in gastritis not associated with Helicobacter pylori colonization.

Marginal-zone B cells of the mucosa-associated lymphoid tissue (MALT) are the normal counterpart of the neoplastic cells in MALT lymphoma. In both cases these lymphocytes express surface immunoglobulins, but are negative when stained for B cell associated antigens like CD10 and CD23. Furthermore, the B cell gene rearrangement has been found in Helicobacter pylori associated chronic gastritis and in extranodal type of marginal-zone lymphoma. The aim of this study was to quantify the number of IgM-, CD10-, and CD23-positive lymphocytes in patients with type B gastritis and to compare the results with the antigen profile of mononuclear cells in patients with gastritis not associated with H. pylori. Additionally, the immunoglobulin heavy-chain (IgH) gene rearrangement in H. pylori positive and H. pylori negative gastritis was studied. From 23 patients with a positive urease test and/or histologically proven H. pylori infection and chronic gastritis and from 22 patients with H. pylori negative chronic gastritis mucosa biopsy specimens were taken. Single-cell suspensions were obtained following enzymatic digestion. For immunocytochemistry, an alkaline phosphatase-antialkaline phosphatase method was applied. IgH gene rearrangement in formalin-fixed, paraffin-embedded specimens was determined by polymerase chain reaction in 11 patients with chronic gastritis. An increase in mu-positive plasma cells and B lymphocytes was detected in patients with H. pylori positive gastritis as compared with patients with H. pylori negative gastritis (10.0 vs. 3.9%, p < 0.001, and 4.3 vs. 1.6%, p < 0.01, respectively). In both groups, the proportion of CD10- and CD23-positive lymphocytes was <1%. IgH gene rearrangement was not restricted to type B gastritis; single bands were also present in 3 of 7 patients with H. pylori negative chronic gastritis. Our finding of IgH gene rearrangement in some of the patients with H. pylori negative chronic gastritis indicates that additional factors may be critical for these genotypical changes and for the pathogenesis of gastric MALT lymphoma.

Molecular genetic basis of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma is a complex genetic disease. As might be expected for a malignancy that is rather homogeneous in clinical presentation and behavior, a distinct subset of genes are found to be genetically inactivated in a majority of the tumors. A yet larger subset of genes experiences genetic inactivation at much lower frequencies. The latter subset could solely reflect a somewhat trivial genetic heterogeneity of the tumor, but more likely will represent the initial insights into pathways whose more widespread importance will be shown in future work. Familial pancreatic cancer susceptibility underlies a significant fraction of the overall incidence. Genetic testing is feasible for many of the causative genes, although the clinical utility remains unsettled. The precursor lesion for pancreatic cancer shares some of the genetic lesions of the more advanced invasive stage, and follows a stepwise progression model both histologically and genetically. Genes Chromosomes Cancer 26:1-12, 1999.

Novel homozygous deletions of chromosomal band 18q22 in pancreatic adenocarcinoma identified by STS marker scanning.

The identification of homozygous deletions in sporadic neoplasms has been pivotal in the positional cloning of several tumor suppressor genes. Chromosomal arm 18q harbors the DPC4, SMAD2, and DCC genes and is suspected on the basis of high frequencies of allelic loss to harbor additional tumor suppressor genes. We applied high-resolution sequence-tagged site (STS) marker scanning to a panel of 106 pancreatic adenocarcinomas to identify novel regions of homozygous deletions on 18q. Three homozygous deletions were identified. Physical mapping of these deletions showed them to be nonoverlapping, but clustered in an approximately 7- to 10-Mb region of chromosome band 18q22. Each deletion spanned physical distances of nearly 1.3 to 3 Mb. A number of transcribed genes map within these deletions. The identification of these homozygous deletions might aid in the identification of novel tumor suppressor genes on chromosomal arm 18q. Genes Chromosomes Cancer 25:370-375, 1999.

High-resolution deletion mapping of chromosome arm 1p in pancreatic cancer identifies a major consensus region at 1p35.

Chromosomal arm 1p has long been suspected, on the basis of loss of heterozygosity (LOH) and other data, to harbor a tumor suppressor gene important in pancreatic carcinomas and other tumors. We constructed a high-resolution map of LOH at I p in a panel of pancreatic adenocarcinomas. Using 44 markers, we identified LOH on I p in 49% of 43 cancers. Breakpoints in 1p were identified in 15 of the carcinomas and could be used to ascertain consensus patterns. We found a major consensus region of LOH at 1p35 between loci D1S233 and D1S247. This region participates in the majority of LOH events on 1 p in pancreatic cancer. These data provide a roadmap for further regional mapping, homozygous deletion searches, comparison to LOH patterns seen in other tumor types, and prioritization of studies using candidate genes.

Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.

The clinical and pathological features of carcinomas of the pancreas with DNA replication errors (RER+) have not been characterized. Eighty-two xenografted carcinomas of the pancreas were screened for DNA replication errors using polymerase chain reaction amplification of microsatellite markers. Cases with microsatellite instability in at least two markers of a minimum of five tested were considered RER+. RER status was correlated with histological appearance, karyotype of the carcinomas when available, K-ras mutational status, and patient outcome. Three (3.7%) of the eighty-two carcinomas were RER+. In contrast to typical gland-forming adenocarcinomas of the pancreas, all three RER+ carcinomas were poorly differentiated and had expanding borders and a prominent syncytial growth pattern. Neither a Crohn's-like lymphoid infiltrate nor extracellular mucin production were prominent. Ductal adenocarcinomas of the pancreas typically contain a mutant K-ras gene, yet all three RER+ carcinomas had wild-type K-ras. One of the three RER+ carcinomas was karyotyped and showed a near diploid pattern. All three of the RER+ tumors were removed via Whipple resection. One of the three patients is free of disease 16 months after pancreaticoduodenectomy, one is alive and free of tumor at 52 months but developed two colon carcinomas during this period, and the third died of pancreatic cancer at 4 months. None of the three patients had a family history of colorectal carcinoma. A review of the K-ras wild-type carcinomas in a previously characterized series of pancreatic carcinomas with known K-ras mutational status identified two additional cancers with poor differentiation, a syncytial growth pattern, and pushing borders. Both of the cancers were diploid and both patients were longterm survivors (over 5 years). The inclusion of such patients in previous prognostic studies of pancreas cancer may explain the failure of histological grade to be a predictor of prognosis. These data suggest that DNA replication errors occur in a small percentage of resected carcinomas of the pancreas and that wild-type K-ras gene status and a medullary phenotype characterized by poor differentiation, and expanding pattern of invasion, and syncytial growth should suggest the possibility of DNA replication errors in carcinomas of the pancreas.

Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene.

Mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) is a component of a stress and cytokine-induced signal transduction pathway involving MAPK proteins. The MKK4 protein has been implicated in activation of JNK1 and p38 MAPK on phosphorylation by conserved kinase pathways. A recent report on the deletion and mutation of the MKK4 gene in human pancreatic, lung, breast, testicle, and colorectal cancer cell lines suggests an additional role for MKK4 in tumor suppression. Both the gene function and the infrequency of mutations might be considered atypical for many human tumor suppressor genes, and constitutional DNA was not previously available to determine whether the reported sequence variants had preceded tumor development. Here, we report that homozygous deletions are detected in 2 of 92 pancreatic adenocarcinomas (2%), 1 of 16 biliary adenocarcinomas (6%), and 1 of 22 breast carcinomas (when combined with reported sequence alterations, 3 of 22 or 14%). In addition, in a panel of 45 pancreatic carcinomas prescreened for loss of heterozygosity, one somatic missense mutation of MKK4 is observed and confirmed in the primary tumor (2%). Mapping of the homozygous deletions further indicated MKK4 to lie at the target of deletion. The finding of a somatic missense mutation in the absence of any other nucleotide polymorphisms or silent nucleotide changes continues to favor MKK4 as a mutationally targeted tumor suppressor gene. Coexistent mutations of other tumor suppressor genes in MKK4-deficient tumors suggest that MKK4 may participate in a tumor suppressive signaling pathway distinct from DPC4, p16, p53, and BRCA2.

Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumors.

PTEN/MMAC1 is a candidate tumor suppressor gene recently identified at chromosomal band 10q23. It is mutated in sporadic brain, breast, and prostate cancer and in the germ line of patients with hereditary Cowden disease. We searched for genetic alterations of the PTEN/MMAC1 gene in 39 primary head and neck cancers (HNSCCs), 42 primary non-small cell lung cancers (NSCLCs), 80 pancreatic cancer xenografts, and 37 cell lines and xenografts from colon, lung, and gastric cancers. Microsatellite analysis revealed loss of heterozygosity at markers near the gene in 41% of primary HNSCCs, 50% of NSCLCs, and 39% of the pancreatic cancers. Three cases of HNSCCs displayed homozygous deletion involving the gene. We sequenced the entire coding region of the PTEN/MMAC1 gene in the remaining tumors displaying loss of heterozygosity and found one terminating mutation in a HNSCC sample. Thus, a second inactivation event was observed in 4 of 39 primary HNSCC cases. By use of a protein truncation assay, one terminating mutation was also identified in one of eight NSCLC cell lines. Our results suggest that PTEN/MMAC1 gene inactivation plays a role in the genesis of some tumor types.

Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas.

The Rb/p16 tumor-suppressive pathway is abrogated frequently in human tumors, either through inactivation of the Rb or p16INK4a/CDKN2/MTS1 tumor-suppressor proteins, or through alteration or overexpression of the cyclin D1 or cyclin-dependent kinase 4 oncoproteins. We reported previously that the p16 gene was genetically inactivated in 82% of pancreatic carcinomas. Nearly half of these inactivations were by intragenic mutation of p16, and the remainder were by homozygous deletion of the gene. Here, we analyzed pancreatic carcinomas for additional mechanisms by which the Rb/p16 pathway might be inactivated. Transcriptional silencing of the p16 gene in association with methylation of its 5'-CpG island was examined by methylation-specific PCR in 18 pancreatic carcinomas. Nine of these were known to harbor an intragenic mutation in p16, and nine had a wild-type p16 coding sequence. Seven of the 18 tumors were hypermethylated, and all 7 were p16 wild-type (P = 0.001). Complete silencing of transcription from methylated wild-type gene sequences was demonstrated. Immunohistochemical analysis revealed normal expression levels of the Rb protein in all carcinomas studied. None of the carcinomas had genomic amplification of the cyclin D1 or CDK4 genes, and none had mutation of the p16-binding domain of CDK4. An additional p16 mutation was identified. In total, the Rb/p16 pathway was abrogated in 49 of the 50 carcinomas (98%) studied, all through inactivation of the p16 gene. Similar results were obtained in an independently analyzed series of 19 pancreatic carcinomas. These data demonstrate the central role of the Rb/p16 pathway in the development of pancreatic carcinoma.

Regulation and role of TGF beta production in breast cancer.

The influence of antiestrogens on the secretion of transforming growth factor beta (TGF beta) proteins that have an autoinhibitory potential for human cancer cells was studied in the estrogen-responsive human breast cancer cell line, MCF-7: Antiestrogens induce the secretion of TGF beta-1 via a nontranscriptional pathway; TGF beta-1 itself induces TGF beta-2 by a direct transcriptional mechanism; and TGF beta-2 is a marker of antiestrogen action. This hypothesis was confirmed in a clinical study with 18 patients with advanced metastatic breast cancer. TGF beta-2 plasma levels were measured before and after 4 weeks of treatment with tamoxifen. In the majority of patients who responded to the treatment, increasing TGF beta-2 concentrations were seen under therapy. Patients who did not respond did not show changes in the TGF beta-2 plasma level after 4 weeks of treatment. These results suggest that the sequential analysis of TGF beta-1 in plasma before and under treatment with tamoxifen allows the early identification of patients with antiestrogen resistance.